Bio

Clinical Focus


  • Plastic Surgery, Pediatric
  • Plastic Surgery

Academic Appointments


Professional Education


  • Residency:UCSF Medical Center (1993) CA
  • Internship:UCSF Medical Center (1985) CA
  • Medical Education:Harvard Medical School (1984) MA
  • Board Certification: General Surgery, American Board of Surgery (1994)
  • Board Recertification, American Board of Sugery, General Sugery (2004)
  • Board Certification, American Board of Plastic Surgery, Plastic Surgery (1998)
  • Board Recertification, American Board of Plastic Surgery, Plastic Surgery and Craniomaxillofacial (2009)
  • Residency:New York University Medical Center (1995) NY
  • Fellowship:UCLA - School of Medicine (1996) CA

Research & Scholarship

Current Research and Scholarly Interests


Deane P. and Louise Mitchell Professor and Vice Chair
Co-Director, Institute of Stem Cell Biology and Regenerative Medicine
Director of the Program in Regenerative Medicine
Director, Children’s Surgical Research
Director of Research, Divison of Plastic & Reconstructive Surgery
Professor, by Courtesy, Department of Bioengineering
Professor, by Courtesy, Department of Materials Science and Engineering

Department of Surgery
Stanford University School of Medicine
Lucile Salter Packard Children’s Hospital

Dr. Michael T. Longaker joined the Stanford University School of Medicine on September 1, 2000 as Director of Children’s Surgical Research in the Department of Surgery, Division of Plastic and Reconstructive Surgery and the Lucile Salter Packard Children’s Hospital. In 2003, he was named the Deane P. and Louise Mitchell Professor and in 2010 became Vice Chair of the Department of Surgery. As Director of Program in Regenerative Medicine and Co-Director of the Institute for Stem Cell Biology and Regenerative Medicine, and Director of the Children’s Surgical Research, Dr. Longaker has the responsibility to develop research programs in the broad areas of developmental biology, epithelial biology and tissue repair, tissue engineering, and stem cell biology. Prior to joining Stanford, Dr. Longaker was the John Marquis Converse Professor of Plastic Surgery and held the positions of Director of Surgical Basic Science and Director of Plastic Surgery Research at the Institute of Reconstructive Plastic Surgery at the New York University School of Medicine.

Michael Longaker’s extensive research experience includes the cellular and molecular biology of extracellular matrix with specific applications to the differences between fetal and post-natal wound healing, the biology of keloids and hypertrophic scars, the cellular and molecular events in craniofacial development and stem cell biology. He brings to Stanford his unique understanding of wound healing, fetal wound healing research, developmental biology, tissue engineering, and stem cell biology.

Dr. Longaker is the recipient of the prestigous Flance-Karl Award from the American Surgical Association, the Jacobson Promising Investigatorn Award from the American College of Surgeons, and was a James IV Traveling Fellow. He is a member Association for Academic Surgeons, the Society of University Surgeons, American Surgical Association and American Society for Clinical Investigation, Association of American Physicians, and the Institute of Medicine of the National Academies of Science. He served as Treasuer and subsequeently President for the Society of University Surgeons. To date, he has published over 1050 publications and has numerous federal grants to support his research. He has recruited 6 faculty to the Children’s Surgical Research Program, all of whom are NIH funded.

Dr. Longaker earned his undergraduate degree at Michigan State University, (where he played varsity basketball and was a member of the 1979 NCAA Men’s Basketball Championship Team) and his medical degree at Harvard Medical School. He completed his general surgical residency at the University of California, San Francisco, a residency in Plastic Surgery at NYU and a craniofacial fellowship at UCLA. The majority of his research training took place while he was a Post Doctoral Research Fellow in the Fetal Treatment Program under Dr. Mike Harrison and in the laboratory of Dr. Michael Banda in Radiobiology, both at UCSF. In December 2003, Dr. Longaker earned his M.B.A. from University of California – Berkeley and Columbia University, in the inaugural class of their combined program and was elected into Columbia University's Beta Gamma Sigma Honor Society.

Teaching

2013-14 Courses


Publications

Journal Articles


  • Paracrine mechanism of angiogenesis in adipose-derived stem cell transplantation. Annals of plastic surgery Suga, H., Glotzbach, J. P., Sorkin, M., Longaker, M. T., Gurtner, G. C. 2014; 72 (2): 234-241

    Abstract

    INTRODUCTION: Adipose-derived stem cells (ASCs) have shown potential for cell-based therapy in the field of plastic surgery. However, the fate of ASCs after transplantation and the mechanism(s) of their biologic capabilities remain unclear. METHODS: We isolated and cultured ASCs from transgenic mice that express both luciferase and green fluorescent protein and injected the cells into the inguinal fat pads of wild-type mice. We tested 4 experimental groups, namely, ischemic fat pads with/without ASCs and control fat pads with/without ASCs. RESULTS: Transplanted ASCs were tracked with bioluminescence imaging. The luminescence gradually decreased over 28 days, indicating cell death after transplantation. More ASCs were retained in ischemic fat pads on day 7 compared to control fat pads. On day 14, adipose tissue vascular density was higher in the ASC transplantation groups compared to those without ASCs. On day 28, there was decreased atrophy of adipose tissue in ASC-treated ischemic fat pads. Transplanted ASCs were detected as nonproliferating green fluorescent protein-positive cells, whereas native endothelial cells adjacent to the transplanted ASCs were proliferative. Protein analysis demonstrated higher expression of hepatocyte growth factor and vascular endothelial growth factor in the ASC transplantation groups, suggesting a paracrine mechanism, which was confirmed by in vitro experiments with conditioned media from ASCs. CONCLUSIONS: Transplanted ASCs are preferentially retained in ischemic adipose tissue, although most of the cells eventually undergo cell death. They exert an angiogenic effect on adipose tissue mainly through a paracrine mechanism. Increased understanding of these effects will help develop ASCs as a tool for cell-based therapy.

    View details for DOI 10.1097/SAP.0b013e318264fd6a

    View details for PubMedID 23636112

  • Molecular analysis and differentiation capacity of adipose-derived stem cells from lymphedema tissue. Plastic and reconstructive surgery Levi, B., Glotzbach, J. P., Sorkin, M., Hyun, J., Januszyk, M., Wan, D. C., Li, S., Nelson, E. R., Longaker, M. T., Gurtner, G. C. 2013; 132 (3): 580-589

    Abstract

    Many breast cancer patients are plagued by the disabling complication of upper limb lymphedema after axillary surgery. Conservative treatments using massage and compression therapy do not offer a lasting relief, as they fail to address the chronic transformation of edema into excess adipose tissue. Liposuction to address the adipose nature of the lymphedema has provided an opportunity for a detailed analysis of the stromal fraction of lymphedema-associated fat to clarify the molecular mechanisms for this adipogenic transformation.Adipose-derived stem cells were harvested from human lipoaspirate of the upper extremity from age-matched patients with lymphedema (n = 3) or subcutaneous adipose tissue from control patients undergoing cosmetic procedures (n = 3). Immediately after harvest, adipose-derived stem cells were analyzed using single-cell transcriptional profiling techniques. Osteogenic, adipogenic, and vasculogenic gene expression and differentiation were assessed by quantitative real-time polymerase chain reaction and standard in vitro differentiation assays.Differential transcriptional clusters of adipose-derived stem cells were found between lymphedema and subcutaneous fat. Interestingly, lymphedema-associated stem cells had a much higher adipogenic gene expression and enhanced ability to undergo adipogenic differentiation. Conversely, they had lower vasculogenic gene expression and diminished capability to form tubules in vitro, whereas the osteogenic differentiation capacity was not significantly altered.Adipose-derived stem cells from extremities affected by lymphedema appear to exhibit transcriptional profiles similar to those of abdominal adipose-derived stem cells; however, their adipogenic differentiation potential is strongly increased and their vasculogenic capacity is compromised. These results suggest that the underlying pathophysiology of lymphedema drives adipose-derived stem cells toward adipogenic differentiation.

    View details for DOI 10.1097/PRS.0b013e31829ace13

    View details for PubMedID 23985633

  • Enhancing stem cell survival in vivo for tissue repair BIOTECHNOLOGY ADVANCES Hyun, J. S., Tran, M. C., Wong, V. W., Chung, M. T., Lo, D. D., Montoro, D. T., Wan, D. C., Longaker, M. T. 2013; 31 (5): 736-743

    Abstract

    The ability to use progenitor cells for regenerative medicine remains an evolving but elusive clinical goal. A serious obstacle towards widespread use of stem cells for tissue regeneration is the challenges that face these cells when they are placed in vivo into a wound for therapy. These environments are hypoxic, acidic, and have an upregulation of inflammatory mediators creating a region that is hostile towards cellular survival. Within this environment, the majority of progenitor cells undergo apoptosis prior to participating in lineage differentiation and cellular integration. In order to maximize the clinical utility of stem cells, strategies must be employed to increase the cell's ability to survive in vivo through manipulation of both the stem cell and the surrounding environment. This review focuses on current advances and techniques being used to increase in vivo stem cell survival for the purpose of tissue regeneration.

    View details for DOI 10.1016/j.biotechadv.2012.11.003

    View details for Web of Science ID 000322058900019

    View details for PubMedID 23153460

  • Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C. K., Lindau, P., Jiang, W., Chen, J. Y., Zhang, L. F., Chen, C., Seita, J., Sahoo, D., Kim, J., Lee, A., Park, S., Nag, D., Gong, Y., Kulkarni, S., Luppen, C. A., Theologis, A. A., Wan, D. C., DeBoer, A., Seo, E. Y., Vincent-Tompkins, J. D., Loh, K., Walmsley, G. G., Kraft, D. L., Wu, J. C., Longaker, M. T., Weissman, I. L. 2013; 110 (31): 12643-12648

    Abstract

    Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

    View details for DOI 10.1073/pnas.1310212110

    View details for Web of Science ID 000322441500042

    View details for PubMedID 23858471

  • Wnt3a reestablishes osteogenic capacity to bone grafts from aged animals. journal of bone and joint surgery. American volume Leucht, P., Jiang, J., Cheng, D., Liu, B., Dhamdhere, G., Fang, M. Y., Monica, S. D., Urena, J. J., Cole, W., Smith, L. R., Castillo, A. B., Longaker, M. T., Helms, J. A. 2013; 95 (14): 1278-1288

    Abstract

    Age-related fatty degeneration of the bone marrow contributes to delayed fracture-healing and osteoporosis-related fractures in the elderly. The mechanisms underlying this fatty change are unknown, but they may relate to the level of Wnt signaling within the aged marrow cavity.Transgenic mice were used in conjunction with a syngeneic bone-graft model to follow the fates of cells involved in the engraftment. Immunohistochemistry along with quantitative assays were used to evaluate Wnt signaling and adipogenic and osteogenic gene expression in bone grafts from young and aged mice. Liposomal Wnt3a protein (L-Wnt3a) was tested for its ability to restore osteogenic potential to aged bone grafts in critical-size defect models created in mice and in rabbits. Radiography, microquantitative computed tomography (micro-CT) reconstruction, histology, and histomorphometric measurements were used to quantify bone-healing resulting from L-Wnt3a or a control substance (liposomal phosphate-buffered saline solution [L-PBS]).Expression profiling of cells in a bone graft demonstrated a shift away from an osteogenic gene profile and toward an adipogenic one with age. This age-related adipogenic shift was accompanied by a significant reduction (p < 0.05) in Wnt signaling and a loss in osteogenic potential. In both large and small animal models, osteogenic competence was restored to aged bone grafts by a brief incubation with the stem-cell factor Wnt3a. In addition, liposomal Wnt3a significantly reduced cell death in the bone graft, resulting in significantly more osseous regenerate in comparison with controls.Liposomal Wnt3a enhances cell survival and reestablishes the osteogenic capacity of bone grafts from aged animals.We developed an effective, clinically applicable, regenerative medicine-based strategy for revitalizing bone grafts from aged patients.

    View details for DOI 10.2106/JBJS.L.01502

    View details for PubMedID 23864176

  • Brg1 governs a positive feedback circuit in the hair follicle for tissue regeneration and repair. Developmental cell Xiong, Y., Li, W., Shang, C., Chen, R. M., Han, P., Yang, J., Stankunas, K., Wu, B., Pan, M., Zhou, B., Longaker, M. T., Chang, C. 2013; 25 (2): 169-181

    Abstract

    Hair follicle stem cells (bulge cells) are essential for hair regeneration and early epidermal repair after wounding. Here we show that Brg1, a key enzyme in the chromatin-remodeling machinery, is dynamically expressed in bulge cells to control tissue regeneration and repair. In mice, sonic hedgehog (Shh) signals Gli to activate Brg1 in bulge cells to begin hair regeneration, whereas Brg1 recruits NF-?B to activate Shh in matrix cells to sustain hair growth. Such reciprocal Brg1-Shh interaction is essential for hair regeneration. Moreover, Brg1 is indispensable for maintaining the bulge cell reservoir. Without Brg1, bulge cells are depleted over time, partly through the ectopic expression of the cell-cycle inhibitor p27(Kip1). Also, bulge Brg1 is activated by skin injury to facilitate early epidermal repair. Our studies demonstrate a molecular circuit that integrates chromatin remodeling (Brg1), transcriptional regulation (NF-?B, Gli), and intercellular signaling (Shh) to control bulge stem cells during tissue regeneration.

    View details for DOI 10.1016/j.devcel.2013.03.015

    View details for PubMedID 23602386

  • CD90 (Thy-1)-Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Chung, M. T., Liu, C., Hyun, J. S., Lo, D. D., Montoro, D. T., Hasegawa, M., Li, S., Sorkin, M., Rennert, R., Keeney, M., Yang, F., Quarto, N., Longaker, M. T., Wan, D. C. 2013; 19 (7-8): 989-997

    Abstract

    Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105(low) cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD(105).Unsorted cells, CD90(+), CD90(-), CD105(high), and CD105(low) cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) calvarial defects in nude mice were treated with the hydroxyapatite-poly(lactic-co-glycolic acid) scaffold seeded with the above-mentioned subpopulations. Healing was followed using micro-CT scans for 8 weeks. Calvaria were harvested at 8 weeks postoperatively, and sections were stained with Movat's Pentachrome.Transcriptional analysis revealed that the CD90(+) subpopulation was enriched for a more osteogenic subtype relative to the CD105(low) subpopulation. Staining at day 7 for ALP was greatest in the CD90(+) cells, followed by the CD105(low) cells. Staining at day 14 for alizarin red demonstrated the greatest amount of mineralized extracellular matrix in the CD90(+) cells, again followed by the CD105(low) cells. Quantification of in vivo healing at 2, 4, 6, and 8weeks postoperatively demonstrated increased bone formation in defects treated with CD90(+) ASCs relative to all other groups. On Movat's Pentachrome-stained sections, defects treated with CD90(+) cells showed the most robust bony regeneration. Defects treated with CD90(-) cells, CD105(high) cells, and CD105(low) cells demonstrated some bone formation, but to a lesser degree when compared with the CD90(+) group.While CD105(low) cells have previously been shown to possess an enhanced osteogenic potential, we found that CD90(+) cells are more capable of forming bone both in vitro and in vivo. These data therefore suggest that CD90 may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.

    View details for DOI 10.1089/ten.tea.2012.0370

    View details for Web of Science ID 000315951500016

    View details for PubMedID 23216074

  • Integration of Multiple Signaling Regulates through Apoptosis the Differential Osteogenic Potential of Neural Crest-Derived and Mesoderm-Derived Osteoblasts PLOS ONE Li, S., Meyer, N. P., Quarto, N., Longaker, M. T. 2013; 8 (3)

    Abstract

    Neural crest-derived (FOb) and mesoderm-derived (POb) calvarial osteoblasts are characterized by distinct differences in their osteogenic potential. We have previously demonstrated that enhanced activation of endogenous FGF and Wnt signaling confers greater osteogenic potential to FOb. Apoptosis, a key player in bone formation, is the main focus of this study. In the current work, we have investigated the apoptotic activity of FOb and POb cells during differentiation. We found that lower apoptosis, as measured by caspase-3 activity is a major feature of neural crest-derived osteoblast which also have higher osteogenic capacity. Further investigation indicated TGF-? signaling as main positive regulator of apoptosis in these two populations of calvarial osteoblasts, while BMP and canonical Wnt signaling negatively regulate the process. By either inducing or inhibiting these signaling pathways we could modulate apoptotic events and improve the osteogenic potential of POb. Taken together, our findings demonstrate that integration of multiple signaling pathways contribute to imparting greater osteogenic potential to FOb by decreasing apoptosis.

    View details for DOI 10.1371/journal.pone.0058610

    View details for Web of Science ID 000317397200008

    View details for PubMedID 23536803

  • Micro-Computed Tomography Evaluation of Human Fat Grafts in Nude Mice TISSUE ENGINEERING PART C-METHODS Chung, M. T., Hyun, J. S., Lo, D. D., Montoro, D. T., Hasegawa, M., Levi, B., Januszyk, M., Longaker, M. T., Wan, D. C. 2013; 19 (3): 227-232

    Abstract

    Although autologous fat grafting has revolutionized the field of soft tissue reconstruction and augmentation, long-term maintenance of fat grafts is unpredictable. Recent studies have reported survival rates of fat grafts to vary anywhere between 10% and 80% over time. The present study evaluated the long-term viability of human fat grafts in a murine model using a novel imaging technique allowing for in vivo volumetric analysis.Human fat grafts were prepared from lipoaspirate samples using the Coleman technique. Fat was injected subcutaneously into the scalp of 10 adult Crl:NU-Foxn1(nu) CD-1 male mice. Micro-computed tomography (CT) was performed immediately following injection and then weekly thereafter. Fat volume was rendered by reconstructing a three-dimensional (3D) surface through cubic-spline interpolation. Specimens were also harvested at various time points and sections were prepared and stained with hematoxylin and eosin (H&E), for macrophages using CD68 and for the cannabinoid receptor 1 (CB1). Finally, samples were explanted at 8- and 12-week time points to validate calculated micro-CT volumes.Weekly CT scanning demonstrated progressive volume loss over the time course. However, volumetric analysis at the 8- and 12-week time points stabilized, showing an average of 62.2% and 60.9% survival, respectively. Gross analysis showed the fat graft to be healthy and vascularized. H&E analysis and staining for CD68 showed minimal inflammatory reaction with viable adipocytes. Immunohistochemical staining with anti-human CB1 antibodies confirmed human origin of the adipocytes.Studies assessing the fate of autologous fat grafts in animals have focused on nonimaging modalities, including histological and biochemical analyses, which require euthanasia of the animals. In this study, we have demonstrated the ability to employ micro-CT for 3D reconstruction and volumetric analysis of human fat grafts in a mouse model. Importantly, this model provides a platform for subsequent study of fat manipulation and soft tissue engineering.

    View details for DOI 10.1089/ten.tec.2012.0371

    View details for Web of Science ID 000314179900006

    View details for PubMedID 22916732

  • Primary cilia act as mechanosensors during bone healing around an implant MEDICAL ENGINEERING & PHYSICS Leucht, P., Monica, S. D., Temiyasathit, S., Lenton, K., Manu, A., Longaker, M. T., Jacobs, C. R., Spilkere, R. L., Guo, H., Brunski, J. B., Helms, J. A. 2013; 35 (3): 392-402

    Abstract

    The primary cilium is an organelle that senses cues in a cell's local environment. Some of these cues constitute molecular signals; here, we investigate the extent to which primary cilia can also sense mechanical stimuli. We used a conditional approach to delete Kif3a in pre-osteoblasts and then employed a motion device that generated a spatial distribution of strain around an intra-osseous implant positioned in the mouse tibia. We correlated interfacial strain fields with cell behaviors ranging from proliferation through all stages of osteogenic differentiation. We found that peri-implant cells in the Col1Cre;Kif3a(fl/fl) mice were unable to proliferate in response to a mechanical stimulus, failed to deposit and then orient collagen fibers to the strain fields caused by implant displacement, and failed to differentiate into bone-forming osteoblasts. Collectively, these data demonstrate that the lack of a functioning primary cilium blunts the normal response of a cell to a defined mechanical stimulus. The ability to manipulate the genetic background of peri-implant cells within the context of a whole, living tissue provides a rare opportunity to explore mechanotransduction from a multi-scale perspective.

    View details for DOI 10.1016/j.medengphy.2012.06.005

    View details for Web of Science ID 000315931400013

    View details for PubMedID 22784673

  • Integration of Multiple Signaling Pathways Determines Differences in the Osteogenic Potential and Tissue Regeneration of Neural Crest-Derived and Mesoderm-Derived Calvarial Bone INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Senarath-Yapa, K., Li, S., Meyer, N. P., Longaker, M. T., Quarto, N. 2013; 14 (3): 5978-5997

    Abstract

    The mammalian skull vault, a product of a unique and tightly regulated evolutionary process, in which components of disparate embryonic origin are integrated, is an elegant model with which to study osteoblast biology. Our laboratory has demonstrated that this distinct embryonic origin of frontal and parietal bones confer differences in embryonic and postnatal osteogenic potential and skeletal regenerative capacity, with frontal neural crest derived osteoblasts benefitting from greater osteogenic potential. We outline how this model has been used to elucidate some of the molecular mechanisms which underlie these differences and place these findings into the context of our current understanding of the key, highly conserved, pathways which govern the osteoblast lineage including FGF, BMP, Wnt and TGF? signaling. Furthermore, we explore recent studies which have provided a tantalizing insight into way these pathways interact, with evidence accumulating for certain transcription factors, such as Runx2, acting as a nexus for cross-talk.

    View details for DOI 10.3390/ijms14035978

    View details for Web of Science ID 000316609800086

    View details for PubMedID 23502464

  • Commentary on the Differential Healing Capacity of Calvarial Bone JOURNAL OF CRANIOFACIAL SURGERY Lo, D. D., McArdle, A., Senarath-Yapa, K., Longaker, M. T. 2013; 24 (2): 344-345

    View details for DOI 10.1097/SCS.0b013e3182802256

    View details for Web of Science ID 000316676300047

    View details for PubMedID 23524689

  • Effective Delivery of Stem Cells Using an Extracellular Matrix Patch Results in Increased Cell Survival and Proliferation and Reduced Scarring in Skin Wound Healing TISSUE ENGINEERING PART A Lam, M. T., Nauta, A., Meyer, N. P., Wu, J. C., Longaker, M. T. 2013; 19 (5-6): 738-747

    Abstract

    Wound healing is one of the most complex biological processes and occurs in all tissues and organs of the body. In humans, fibrotic tissue, or scar, hinders function and is aesthetically unappealing. Stem cell therapy offers a promising new technique for aiding in wound healing; however, current findings show that stem cells typically die and/or migrate from the wound site, greatly decreasing efficacy of the treatment. Here, we demonstrate effectiveness of a stem cell therapy for improving wound healing in the skin and reducing scarring by introducing stem cells using a natural patch material. Adipose-derived stromal cells were introduced to excisional wounds created in mice using a nonimmunogenic extracellular matrix (ECM) patch material derived from porcine small-intestine submucosa (SIS). The SIS served as an attractive delivery vehicle because of its natural ECM components, including its collagen fiber network, providing the stem cells with a familiar structure. Experimental groups consisted of wounds with stem cell-seeded patches removed at different time points after wounding to determine an optimal treatment protocol. Stem cells delivered alone to skin wounds did not survive post-transplantation as evidenced by bioluminescence in vivo imaging. In contrast, delivery with the patch enabled a significant increase in stem cell proliferation and survival. Wound healing rates were moderately improved by treatment with stem cells on the patch; however, areas of fibrosis, indicating scarring, were significantly reduced in wounds treated with the stem cells on the patch compared to untreated wounds.

    View details for DOI 10.1089/ten.tea.2012.0480

    View details for Web of Science ID 000314581100015

    View details for PubMedID 23072446

  • Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing MOLECULAR THERAPY Nauta, A., Seidel, C., Deveza, L., Montoro, D., Grova, M., Ko, S. H., Hyun, J., Gurtner, G. C., Longaker, M. T., Yang, F. 2013; 21 (2): 445-455

    Abstract

    Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (?-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.

    View details for DOI 10.1038/mt.2012.234

    View details for Web of Science ID 000314434600021

    View details for PubMedID 23164936

  • Discussion: A Report of the ASPS Task Force on Regenerative Medicine: Opportunities for Plastic Surgery PLASTIC AND RECONSTRUCTIVE SURGERY McArdle, A., Lo, D. D., Hyun, J. S., Senarath-Yapa, K., Chung, M. T., Wan, D. C., Longaker, M. T. 2013; 131 (2): 400-403

    View details for DOI 10.1097/PRS.0b013e318278d88c

    View details for Web of Science ID 000314355700076

    View details for PubMedID 23358002

  • The Seed and the Soil Optimizing Stem Cells and Their Environment for Tissue Regeneration ANNALS OF PLASTIC SURGERY Hyun, J. S., Montoro, D. T., Lo, D. D., Flynn, R. A., Wong, V., Chung, M. T., Longaker, M. T., Wan, D. C. 2013; 70 (2): 235-239

    Abstract

    The potential for stem cells to serve as cellular building blocks for reconstruction of complex defects has prompted significant enthusiasm in the field of regenerative medicine. Clinical application, however, is still limited, as implantation of cells into hostile wound environments may greatly hinder their tissue forming capacity. To circumvent this obstacle, novel approaches have been developed to manipulate both the stem cell itself and its surrounding environmental niche. By understanding this paradigm of seed and soil optimization, innovative strategies may thus be developed to harness the true promise of stem cells for tissue regeneration.

    View details for DOI 10.1097/SAP.0b013e31826a18fb

    View details for Web of Science ID 000313964300024

    View details for PubMedID 23295233

  • Abnormal Calcium Handling Properties Underlie Familial Hypertrophic Cardiomyopathy Pathology in Patient-Specific Induced Pluripotent Stem Cells CELL STEM CELL Lan, F., Lee, A. S., Liang, P., Sanchez-Freire, V., Nguyen, P. K., Wang, L., Han, L., Yen, M., Wang, Y., Sun, N., Abilez, O. J., Hu, S., Ebert, A. D., Navarrete, E. G., Simmons, C. S., Wheeler, M., Pruitt, B., Lewis, R., Yamaguchi, Y., Ashley, E. A., Bers, D. M., Robbins, R. C., Longaker, M. T., Wu, J. C. 2013; 12 (1): 101-113

    Abstract

    Familial hypertrophic cardiomyopathy (HCM) is a prevalent hereditary cardiac disorder linked to arrhythmia and sudden cardiac death. While the causes of HCM have been identified as genetic mutations in the cardiac sarcomere, the pathways by which sarcomeric mutations engender myocyte hypertrophy and electrophysiological abnormalities are not understood. To elucidate the mechanisms underlying HCM development, we generated patient-specific induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from a ten-member family cohort carrying a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Diseased iPSC-CMs recapitulated numerous aspects of the HCM phenotype including cellular enlargement and contractile arrhythmia at the single-cell level. Calcium (Ca(2+)) imaging indicated dysregulation of Ca(2+) cycling and elevation in intracellular Ca(2+) ([Ca(2+)](i)) are central mechanisms for disease pathogenesis. Pharmacological restoration of Ca(2+) homeostasis prevented development of hypertrophy and electrophysiological irregularities. We anticipate that these findings will help elucidate the mechanisms underlying HCM development and identify novel therapies for the disease.

    View details for DOI 10.1016/j.stem.2012.10.010

    View details for Web of Science ID 000313839500014

    View details for PubMedID 23290139

  • Absence of endochondral ossification and craniosynostosis in posterior frontal cranial sutures of axin2(-/-) mice. PloS one Behr, B., Longaker, M. T., Quarto, N. 2013; 8 (8)

    Abstract

    During the first month of life, the murine posterior-frontal suture (PF) of the cranial vault closes through endochondral ossification, while other sutures remain patent. These processes are tightly regulated by canonical Wnt signaling. Low levels of active canonical Wnt signaling enable endochondral ossification and therefore PF-suture closure, whereas constitutive activation of canonical Wnt causes PF-suture patency. We therefore sought to test this concept with a knockout mouse model. PF-sutures of Axin2(-/-) mice, which resemble a state of constantly activated canonical Wnt signaling, were investigated during the physiological time course of PF-suture closure and compared in detail with wild type littermates. Histological analysis revealed that the architecture in Axin2(-/-) PF-sutures was significantly altered in comparison to wild type. The distance between the endocranial layers was dramatically increased and suture closure was significantly delayed. Moreover, physiological endochondral ossification did not occur, rather an ectopic cartilage appeared between the endocranial and ectocranial bone layers at P7 which eventually involutes at P13. Quantitative PCR analysis showed the lack of Col10α1 upregulation in Axin2(-/-) PF-suture. Immunohistochemistry and gene expression analysis also revealed high levels of type II collagen as compared to type I collagen and absence of Mmp-9 in the cartilage of Axin2(-/-) PF-suture. Moreover, TUNEL staining showed a high percentage of apoptotic chondrocytes in Axin2(-/-) PF-sutures at P9 and P11 as compared to wild type. These data indicated that Axin2(-/-) PF-sutures lack physiological endochondral ossification, contain ectopic cartilage and display delayed suture closure.

    View details for DOI 10.1371/journal.pone.0070240

    View details for PubMedID 23936395

  • Evidence that mast cells are not required for healing of splinted cutaneous excisional wounds in mice. PloS one Nauta, A. C., Grova, M., Montoro, D. T., Zimmermann, A., Tsai, M., Gurtner, G. C., Galli, S. J., Longaker, M. T. 2013; 8 (3)

    Abstract

    Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-Kit(W-sh/W-sh), WBB6F1-Kit(W/W-v), and Cpa3-Cre; Mcl-1(fl/fl) mice re-epithelialized splinted excisional skin wounds at rates very similar to those in the corresponding wild type or control mice. Furthermore, at the time of closure, scars were similar in the genetically mast cell-deficient mice and the corresponding wild type or control mice in both quantity of collagen deposition and maturity of collagen fibers, as evaluated by Masson's Trichrome and Picro-Sirius red staining. These data indicate that mast cells do not play a significant non-redundant role in these features of the healing of splinted full thickness excisional cutaneous wounds in mice.

    View details for DOI 10.1371/journal.pone.0059167

    View details for PubMedID 23544053

  • Adult stem cells in small animal wound healing models. Methods in molecular biology (Clifton, N.J.) Nauta, A. C., Gurtner, G. C., Longaker, M. T. 2013; 1037: 81-98

    Abstract

    This chapter broadly reviews the use of stem cells as a means to accelerate wound healing, focusing first on the properties of stem cells that make them attractive agents to influence repair, both alone and as vehicles for growth factor delivery. Major stem cell reservoirs are described, including adult, embryonic, and induced pluripotent cell sources, outlining the advantages and limitations of each source as wound healing agents, as well as the possible mechanisms responsible for wound healing acceleration. Finally, the chapter includes a materials and methods section that provides an in-depth description of adult tissue harvest techniques.

    View details for DOI 10.1007/978-1-62703-505-7_5

    View details for PubMedID 24029931

  • In vivo directed differentiation of pluripotent stem cells for skeletal regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Levi, B., Hyun, J. S., Montoro, D. T., Lo, D. D., Chan, C. K., Hu, S., Sun, N., Lee, M., Grova, M., Connolly, A. J., Wu, J. C., Gurtner, G. C., Weissman, I. L., Wan, D. C., Longaker, M. T. 2012; 109 (50): 20379-20384

    Abstract

    Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

    View details for DOI 10.1073/pnas.1218052109

    View details for Web of Science ID 000312605600055

    View details for PubMedID 23169671

  • Femtosecond plasma mediated laser ablation has advantages over mechanical osteotomy of cranial bone LASERS IN SURGERY AND MEDICINE Lo, D. D., Mackanos, M. A., Chung, M. T., Hyun, J. S., Montoro, D. T., Grova, M., Liu, C., Wang, J., Palanker, D., Connolly, A. J., Longaker, M. T., Contag, C. H., Wan, D. C. 2012; 44 (10): 805-814

    Abstract

    Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma-mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration.Critical-size circular calvarial defects were created with a trephine drill bit or with a Ti:Sapphire femtosecond pulsed laser. Healing was followed using micro-CT scans for 8 weeks. Calvaria were also harvested at various time points for histological analysis. Finally, scanning electron microscopy was used to analyze the microstructure of bone tissue treated with the Ti:Sapphire laser, and compared to that treated with the trephine bur.Laser-created defects healed significantly faster than those created mechanically at 2, 4, and 6 weeks post-surgery. However, at 8 weeks post-surgery, there was no significant difference. In the drill osteotomy treatment group, empty osteocyte lacunae were seen to extend 699?±?27?µm away from the edge of the defect. In marked contrast, empty osteocyte lacunae were seen to extend only 182?±?22?µm away from the edge of the laser-created craters. Significantly less ossification and formation of irregular woven bone was noted on histological analysis for drill defects.We demonstrate accelerated bone healing after femtosecond laser ablation in a calvarial defect model compared to traditional mechanical drilling techniques. Improved rates of early regeneration make plasma-mediated ablation of the craniofacial skeleton advantageous for applications to osteotomy.

    View details for DOI 10.1002/lsm.22098

    View details for Web of Science ID 000312941600004

    View details for PubMedID 23184427

  • Exogenous Activation of BMP-2 Signaling Overcomes TGF beta-Mediated Inhibition of Osteogenesis in Marfan Embryonic Stem Cells and Marfan Patient-Specific Induced Pluripotent Stem Cells STEM CELLS Quarto, N., Li, S., Renda, A., Longaker, M. T. 2012; 30 (12): 2709-2719

    Abstract

    Marfan syndrome (MFS) is a hereditary disease caused by mutations in the gene encoding Fibrillin-1 (FBN1) and characterized by a number of skeletal abnormalities, aortic root dilatation, and sometimes ectopia lentis. Although the molecular pathogenesis of MFS was attributed initially to a structural weakness of the fibrillin-rich microfibrils within the extracellular matrix, more recent results have documented that many of the pathogenic abnormalities in MFS are the result of alterations in TGF? signaling. Mutations in FBN1 are therefore associated with increased activity and bioavailability of TGF-?1, which is suspected to be the basis for phenotypical similarities of FBN1 mutations in MFS and mutations in the receptors for TGF? in Marfan syndrome-related diseases. We have previously demonstrated that unique skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells (MFSiPS) derived independently from MFS patient fibroblasts. In this study, we aimed to determine further the biochemical features of transducing signaling(s) in MFS stem cells and MFSiPS cells highlighting a crosstalk between TGF? and BMP signaling. Our results revealed that enhanced activation of TGF? signaling observed in MFS cells decreased their endogenous BMP signaling. Moreover, exogenous BMP antagonized the enhanced TGF? signaling in both MFS stem cells and MFSiPS cells therefore, rescuing their ability to undergo osteogenic differentiation. This study advances our understanding of molecular mechanisms underlying the pathogenesis of bone loss/abnormal skeletogenesis in human diseases caused by mutations in FBN1.

    View details for DOI 10.1002/stem.1250

    View details for Web of Science ID 000311493600011

    View details for PubMedID 23037987

  • Introduction: Wound repair SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Longaker, M. T. 2012; 23 (9): 945-945

    View details for DOI 10.1016/j.semcdb.2012.10.002

    View details for Web of Science ID 000311962800001

    View details for PubMedID 23059792

  • Soft tissue mechanotransduction in wound healing and fibrosis SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Wong, V. W., Longaker, M. T., Gurtner, G. C. 2012; 23 (9): 981-986

    Abstract

    Recent evidence suggests that mechanical forces can significantly impact the biologic response to injury. Integrated mechanical and chemical signaling networks have been discovered that enable physical cues to regulate disease processes such as pathologic scar formation. Distinct molecular mechanisms control how tensional forces influence wound healing and fibrosis. Conceptual frameworks to understand cutaneous repair have expanded beyond traditional cell-cytokine models to include dynamic interactions driven by mechanical force and the extracellular matrix. Strategies to manipulate these biomechanical signaling networks have tremendous therapeutic potential to reduce scar formation and promote skin regeneration.

    View details for DOI 10.1016/j.semcdb.2012.09.010

    View details for Web of Science ID 000311962800006

    View details for PubMedID 23036529

  • Comparison of several attachment methods for human iPS, embryonic and adipose-derived stem cells for tissue engineering JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE Lam, M. T., Longaker, M. T. 2012; 6: s80-s86

    Abstract

    As actual stem cell application quickly approaches tissue engineering and regenerative medicine, aspects such as cell attachment to scaffolds and biomaterials become important and are often overlooked. Here, we compare the effects of several attachment proteins on the adhesion, proliferation and stem cell identity of three promising human stem cell types: human adipose-derived stem cells (hASCs), human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Traditional tissue culture polystyrene plates (TCPS), Matrigel (Mat), laminin (Lam), fibronectin (FN) and poly-L-lysine (PLL) were investigated as attachment protein surfaces. For hASCs typically cultured on TCPS, laminin resulted in the greatest cell attachment and proliferation with largest cell areas, indicating favourability by cell spreading. However, mesenchymal stem cell markers indicative of hASCs were slightly more expressed on surfaces with lowest cell attachment, corresponding to increased cell roundness, a newly observed attribute in hASCs possibly indicating a more stem cell-like character. hESCs preferred Matrigel as a feeder-free culture surface. Interestingly, hiPSCs favoured laminin over Matrigel for colony expansion, shown by larger cell colony area and perimeter lengths, although cell numbers and stem cell marker expression level remained highest on Matrigel. These data provide a practical reference guide for selecting a suitable attachment method for using human induced pluripotent, embryonic or adipose stem cells in tissue engineering and regenerative medicine applications.

    View details for DOI 10.1002/term.1499

    View details for Web of Science ID 000313431100009

    View details for PubMedID 22610948

  • Models of cranial suture biology. journal of craniofacial surgery Grova, M., Lo, D. D., Montoro, D., Hyun, J. S., Chung, M. T., Wan, D. C., Longaker, M. T. 2012; 23 (7): 1954-1958

    Abstract

    Craniosynostosis is a common congenital defect caused by premature fusion of cranial sutures. The severe morphologic abnormalities and cognitive deficits resulting from craniosynostosis and the potential morbidity of surgical correction espouse the need for a deeper understanding of the complex etiology for this condition. Work in animal models for the past 20 years has been pivotal in advancing our understanding of normal suture biology and elucidating pathologic disease mechanisms. This article provides an overview of milestone studies in suture development, embryonic origins, and signaling mechanisms from an array of animal models including transgenic mice, rats, rabbits, fetal sheep, zebrafish, and frogs. This work contributes to an ongoing effort toward continued development of novel treatment strategies.

    View details for DOI 10.1097/SCS.0b013e318258ba53

    View details for PubMedID 23154351

  • Pierre Robin sequence and Treacher Collins hypoplastic mandible comparison using three-dimensional morphometric analysis. journal of craniofacial surgery Chung, M. T., Levi, B., Hyun, J. S., Lo, D. D., Montoro, D. T., Lisiecki, J., Bradley, J. P., Buchman, S. R., Longaker, M. T., Wan, D. C. 2012; 23 (7): 1959-1963

    Abstract

    Pierre Robin sequence and Treacher Collins syndrome are both associated with mandibular hypoplasia. It has been hypothesized, however, that the mandible may be differentially affected. The purpose of this study was to therefore compare mandibular morphology in children with Pierre Robin sequence with children with Treacher Collins syndrome using three-dimensional analysis of computed tomographic scans. A retrospective analysis was performed identifying children with Pierre Robin sequence and Treacher Collins syndrome undergoing computed tomography. Three-dimensional reconstruction was performed, and ramus height, mandibular body length, and gonial angle were measured. These were then compared with those in control children with normal mandibles and with the clinical norms corrected for age and sex based on previously published measurements. Mandibular body length was found to be significantly shorter for children with Pierre Robin sequence, whereas ramus height was significantly shorter for children with Treacher Collins syndrome. This resulted in distinctly different ramus height-mandibular body length ratios. In addition, the gonial angle was more obtuse in both the Pierre Robin sequence and Treacher Collins syndrome groups compared with the controls. Three-dimensional mandibular morphometric analysis in patients with Pierre Robin sequence and Treacher Collins syndrome thus revealed distinctly different patterns of mandibular hypoplasia relative to normal controls. These findings underscore distinct considerations that must be made in surgical planning for reconstruction.

    View details for DOI 10.1097/SCS.0b013e318258bcf1

    View details for PubMedID 23154353

  • Regenerative Surgery: Tissue Engineering in General Surgical Practice WORLD JOURNAL OF SURGERY Wong, V. W., Wan, D. C., Gurtner, G. C., Longaker, M. T. 2012; 36 (10): 2288-2299

    Abstract

    Tissue engineering is a broad interdisciplinary field that aims to develop complex tissue and organ constructs through a combination of cell-, biomaterial-, and molecular-based approaches. This approach has the potential to transform the surgical treatment for diseases including trauma, cancer, and congenital malformations. A fundamental knowledge of key concepts in regenerative medicine is imperative for surgeons to maintain a leading role in developing and implementing these technologies. Researchers have started to elucidate the biologic mechanisms that maintain organ homeostasis throughout life, indicating that humans may have the latent capacity to regenerate complex tissues. By exploiting this intrinsic potential of the body, we can move even closer to developing functional, autologous replacement parts for a wide range of surgical diseases.

    View details for DOI 10.1007/s00268-012-1710-1

    View details for Web of Science ID 000309559800003

    View details for PubMedID 22777416

  • Craniosynostosis Molecular pathways and future pharmacologic therapy ORGANOGENESIS Senarath-Yapa, K., Chung, M. T., McArdle, A., Wong, V. W., Quarto, N., Longaker, M. T., Wan, D. C. 2012; 8 (4): 103-113

    View details for DOI 10.4161/org.23307

    View details for Web of Science ID 000314500600002

  • Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell-Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation CIRCULATION RESEARCH Gu, M., Nguyen, P. K., Lee, A. S., Xu, D., Hu, S., Plews, J. R., Han, L., Huber, B. C., Lee, W. H., Gong, Y., de Almeida, P. E., Lyons, J., Ikeno, F., Pacharinsak, C., Connolly, A. J., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2012; 111 (7): 882-893

    Abstract

    Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models.To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia.Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery.In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

    View details for DOI 10.1161/CIRCRESAHA.112.269001

    View details for Web of Science ID 000308868800015

    View details for PubMedID 22821929

  • Scarless fetal skin wound healing update BIRTH DEFECTS RESEARCH PART C-EMBRYO TODAY-REVIEWS Lo, D. D., Zimmermann, A. S., Nauta, A., Longaker, M. T., Lorenz, H. P. 2012; 96 (3): 237-247

    Abstract

    Scar formation, a physiologic process in adult wound healing, can have devastating effects for patients; a multitude of pathologic outcomes, affecting all organ systems, stems from an amplification of this process. In contrast to adult wound repair, the early-gestation fetal skin wound heals without scar formation, a phenomenon that appears to be intrinsic to fetal skin. An intensive research effort has focused on unraveling the mechanisms that underlie scarless fetal wound healing in an attempt to improve the quality of healing in both children and adults. Unique properties of fetal cells, extracellular matrix, cytokine profile, and gene expression contribute to this scarless repair. Despite the great increase in knowledge gained over the past decades, the precise mechanisms regulating scarless fetal healing remain unknown. Herein, we describe the current proposed mechanisms underlying fetal scarless wound healing in an effort to recapitulate the fetal phenotype in the postnatal environment.

    View details for DOI 10.1002/bdrc.21018

    View details for Web of Science ID 000310475000003

    View details for PubMedID 23109319

  • Fetal Mouse Skin Heals Scarlessly in a Chick Chorioallantoic Membrane Model System ANNALS OF PLASTIC SURGERY Carre, A. L., Larson, B. J., Knowles, J. A., Kawai, K., Longaker, M. T., Lorenz, H. P. 2012; 69 (1): 85-90

    Abstract

    In mammals, the early-gestation fetus has the regenerative ability to heal skin wounds without scar formation. This observation was first reported more than 3 decades ago, and has been confirmed in a number of in vivo animal models. Although an intensive research effort has focused on unraveling the mechanisms underlying scarless fetal wound repair, no suitable model of in vitro fetal skin healing has been developed. In this article, we report a novel model for the study of fetal wound healing. Fetal skin from gestational day 16.5 Balb/c mice (total gestation, 20 days) was grafted onto the chorioallantoic membrane of 12-day-old chicken embryos and cultured for up to 7 days. At 48 hours postengraftment, circular wounds (diameter = 1 mm) were made in the fetal skin using a rotating titanium sapphire laser (N = 45). The tissue was examined daily by visual inspection to look for signs of infection and ischemia. The grafts and the surrounding host tissue were examined histologically. In all fetal skin grafts, the wounds completely reepithelialized by postinjury day 7, with regeneration of the dermis. Fetal mouse skin xenografts transplanted onto the chorioallantoic membrane of fertilized chicken eggs provides a useful model for the study of fetal wound healing. This model can be used as an adjunct to traditional in vivo mammalian models of fetal repair.

    View details for DOI 10.1097/SAP.0b013e31822128a9

    View details for Web of Science ID 000305485200020

    View details for PubMedID 21712703

  • Large animal induced pluripotent stem cells as pre-clinical models for studying human disease JOURNAL OF CELLULAR AND MOLECULAR MEDICINE Plews, J. R., Gu, M., Longaker, M. T., Wu, J. C. 2012; 16 (6): 1196-1202

    Abstract

    The path to induced pluripotency Discovery of a pan-species pluripotency network Animal iPSCs and disease modelling Issues with large animal iPSCs Conclusions The derivation of human embryonic stem cells and subsequently human induced pluripotent stem cells (iPSCs) has energized regenerative medicine research and enabled seemingly limitless applications. Although small animal models, such as mouse models, have played an important role in the progression of the field, typically, they are poor representations of the human disease phenotype. As an alternative, large animal models should be explored as a potentially better approach for clinical translation of cellular therapies. However, only fragmented information regarding the derivation, characterization and clinical usefulness of pluripotent large animal cells is currently available. Here, we briefly review the latest advances regarding the derivation and use of large animal iPSCs.

    View details for DOI 10.1111/j.1582-4934.2012.01521.x

    View details for Web of Science ID 000304468600005

    View details for PubMedID 22212700

  • Craniofacial Reconstruction With Induced Pluripotent Stem Cells JOURNAL OF CRANIOFACIAL SURGERY Wan, D. C., Wong, V. W., Longaker, M. T. 2012; 23 (3): 623-626

    View details for DOI 10.1097/SCS.0b013e318252f41b

    View details for Web of Science ID 000304479600041

    View details for PubMedID 22627398

  • Rethinking the Blastema PLASTIC AND RECONSTRUCTIVE SURGERY Hyun, J. S., Chung, M. T., Wong, V. W., Montoro, D., Longaker, M. T., Wan, D. C. 2012; 129 (5): 1097-1103

    Abstract

    The phenomenon of tissue regeneration has been well documented across many species. Although some possess the capacity to completely restore an entire amputated limb, others are limited to just the distal digit tip. Initiation of limb regeneration has been described to start with the formation of a blastema, the composition of which has long been thought to consist of undifferentiated pluripotent cells derived through the process of dedifferentiation. Competing theories have been proposed, however, including cellular contributions through transdifferentiation and tissue-specific stem cells. Recent studies have now begun to shed light on this controversy, demonstrating tissue resident stem cells to be an evolutionarily conserved measure for limb regeneration.

    View details for DOI 10.1097/PRS.0b013e31824a2c49

    View details for Web of Science ID 000303497300059

    View details for PubMedID 22544093

  • A Comparative Analysis of the Osteogenic Effects of BMP-2, FGF-2, and VEGFA in a Calvarial Defect Model TISSUE ENGINEERING PART A Behr, B., Sorkin, M., Lehnhardt, M., Renda, A., Longaker, M. T., Quarto, N. 2012; 18 (9-10): 1079-1086

    Abstract

    The utilization of growth factors for bone regeneration is a widely studied field. Since the approval of bone morphogenetic protein-2 (BMP-2) for therapeutic use in humans, the concept of utilizing growth factors for bone regeneration in translational medicine has become even more attractive. Despite many studies published on individual growth factors in various bone models, comparative analysis is largely lacking. The aim of our study was to compare three different proosteogenic factors under identical in vivo conditions. Thus, we tested the bone regeneration capacity of the three different growth factors BMP-2, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor A (VEGFA) in a calvarial defect model. We demonstrated that BMP-2 and VEGFA had similar bone healing capacities, resulting in complete calvarial healing as early as week 3. FGF-2 also showed a significantly higher bone regeneration capacity; however, the healing rate was lower than with BMP-2 and VEGFA. Interestingly, these findings were paralleled by an increased angiogenic response upon healing in BMP-2- and VEGFA-treated calvarial defects as compared with FGF-2. Immunohistochemistry for proliferating and osteoprogenitor cells revealed activity at different points after surgery among the groups. In conclusion, we demonstrated an efficient bone regeneration capacity of both BMP-2 and VEGFA, which was superior to FGF-2. Moreover, this study highlights the efficient bone regeneration of VEGFA, which was comparable with BMP-2. These data provide a valuable comparative analysis, which can be used to further optimize growth factor-based strategies in skeletal tissue engineering.

    View details for DOI 10.1089/ten.tea.2011.0537

    View details for Web of Science ID 000303540400019

    View details for PubMedID 22195699

  • Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy SCIENCE TRANSLATIONAL MEDICINE Sun, N., Yazawa, M., Liu, J., Han, L., Sanchez-Freire, V., Abilez, O. J., Navarrete, E. G., Hu, S., Wang, L., Lee, A., Pavlovic, A., Lin, S., Chen, R., Hajjar, R. J., Snyder, M. P., Dolmetsch, R. E., Butte, M. J., Ashley, E. A., Longaker, M. T., Robbins, R. C., Wu, J. C. 2012; 4 (130)

    Abstract

    Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca(2+)), decreased contractility, and abnormal distribution of sarcomeric ?-actinin. When stimulated with a ?-adrenergic agonist, DCM iPSC-derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric ?-actinin distribution. Treatment with ?-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

    View details for DOI 10.1126/scitranslmed.3003552

    View details for Web of Science ID 000303045900004

    View details for PubMedID 22517884

  • Training the Contemporary Surgeon-Scientist PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Wang, K. C., Longaker, M. T. 2012; 129 (4): 1023-1025

    View details for DOI 10.1097/PRS.0b013e31824421e8

    View details for Web of Science ID 000302227100076

    View details for PubMedID 22456371

  • Delivery Strategies for Stem Cell-Based Therapy JOURNAL OF HEALTHCARE ENGINEERING Glotzbach, J. P., Wong, V. W., Levi, B., Longaker, M. T., Gurtner, G. C. 2012; 3 (1): 1-20
  • The angiogenic factor Dell prevents apoptosis of endothelial cells through integrin binding SURGERY Wang, Z., Kundu, R. K., Longaker, M. T., Quertermous, T., Yang, G. P. 2012; 151 (2): 296-305

    Abstract

    Del1 is a secreted protein that is expressed in the endothelium during development and can stimulate angiogenesis through integrin binding and signaling. We were interested in the specific effects of del1 on endothelial cell biology to gain insight into its biologic role during angiogenesis.Primary endothelial cells were treated with a variety of inducers of apoptosis and anoikis followed by assays for numbers of apoptotic cells, and harvest of total protein for immunoblot analysis.Del1 prevented endothelial cell apoptosis in response to TNF?/IFN?, etoposide, and anoikis, but had no effect on proliferation. The anti-apoptotic effect was mediated specifically through binding of integrin ?v?3 by the RGD motif. FAK/ERK and Akt signaling were both necessary to mediate the anti-apoptotic effect of Del1 with the exception of anoikis, which required only Akt activation.Del1 has been previously shown to promote vascular smooth muscle cell adhesion, migration, and proliferation. We demonstrate here that Del1 prevented apoptosis of endothelial cells in cell culture through integrin binding without any effect on proliferation.

    View details for DOI 10.1016/j.surg.2011.07.013

    View details for Web of Science ID 000299607800019

    View details for PubMedID 21893328

  • Skeletogenic phenotype of human Marfan embryonic stem cells faithfully phenocopied by patient-specific induced-pluripotent stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Quarto, N., Leonard, B., Li, S., Marchand, M., Anderson, E., Behr, B., Francke, U., Reijo-Pera, R., Chiao, E., Longaker, M. T. 2012; 109 (1): 215-220

    Abstract

    Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1. We demonstrate that, as a biological consequence of the activation of TGF-? signaling, osteogenic differentiation of embryonic stem cells with a FBN1 mutation is inhibited; osteogenesis is rescued by inhibition of TGF-? signaling. In contrast, chondrogenesis is not perturbated and occurs in a TGF-? cell-autonomous fashion. Importantly, skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells derived independently from MFS patient fibroblasts. Results indicate a unique phenotype uncovered by examination of mutant pluripotent stem cells and further demonstrate the faithful alignment of phenotypes in differentiated cells obtained from both human embryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into human molecular pathogenesis, especially of MFS.

    View details for DOI 10.1073/pnas.1113442109

    View details for Web of Science ID 000298876500045

    View details for PubMedID 22178754

  • Enhancement of Human Adipose-Derived Stromal Cell Angiogenesis through Knockdown of a BMP-2 Inhibitor PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., Nelson, E. R., Hyun, J. S., Glotzbach, J. P., Li, S., Nauta, A., Montoro, D. T., Lee, M., Commons, G. C., Hu, S., Wu, J. C., Gurtner, G. C., Longaker, M. T. 2012; 129 (1): 53-66

    Abstract

    Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone.Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry.Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2-releasing scaffolds significantly enhanced vascular signaling in the defect site.Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.

    View details for DOI 10.1097/PRS.0b013e3182361ff5

    View details for Web of Science ID 000298857100075

    View details for PubMedID 21915082

  • Enhancement of mesenchymal stem cell angiogenic capacity and stemness by a biomimetic hydrogel scaffold BIOMATERIALS Rustad, K. C., Wong, V. W., Sorkin, M., Glotzbach, J. P., Major, M. R., Rajadas, J., Longaker, M. T., Gurtner, G. C. 2012; 33 (1): 80-90

    Abstract

    In this study, we examined the capacity of a biomimetic pullulan-collagen hydrogel to create a functional biomaterial-based stem cell niche for the delivery of mesenchymal stem cells (MSCs) into wounds. Murine bone marrow-derived MSCs were seeded into hydrogels and compared to MSCs grown in standard culture conditions. Hydrogels induced MSC secretion of angiogenic cytokines and expression of transcription factors associated with maintenance of pluripotency and self-renewal (Oct4, Sox2, Klf4) when compared to MSCs grown in standard conditions. An excisonal wound healing model was used to compare the ability of MSC-hydrogel constructs versus MSC injection alone to accelerate wound healing. Injection of MSCs did not significantly improve time to wound closure. In contrast, wounds treated with MSC-seeded hydrogels showed significantly accelerated healing and a return of skin appendages. Bioluminescence imaging and FACS analysis of luciferase+/GFP+ MSCs indicated that stem cells delivered within the hydrogel remained viable longer and demonstrated enhanced engraftment efficiency than those delivered via injection. Engrafted MSCs were found to differentiate into fibroblasts, pericytes and endothelial cells but did not contribute to the epidermis. Wounds treated with MSC-seeded hydrogels demonstrated significantly enhanced angiogenesis, which was associated with increased levels of VEGF and other angiogenic cytokines within the wounds. Our data suggest that biomimetic hydrogels provide a functional niche capable of augmenting MSC regenerative potential and enhancing wound healing.

    View details for DOI 10.1016/j.biomaterials.2011.09.041

    View details for Web of Science ID 000297399700009

    View details for PubMedID 21963148

  • Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling. Nature medicine Wong, V. W., Rustad, K. C., Akaishi, S., Sorkin, M., Glotzbach, J. P., Januszyk, M., Nelson, E. R., Levi, K., Paterno, J., Vial, I. N., Kuang, A. A., Longaker, M. T., Gurtner, G. C. 2012; 18 (1): 148-152

    Abstract

    Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK-ERK-MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

    View details for DOI 10.1038/nm.2574

    View details for PubMedID 22157678

  • Craniofacial Surgery: Innovation, Design, and Strategy JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Longaker, M. T. 2012; 23 (1): 7-7

    View details for DOI 10.1097/SCS.0b013e31824209e8

    View details for Web of Science ID 000300234900029

    View details for PubMedID 22337364

  • Cranial Suture Biology: From Pathways to Patient Care JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Wan, D. C., Wong, V. W., Nelson, E., Hyun, J., Longaker, M. T. 2012; 23 (1): 13-19

    Abstract

    Craniosynostosis describes the premature pathologic partial or complete fusion of 1 or more of the cranial sutures. Over the past few decades, research on craniosynostosis has progressed from gross description of deformities to an understanding of some of the molecular etiologies behind premature suture fusion. Studies on patients with syndromic craniosynostosis have resulted in the identification of several genes, molecular events, and deformational forces involved in abnormal growth and development of the cranial vault. Conservation of craniofacial development and sequence homology between humans and other species have also led to insightful discoveries in cranial suture development. In this review, we discuss the development of the cranial vault and explain the basic science behind craniosynostosis in humans as well as in animal models and how these studies may lead to future advances in craniosynostosis treatments.

    View details for DOI 10.1097/SCS.0b013e318240c6c0

    View details for Web of Science ID 000300234900033

    View details for PubMedID 22337368

  • Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate. Journal of visualized experiments : JoVE Lo, D. D., Hyun, J. S., Chung, M. T., Montoro, D. T., Zimmermann, A., Grova, M. M., Lee, M., Wan, D. C., Longaker, M. T. 2012

    Abstract

    Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

    View details for DOI 10.3791/4221

    View details for PubMedID 23149856

  • Stem Cells: Update and Impact on Craniofacial Surgery JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Glotzbach, J. P., Wong, V. W., Nelson, E. R., Hyun, J., Wan, D. C., Gurtner, G. C., Longaker, M. T. 2012; 23 (1): 319-322

    View details for DOI 10.1097/SCS.0b013e318241dbaf

    View details for Web of Science ID 000300234900099

    View details for PubMedID 22337434

  • Stem cell niches for skin regeneration. International journal of biomaterials Wong, V. W., Levi, B., Rajadas, J., Longaker, M. T., Gurtner, G. C. 2012; 2012: 926059-?

    Abstract

    Stem cell-based therapies offer tremendous potential for skin regeneration following injury and disease. Functional stem cell units have been described throughout all layers of human skin and the collective physical and chemical microenvironmental cues that enable this regenerative potential are known as the stem cell niche. Stem cells in the hair follicle bulge, interfollicular epidermis, dermal papillae, and perivascular space have been closely investigated as model systems for niche-driven regeneration. These studies suggest that stem cell strategies for skin engineering must consider the intricate molecular and biologic features of these niches. Innovative biomaterial systems that successfully recapitulate these microenvironments will facilitate progenitor cell-mediated skin repair and regeneration.

    View details for DOI 10.1155/2012/926059

    View details for PubMedID 22701121

  • Craniosynostosis: Molecular pathways and future pharmacologic therapy. Organogenesis Senarath-Yapa, K., Chung, M. T., McArdle, A., Wong, V. W., Quarto, N., Longaker, M. T., Wan, D. C. 2012; 8 (4)

    Abstract

    Craniosynostosis describes the premature fusion of one or more cranial sutures and can lead to dramatic manifestations in terms of appearance and functional impairment. Contemporary approaches for this condition are primarily surgical and are associated with considerable morbidity and mortality. The additional post-operative problems of suture refusion and bony relapse may also necessitate repeated surgeries with their own attendant risks. Therefore, a need exists to not only optimize current strategies but also to develop novel biological therapies which could obviate the need for surgery and potentially treat or even prevent premature suture fusion. Clinical studies of patients with syndromic craniosynostosis have provided some useful insights into the important signaling pathways and molecular events guiding suture fate. Furthermore, the highly conserved nature of craniofacial development between humans and other species have permitted more focused and step-wise elucidation of the molecular underpinnings of craniosynostosis. This review will describe the clinical manifestations of craniosynostosis, reflect on our understanding of syndromic and non-syndromic craniosynostoses and outline the different approaches that have been adopted in our laboratory and elsewhere to better understand the pathogenesis of premature suture fusion. Finally, we will assess to what extent our improved understanding of the pathogenesis of craniosynostosis, achieved through laboratory-based and clinical studies, have made the possibility of a non-surgical pharmacological approach both realistic and tangible.

    View details for PubMedID 23249483

  • Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose-Derived Stem Cells in Skeletal Repair STEM CELLS Levi, B., Hyun, J. S., Nelson, E. R., Li, S., Montoro, D. T., Wan, D. C., Jia, F. J., Glotzbach, J. C., James, A. W., Lee, M., Huang, M., Quarto, N., Gurtner, G. C., Wu, J. C., Longaker, M. T. 2011; 29 (12): 2018-2029

    Abstract

    An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose-derived stem cells (hASCs) represent a readily available, autogenous cell source with well-documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP-releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine.

    View details for DOI 10.1002/stem.757

    View details for Web of Science ID 000297220000012

    View details for PubMedID 21997852

  • Mechanical force prolongs acute inflammation via T-cell-dependent pathways during scar formation FASEB JOURNAL Wong, V. W., Paterno, J., Sorkin, M., Glotzbach, J. P., Levi, K., Januszyk, M., Rustad, K. C., Longaker, M. T., Gurtner, G. C. 2011; 25 (12): 4498-4510

    Abstract

    Mechanical force significantly modulates both inflammation and fibrosis, yet the fundamental mechanisms that regulate these interactions remain poorly understood. Here we performed microarray analysis to compare gene expression in mechanically loaded wounds vs. unloaded control wounds in an established murine hypertrophic scar (HTS) model. We identified 853 mechanically regulated genes (false discovery rate <2) at d 14 postinjury, a subset of which were enriched for T-cell-regulated pathways. To substantiate the role of T cells in scar mechanotransduction, we applied the HTS model to T-cell-deficient mice and wild-type mice. We found that scar formation in T-cell-deficient mice was reduced by almost 9-fold (P < 0.001) with attenuated epidermal (by 2.6-fold, P < 0.01) and dermal (3.9-fold, P < 0.05) proliferation. Mechanical stimulation was highly associated with sustained T-cell-dependent Th2 cytokine (IL-4 and IL-13) and chemokine (MCP-1) signaling. Further, T-cell-deficient mice failed to recruit systemic inflammatory cells such as macrophages or monocytic fibroblast precursors in response to mechanical loading. These findings indicate that T-cell-regulated fibrogenic pathways are highly mechanoresponsive and suggest that mechanical forces induce a chronic-like inflammatory state through immune-dependent activation of both local and systemic cell populations.

    View details for DOI 10.1096/fj.10-178087

    View details for Web of Science ID 000298138100040

    View details for PubMedID 21911593

  • Antimycotic Ciclopirox Olamine in the Diabetic Environment Promotes Angiogenesis and Enhances Wound Healing PLOS ONE Ko, S. H., Nauta, A., Morrison, S. D., Zhou, H., Zimmermann, A., Gurtner, G. C., Ding, S., Longaker, M. T. 2011; 6 (11)

    Abstract

    Diabetic wounds remain a major medical challenge with often disappointing outcomes despite the best available care. An impaired response to tissue hypoxia and insufficient angiogenesis are major factors responsible for poor healing in diabetic wounds. Here we show that the antimycotic drug ciclopirox olamine (CPX) can induce therapeutic angiogenesis in diabetic wounds. Treatment with CPX in vitro led to upregulation of multiple angiogenic genes and increased availability of HIF-1?. Using an excisional wound splinting model in diabetic mice, we showed that serial topical treatment with CPX enhanced wound healing compared to vehicle control treatment, with significantly accelerated wound closure, increased angiogenesis, and increased dermal cellularity. These findings offer a promising new topical pharmacologic therapy for the treatment of diabetic wounds.

    View details for DOI 10.1371/journal.pone.0027844

    View details for Web of Science ID 000297789200029

    View details for PubMedID 22125629

  • CD105 Protein Depletion Enhances Human Adipose-derived Stromal Cell Osteogenesis through Reduction of Transforming Growth Factor beta 1 (TGF-beta 1) Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Levi, B., Wan, D. C., Glotzbach, J. P., Hyun, J., Januszyk, M., Montoro, D., Sorkin, M., James, A. W., Nelson, E. R., Li, S., Quarto, N., Lee, M., Gurtner, G. C., Longaker, M. T. 2011; 286 (45): 39497-39509

    Abstract

    Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-?1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

    View details for DOI 10.1074/jbc.M111.256529

    View details for Web of Science ID 000296759800067

    View details for PubMedID 21949130

  • Pullulan Hydrogels Improve Mesenchymal Stem Cell Delivery into High-Oxidative-Stress Wounds MACROMOLECULAR BIOSCIENCE Wong, V. W., Rustad, K. C., Glotzbach, J. P., Sorkin, M., Inayathullah, M., Major, M. R., Longaker, M. T., Rajadas, J., Gurtner, G. C. 2011; 11 (11): 1458-1466

    Abstract

    Cell-based therapies for wound repair are limited by inefficient delivery systems that fail to protect cells from the acute inflammatory environment. Here, a biomimetic hydrogel system is described that is based on the polymer pullulan, a carbohydrate glucan known to exhibit potent antioxidant capabilities. It is shown that pullulan hydrogels are an effective cell delivery system and improve mesenchymal stem cell survival and engraftment in high-oxidative-stress environments. The results suggest that glucan hydrogel systems may prove beneficial for progenitor-cell-based approaches to skin regeneration.

    View details for DOI 10.1002/mabi.201100180

    View details for Web of Science ID 000297555500002

    View details for PubMedID 21994074

  • Calcium-Based Nanoparticles Accelerate Skin Wound Healing PLOS ONE Kawai, K., Larson, B. J., Ishise, H., Carre, A. L., Nishimoto, S., Longaker, M., Lorenz, H. P. 2011; 6 (11)

    Abstract

    Nanoparticles (NPs) are small entities that consist of a hydroxyapatite core, which can bind ions, proteins, and other organic molecules from the surrounding environment. These small conglomerations can influence environmental calcium levels and have the potential to modulate calcium homeostasis in vivo. Nanoparticles have been associated with various calcium-mediated disease processes, such as atherosclerosis and kidney stone formation. We hypothesized that nanoparticles could have an effect on other calcium-regulated processes, such as wound healing. In the present study, we synthesized pH-sensitive calcium-based nanoparticles and investigated their ability to enhance cutaneous wound repair.Different populations of nanoparticles were synthesized on collagen-coated plates under various growth conditions. Bilateral dorsal cutaneous wounds were made on 8-week-old female Balb/c mice. Nanoparticles were then either administered intravenously or applied topically to the wound bed. The rate of wound closure was quantified. Intravenously injected nanoparticles were tracked using a FLAG detection system. The effect of nanoparticles on fibroblast contraction and proliferation was assessed.A population of pH-sensitive calcium-based nanoparticles was identified. When intravenously administered, these nanoparticles acutely increased the rate of wound healing. Intravenously administered nanoparticles were localized to the wound site, as evidenced by FLAG staining. Nanoparticles increased fibroblast calcium uptake in vitro and caused contracture of a fibroblast populated collagen lattice in a dose-dependent manner. Nanoparticles also increased the rate of fibroblast proliferation.Intravenously administered, calcium-based nanoparticles can acutely decrease open wound size via contracture. We hypothesize that their contraction effect is mediated by the release of ionized calcium into the wound bed, which occurs when the pH-sensitive nanoparticles disintegrate in the acidic wound microenvironment. This is the first study to demonstrate that calcium-based nanoparticles can have a therapeutic benefit, which has important implications for the treatment of wounds.

    View details for DOI 10.1371/journal.pone.0027106

    View details for Web of Science ID 000297154900079

    View details for PubMedID 22073267

  • Pushing Back: Wound Mechanotransduction in Repair and Regeneration JOURNAL OF INVESTIGATIVE DERMATOLOGY Wong, V. W., Akaishi, S., Longaker, M. T., Gurtner, G. C. 2011; 131 (11): 2186-2196

    Abstract

    Human skin is a highly specialized mechanoresponsive interface separating our bodies from the external environment. It must constantly adapt to dynamic physical cues ranging from rapid expansion during embryonic and early postnatal development to ubiquitous external forces throughout life. Despite the suspected role of the physical environment in cutaneous processes, the fundamental molecular mechanisms responsible for how skin responds to force remain unclear. Intracellular pathways convert mechanical cues into biochemical responses (in a process known as mechanotransduction) via complex mechanoresponsive elements that often blur the distinction between physical and chemical signaling. For example, cellular focal adhesion components exhibit dual biochemical and scaffolding functions that are critically modulated by force. Moreover, the extracellular matrix itself is increasingly recognized to mechanically regulate the spatiotemporal distribution of soluble and matrix-bound ligands, underscoring the importance of bidirectional crosstalk between cells and their physical environment. It seems likely that a structural hierarchy exists to maintain both cells and matrix in mechanical homeostasis and that dysregulation of this architectural integrity may underlie or contribute to various skin disorders. An improved understanding of these interactions will facilitate the development of novel biophysical materials and mechanomodulatory approaches to augment wound repair and regeneration.

    View details for DOI 10.1038/jid.2011.212

    View details for Web of Science ID 000296240100012

    View details for PubMedID 21776006

  • Role of GSK-3 beta in the Osteogenic Differentiation of Palatal Mesenchyme PLOS ONE Nelson, E. R., Levi, B., Sorkin, M., James, A. W., Liu, K. J., Quarto, N., Longaker, M. T. 2011; 6 (10)

    Abstract

    The function of Glycogen Synthase Kinases 3? (GSK-3?) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification.Palates were harvested from GSK-3?, embryonic days 15.0-18.5 (e15.0-e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3? null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3? and Ihh +/+ and -/- e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists.GSK-3? null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3? null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3? -/- palate cultures were "rescued" with the Wnt inhibitor, Dkk-1.Here, we identify a critical role for GSK-3? in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation.

    View details for DOI 10.1371/journal.pone.0025847

    View details for Web of Science ID 000295981600015

    View details for PubMedID 22022457

  • Indian Hedgehog Positively Regulates Calvarial Ossification and Modulates Bone Morphogenetic Protein Signaling GENESIS Lenton, K., James, A. W., Manu, A., Brugmann, S. A., Birker, D., Nelson, E. R., Leucht, P., Helms, J. A., Longaker, M. T. 2011; 49 (10): 784-796

    Abstract

    Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh-/- phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh-/- mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro-osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh-/- calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification.

    View details for DOI 10.1002/dvg.20768

    View details for Web of Science ID 000296420300003

    View details for PubMedID 21557453

  • Palatogenesis Engineering, pathways and pathologies ORGANOGENESIS Levi, B., Brugman, S., Wong, V. W., Grova, M., Longaker, M. T., Wan, D. C. 2011; 7 (4): 242-254

    Abstract

    Cleft palate represents the second most common birth defect and carries substantial physiologic and social challenges for affected patients, as they often require multiple surgical interventions during their lifetime. A number of genes have been identified to be associated with the cleft palate phenotype, but etiology in the majority of cases remains elusive. In order to better understand cleft palate and both surgical and potential tissue engineering approaches for repair, we have performed an in-depth literature review into cleft palate development in humans and mice, as well as into molecular pathways underlying these pathologic developments. We summarize the multitude of pathways underlying cleft palate development, with the transforming growth factor beta superfamily being the most commonly studied. Furthermore, while the majority of cleft palate studies are performed using a mouse model, studies focusing on tissue engineering have also focused heavily on mouse models. A paucity of human randomized controlled studies exists for cleft palate repair, and so far, tissue engineering approaches are limited. In this review, we discuss the development of the palate, explain the basic science behind normal and pathologic palate development in humans as well as mouse models and elaborate on how these studies may lead to future advances in palatal tissue engineering and cleft palate treatments.

    View details for DOI 10.4161/org.7.4.17926

    View details for Web of Science ID 000299593000002

    View details for PubMedID 21964245

  • Preclinical Derivation and Imaging of Autologously Transplanted Canine Induced Pluripotent Stem Cells JOURNAL OF BIOLOGICAL CHEMISTRY Lee, A. S., Xu, D., Plews, J. R., Nguyen, P. K., Nag, D., Lyons, J. K., Han, L., Hu, S., Lan, F., Liu, J., Huang, M., Narsinh, K. H., Long, C. T., de Almeida, P. E., Levi, B., Kooreman, N., Bangs, C., Pacharinsak, C., Ikeno, F., Yeung, A. C., Gambhir, S. S., Robbins, R. C., Longaker, M. T., Wu, J. C. 2011; 286 (37): 32697-32704

    Abstract

    Derivation of patient-specific induced pluripotent stem cells (iPSCs) opens a new avenue for future applications of regenerative medicine. However, before iPSCs can be used in a clinical setting, it is critical to validate their in vivo fate following autologous transplantation. Thus far, preclinical studies have been limited to small animals and have yet to be conducted in large animals that are physiologically more similar to humans. In this study, we report the first autologous transplantation of iPSCs in a large animal model through the generation of canine iPSCs (ciPSCs) from the canine adipose stromal cells and canine fibroblasts of adult mongrel dogs. We confirmed pluripotency of ciPSCs using the following techniques: (i) immunostaining and quantitative PCR for the presence of pluripotent and germ layer-specific markers in differentiated ciPSCs; (ii) microarray analysis that demonstrates similar gene expression profiles between ciPSCs and canine embryonic stem cells; (iii) teratoma formation assays; and (iv) karyotyping for genomic stability. Fate of ciPSCs autologously transplanted to the canine heart was tracked in vivo using clinical positron emission tomography, computed tomography, and magnetic resonance imaging. To demonstrate clinical potential of ciPSCs to treat models of injury, we generated endothelial cells (ciPSC-ECs) and used these cells to treat immunodeficient murine models of myocardial infarction and hindlimb ischemia.

    View details for DOI 10.1074/jbc.M111.235739

    View details for Web of Science ID 000294726800078

    View details for PubMedID 21719696

  • Wound healing and regenerative strategies ORAL DISEASES Nauta, A., Gurtner, G. C., Longaker, M. T. 2011; 17 (6): 541-549

    Abstract

    Wound healing is a complex biological process that affects multiple tissue types. Wounds in the oral cavity are particularly challenging given the variety of tissue types that exist in close proximity to one another. The goal of regenerative medicine is to facilitate the rapid replacement of lost or damaged tissue with tissue that is functional, and physiologically similar to what previously existed. This review provides a general overview of wound healing and regenerative medicine, focusing specifically on how recent advances in the fields of stem cell biology, tissue engineering, and oral disease could translate into improved clinical outcomes.

    View details for DOI 10.1111/j.1601-0825.2011.01787.x

    View details for Web of Science ID 000292985000001

    View details for PubMedID 21332599

  • Recommendations on clinical proof of efficacy for potential scar prevention and reduction therapies WOUND REPAIR AND REGENERATION Bush, J. A., McGrouther, D. A., Young, V. L., Herndon, D. N., Longaker, M. T., Mustoe, T. A., Ferguson, M. W. 2011; 19: S32-S37

    Abstract

    Cutaneous scarring is an enormous medical problem with approximately 100 million patients acquiring scars each year. Scar prevention/reduction represents a significant, and largely unmet, clinical need. Research into the prophylactic modulation of scar outcome differs from research into other disease processes as the scar is not present at the start of the study; measurements of changes from baseline are impossible. Final scar morphology is influenced by many variables. A fundamental principle that should be observed in the prospective evaluation of scar prevention/reduction therapies is that, if left untreated, wounds in treatment and control groups should have healed with identical scars. Observation of this principle will allow the detection of true treatment effects. The many variables that influence scar morphology mean that the evaluation of potential pharmaceutical products for this indication favors the use of self-controlled designs in clinical trials. In this article, we review variables that affect scar morphology and recommend the self-controlled design for clinical trials aiming to establish proof of efficacy of scar prevention and reduction pharmaceuticals. With no pharmaceutical products currently licensed for this indication, this represents a new therapeutic area. The principles discussed will also have direct relevance to the wider fields of wound healing and regenerative medicine.

    View details for DOI 10.1111/j.1524-475X.2010.00607.x

    View details for Web of Science ID 000293237400006

    View details for PubMedID 21793964

  • Commentary on Role of Apoptosis in Retinoic Acid-Induced Cleft Palate JOURNAL OF CRANIOFACIAL SURGERY Nelson, E. R., Levi, B., Longaker, M. T. 2011; 22 (5): 1572-1573

    View details for DOI 10.1097/SCS.0b013e31822e5ea6

    View details for Web of Science ID 000295398700007

    View details for PubMedID 21959389

  • Vascular anastomosis using controlled phase transitions in poloxamer gels NATURE MEDICINE Chang, E. I., Galvez, M. G., Glotzbach, J. P., Hamou, C. D., El-Ftesi, S., Rappleye, C. T., Sommer, K., Rajadas, J., Abilez, O. J., Fuller, G. G., Longaker, M. T., Gurtner, G. C. 2011; 17 (9): 1147-U160

    Abstract

    Vascular anastomosis is the cornerstone of vascular, cardiovascular and transplant surgery. Most anastomoses are performed with sutures, which are technically challenging and can lead to failure from intimal hyperplasia and foreign body reaction. Numerous alternatives to sutures have been proposed, but none has proven superior, particularly in small or atherosclerotic vessels. We have developed a new method of sutureless and atraumatic vascular anastomosis that uses US Food and Drug Administration (FDA)-approved thermoreversible tri-block polymers to temporarily maintain an open lumen for precise approximation with commercially available glues. We performed end-to-end anastomoses five times more rapidly than we performed hand-sewn controls, and vessels that were too small (<1.0 mm) to sew were successfully reconstructed with this sutureless approach. Imaging of reconstructed rat aorta confirmed equivalent patency, flow and burst strength, and histological analysis demonstrated decreased inflammation and fibrosis at up to 2 years after the procedure. This new technology has potential for improving efficiency and outcomes in the surgical treatment of cardiovascular disease.

    View details for DOI 10.1038/nm.2424

    View details for Web of Science ID 000294605100038

    View details for PubMedID 21873986

  • Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip NATURE Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M. T., Weissman, I. L. 2011; 476 (7361): 409-U53

    Abstract

    The regrowth of amputated limbs and the distal tips of digits represent models of tissue regeneration in amphibians, fish and mice. For decades it had been assumed that limb regeneration derived from the blastema, an undifferentiated pluripotent cell population thought to be derived from mature cells via dedifferentiation. Here we show that a wide range of tissue stem/progenitor cells contribute towards the restoration of the mouse distal digit. Genetic fate mapping and clonal analysis of individual cells revealed that these stem cells are lineage restricted, mimicking digit growth during development. Transplantation of cyan-fluorescent-protein-expressing haematopoietic stem cells, and parabiosis between genetically marked mice, confirmed that the stem/progenitor cells are tissue resident, including the cells involved in angiogenesis. These results, combined with those from appendage regeneration in other vertebrate subphyla, collectively demonstrate that tissue stem cells rather than pluripotent blastema cells are an evolutionarily conserved cellular mode for limb regeneration after amputation.

    View details for DOI 10.1038/nature10346

    View details for Web of Science ID 000294209400027

    View details for PubMedID 21866153

  • Differences in Osteogenic Differentiation of Adipose-Derived Stromal Cells from Murine, Canine, and Human Sources In Vitro and In Vivo PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., Nelson, E. R., Brown, K., James, A. W., Xu, D., Dunlevie, R., Wu, J. C., Lee, M., Wu, B., Commons, G. W., Vistnes, D., Longaker, M. T. 2011; 128 (2): 373-386

    Abstract

    Given the diversity of species from which adipose-derived stromal cells are derived and studied, the authors set out to delineate the differences in the basic cell biology that may exist across species. Briefly, the authors found that significant differences exist with regard to proliferation and osteogenic potentials of adipose-derived stromal cells across species.Adipose-derived stromal cells were derived from human, mouse, and canine sources as previously described. Retinoic acid, insulin-like growth factor-1, and bone morphogenetic protein-2 were added to culture medium; proliferation and osteogenic differentiation were assessed by standardized assays. In vivo methods included seeding 150,000 adipose-derived stromal cells on a biomimetic scaffold and analyzing healing by micro-computed tomography and histology.Adipose-derived stromal cells from all species had the capability to undergo osteogenic differentiation. Canine adipose-derived stromal cells were the most proliferative, whereas human adipose-derived stromal cells were the most osteogenic (p < 0.05). Human cells, however, had the most significant osteogenic response to osteogenic media. Retinoic acid stimulated osteogenesis in mouse and canine cells but not in human adipose-derived stromal cells. Insulin-like growth factor-1 enhanced osteogenesis across all species, most notably in human- and canine-derived cells.Adipose-derived stromal cells derived from human, mouse, and canine all have the capacity to undergo osteogenic differentiation. Canine adipose-derived stromal cells appear to be the most proliferative, whereas human adipose-derived stromal cells appear to be the most osteogenic. Different cytokines and chemicals can be used to modulate this osteogenic response. These results are promising as attempts are made to optimize tissue-engineered bone using adipose-derived stromal cells.

    View details for DOI 10.1097/PRS.0b013e31821e6e49

    View details for Web of Science ID 000293464400032

    View details for PubMedID 21788829

  • Improving Cutaneous Scar Formation by Controlling the Mechanical Environment Large Animal and Phase I Studies ANNALS OF SURGERY Gurtner, G. C., Dauskardt, R. H., Wong, V. W., Bhatt, K. A., Wu, K., Vial, I. N., Padois, K., Korman, J. M., Longaker, M. T. 2011; 254 (2): 217-225

    Abstract

    To test the hypothesis that the mechanical environment of cutaneous wounds can control scar formation.Mechanical forces have been recognized to modulate myriad biologic processes, but the role of physical force in scar formation remains unclear. Furthermore, the therapeutic benefits of offloading cutaneous wounds with a device have not been rigorously tested.A mechanomodulating polymer device was utilized to manipulate the mechanical environment of closed cutaneous wounds in red Duroc swine. After 8 weeks, wounds subjected to different mechanical stress states underwent immunohistochemical analysis for fibrotic markers. In a phase I clinical study, 9 human patients undergoing elective abdominal surgery were treated postoperatively with a stress-shielding polymer on one side whereas the other side was treated as standard of care. Professional photographs were taken between 8 and 12 months postsurgery and evaluated using a visual analog scale by lay and professional panels. This study is registered with ClinicalTrials.gov, number NCT00766727.Stress shielding of swine incisions reduced histologic scar area by 6- and 9-fold compared to control and elevated stress states, respectively (P < 0.01 for both) and dramatically decreased the histologic expression of profibrotic markers. Closure of high-tension wounds induced human-like scar formation in the red Duroc, a phenotype effectively mitigated with stress shielding of wounds. In the study on humans, stress shielding of abdominal incisions significantly improved scar appearance (P = 0.004) compared with within-patient controls.These results indicate that mechanical manipulation of the wound environment with a dynamic stress-shielding polymer device can significantly reduce scar formation.

    View details for DOI 10.1097/SLA.0b013e318220b159

    View details for Web of Science ID 000292908700007

    View details for PubMedID 21606834

  • Dura Mater Stimulates Human Adipose-Derived Stromal Cells to Undergo Bone Formation in Mouse Calvarial Defects STEM CELLS Levi, B., Nelson, E. R., Li, S., James, A. W., Hyun, J. S., Montoro, D. T., Lee, M., Glotzbach, J. P., Commons, G. W., Longaker, M. T. 2011; 29 (8): 1241-1255

    Abstract

    Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2-Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.

    View details for DOI 10.1002/stem.670

    View details for Web of Science ID 000293133900009

    View details for PubMedID 21656608

  • Fgf-18 Is Required for Osteogenesis But Not Angiogenesis During Long Bone Repair TISSUE ENGINEERING PART A Behr, B., Sorkin, M., Manu, A., Lehnhardt, M., Longaker, M. T., Quarto, N. 2011; 17 (15-16): 2061-2069

    Abstract

    Bone regeneration is a complex event that requires the interaction of numerous growth factors. Fibroblast growth factor (Fgf)-ligands have been previously described for their importance in osteogenesis during development. In the current study, we investigated the role of Fgf-18 during bone regeneration. By utilizing a unicortical tibial defect model, we revealed that mice haploinsufficient for Fgf-18 have a markedly reduced healing capacity as compared with wild-type mice. Reduced levels of Runx2 and Osteocalcin but not Vegfa accompanied the impaired bone regeneration. Interestingly, our data indicated that upon injury angiogenesis was not impaired in Fgf-18(+/-) mice. Moreover, other Fgf-ligands and Bmp-2 could not compensate for the loss of Fgf-18. Finally, application of FGF-18 protein was able to rescue the impaired healing in Fgf-18(+/-) mice. Thus, we identified Fgf-18 as an important mediator of bone regeneration, which is required during later stages of bone regeneration. This study provides hints on how to engineering efficiently programmed bony tissue for long bone repair.

    View details for DOI 10.1089/ten.tea.2010.0719

    View details for Web of Science ID 000293217700015

    View details for PubMedID 21457097

  • An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells PLOS ONE Glotzbach, J. P., Januszyk, M., Vial, I. N., Wong, V. W., Gelbard, A., Kalisky, T., Thangarajah, H., Longaker, M. T., Quake, S. R., Chu, G., Gurtner, G. C. 2011; 6 (6)

    Abstract

    An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

    View details for DOI 10.1371/journal.pone.0021211

    View details for Web of Science ID 000292033700046

    View details for PubMedID 21731674

  • Unfolded Protein Response Regulation in Keloid Cells JOURNAL OF SURGICAL RESEARCH Butler, P. D., Wang, Z., Ly, D. P., Longaker, M. T., Koong, A. C., Yang, G. P. 2011; 167 (1): 151-157

    Abstract

    Keloids are a common form of pathologic wound healing characterized by excessive production of extracellular matrix. The unfolded protein response (UPR) is a cellular response to hypoxia, a component of the wound microenvironment, capable of protecting cells from the effects of over-accumulation of misfolded proteins. Since keloids have hypersecretion of extracellular matrix, we hypothesized that keloid fibroblasts (KFs) may have enhanced activation of the UPR compared with normal fibroblasts (NFs).KFs and NFs were placed in a hypoxia chamber for 0, 24, and 48h. We also used tunicamycin to specifically up-regulate the UPR. UPR activation was assayed by PCR for xbp-1 splicing and by immunoblotting with specific antibodies for the three UPR transducers. Nuclear localization of XBP-1 protein in KFs was confirmed by immunofluorescence.There is increased activation of XBP-1 protein in KFs compared with NFs following exposure to hypoxia. Pancreatic ER kinase (PERK) and ATF-6, two other pathways activated by the UPR, show comparable activation between KFs and NFs. We confirmed that there is enhanced activation of XBP-1 by demonstrating increased nuclear localization of XBP-1 using immunofluorescence.In contrast to our initial hypothesis that keloids would have broad activation of the UPR, we demonstrate here that there is a specific up-regulation of one facet of the UPR response. This may represent a specific molecular defect in KFs compared with NFs, and also suggests modulation of the UPR can be used in wound healing therapy.

    View details for DOI 10.1016/j.jss.2009.04.036

    View details for Web of Science ID 000288744100029

    View details for PubMedID 19631342

  • Integrating Cultural Competency and Humility Training into Clinical Clerkships: Surgery as a Model JOURNAL OF SURGICAL EDUCATION Butler, P. D., Swift, M., Kothari, S., Nazeeri-Simmons, I., Friel, C. M., Longaker, M. T., Britt, L. D. 2011; 68 (3): 222-230

    Abstract

    Cultural competency is gaining recognition as an essential strategy by which to address health care disparities. A closer examination of medical school curriculums was undertaken to determine how the need for cultural competency and humility (CCH) training in medical education is being addressed.A MEDLINE review of published literature regarding CCH training in medical education was performed. Additionally, key informant interviews with influential faculty members from prominent medical institutions were completed.Many academic medical institutions recognize the need for CCH and have successfully integrated it into the first 2 years of their curriculums. However, there seems to be a uniform deficit in CCH training in the third and fourth years of their education.Recognizing the need for CCH training during the third and fourth years of medical education, we explored the issues inherent to the integration of CCH training in clinical education. Using surgery as a model, we established a set of recommendations to assist clerkship directors and curriculum committees in their efforts to ensure CCH training in the last 2 years of medical education.

    View details for DOI 10.1016/j.jsurg.2011.01.002

    View details for Web of Science ID 000289877700014

    View details for PubMedID 21481809

  • Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation FEBS LETTERS Lahti, J. L., Lui, B. H., Beck, S. E., Lee, S. S., Ly, D. P., Longaker, M. T., Yang, G. P., Cochran, J. R. 2011; 585 (8): 1135-1139

    Abstract

    Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity.

    View details for DOI 10.1016/j.febslet.2011.03.044

    View details for Web of Science ID 000289505400004

    View details for PubMedID 21439278

  • Concise Review: Adipose-Derived Stromal Cells for Skeletal Regenerative Medicine STEM CELLS Levi, B., Longaker, M. T. 2011; 29 (4): 576-582

    Abstract

    As the average age of the population grows, the incidence of osteoporosis and skeletal diseases continues to rise. Current treatment options for skeletal repair include immobilization, rigid fixation, alloplastic materials, and bone grafts, all which have significant limitations, especially in the elderly. Adipose-derived stromal cells (ASCs) represent a readily available abundant supply of mesenchymal stem cells, which demonstrate the ability to undergo osteogenesis in vitro and in vivo, making ASCs a promising source of skeletal progenitor cells. Current protocols allow for the harvest of over one million cells from only 15 ml of lipoaspirate. Despite the clinical use of ASCs to treat systemic inflammatory diseases, no large human clinical trials exist using ASCs for skeletal tissue engineering. The aim of this review is to define ASCs, to describe the isolation procedure of ASCs, to review the basic biology of their osteogenic differentiation, discuss cell types and scaffolds available for bone tissue engineering, and finally, to explore imaging of ASCs and their potential future role in human skeletal tissue engineering efforts.

    View details for DOI 10.1002/stem.612

    View details for Web of Science ID 000289157100004

    View details for PubMedID 21305671

  • Chemical Control of FGF-2 Release for Promoting Calvarial Healing with Adipose Stem Cells JOURNAL OF BIOLOGICAL CHEMISTRY Kwan, M. D., Sellmyer, M. A., Quarto, N., Ho, A. M., Wandless, T. J., Longaker, M. T. 2011; 286 (13): 11307-11313

    Abstract

    Chemical control of protein secretion using a small molecule approach provides a powerful tool to optimize tissue engineering strategies by regulating the spatial and temporal dimensions that are exposed to a specific protein. We placed fibroblast growth factor 2 (FGF-2) under conditional control of a small molecule and demonstrated greater than 50-fold regulation of FGF-2 release as well as tunability, reversibility, and functionality in vitro. We then applied conditional control of FGF-2 secretion to a cell-based, skeletal tissue engineering construct consisting of adipose stem cells (ASCs) on a biomimetic scaffold to promote bone formation in a murine critical-sized calvarial defect model. ASCs are an easily harvested and abundant source of postnatal multipotent cells and have previously been demonstrated to regenerate bone in critical-sized defects. These results suggest that chemically controlled FGF-2 secretion can significantly increase bone formation by ASCs in vivo. This study represents a novel approach toward refining protein delivery for tissue engineering applications.

    View details for DOI 10.1074/jbc.M110.180042

    View details for Web of Science ID 000288797100043

    View details for PubMedID 21262969

  • Research Training in Plastic Surgery JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Longaker, M. T. 2011; 22 (2): 383-384

    View details for DOI 10.1097/SCS.0b013e318208ba73

    View details for Web of Science ID 000288535800004

    View details for PubMedID 21415623

  • Studies in Adipose-Derived Stromal Cells: Migration and Participation in Repair of Cranial Injury after Systemic Injection PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Nelson, E. R., Hu, S., Sun, N., Peng, M., Wu, J., Longaker, M. T. 2011; 127 (3): 1130-1140

    Abstract

    Adipose-derived stromal cells are a multipotent cell type with the ability to undergo osteogenic differentiation. The authors sought to examine whether systemically administered adipose-derived stromal cells would migrate to and heal surgically created defects of the mouse cranial skeleton.Mouse adipose-derived stromal cells were harvested from luciferase-positive transgenic mice; human adipose-derived stromal cells were harvested from human lipoaspirate and labeled with luciferase and green fluorescent protein. A 4-mm calvarial defect (critical sized) was made in the mouse parietal bone; skin incisions alone were used as a control (n = 5 per group). Adipose-derived stromal cells were injected intravenously (200,000 cells per animal) and compared with saline injection only. Methods of analyses included micro-computed tomographic scanning, in vivo imaging system detection of luciferase activity, and standard histology.Migration of adipose-derived stromal cells to calvarial defect sites was confirmed by accumulation of luciferase activity and green fluorescent protein stain as early as 4 days and persisting up to 4 weeks. Little activity was observed among control groups. Intravenous administration of either mouse or human adipose-derived stromal cells resulted in histologic evidence of bone formation within the defect site, in comparison with an absence of bone among control defects. By micro-computed tomographic analysis, human but not mouse adipose-derived stromal cells stimulated significant osseous healing.Intravenously administered adipose-derived stromal cells migrate to sites of calvarial injury. Thereafter, intravenous human adipose-derived stromal cells contribute to bony calvarial repair. Intravenous administration of adipose-derived stromal cells may be an effective delivery method for future efforts in skeletal regeneration.

    View details for DOI 10.1097/PRS.0b013e3182043712

    View details for Web of Science ID 000287680200013

    View details for PubMedID 21364416

  • Osteogenic Differentiation of Adipose-Derived Stromal Cells in Mouse and Human: In Vitro and In Vivo Methods JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Longaker, M. T. 2011; 22 (2): 388-391

    View details for DOI 10.1097/SCS.0b013e318207b72b

    View details for Web of Science ID 000288535800006

    View details for PubMedID 21415625

  • Role of Indian Hedgehog Signaling in Palatal Osteogenesis PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Nelson, E. R., Brugmann, S. A., Sorkin, M., Manu, A., Longaker, M. T. 2011; 127 (3): 1182-1190

    Abstract

    Cleft lip-cleft palate is a common congenital disability and represents a large biomedical burden. Through the use of animal models, the molecular underpinnings of cleft palate are becoming increasingly clear. Indian hedgehog (Ihh) has been shown to be associated with craniofacial development and to be active in the palatine bone. The authors hypothesize that Indian hedgehog activity plays a role in osteogenesis within the secondary palate and that defects in this pathway may inhibit osteogenesis of the secondary palate.Palates were isolated from wild-type mice during the period of palate development (embryonic days 9.5 to 17.5). Quantitative real-time polymerase chain reaction was used for detecting gene expression during osteogenic differentiation and cellular differentiation (Shh, Ihh, Ptc1, Gli1, Gli2, Gli3, Runx2, Alp, and Col1a1). Next, palates were analyzed by hematoxylin and eosin, aniline blue, pentachrome, and in situ hybridization to assess osteogenesis of the palatal shelf and expression of hedgehog pathway genes. Finally, the palates of Indian hedgehog-null mice were analyzed to determine the effect of genetic deficiency on palatal development osteogenesis.Increased Indian hedgehog and osteogenic signaling coincided with ossification and fusion of the palate in wild-type mice. This included a fivefold to 150-fold peak in expression of hedgehog elements, including Ihh, at embryonic day 15.5 as compared with embryonic day 9.5. Contrarily, loss of Indian hedgehog by genetic knockout (Ihh-/-) resulted in decreased secondary palate ossification.The authors' results suggest a role for hedgehog signaling during palatal ossification. The hedgehog pathway is activated during palatal fusion, and deletion of Indian hedgehog leads to diminished ossification of the secondary hard palate.

    View details for DOI 10.1097/PRS.0b013e3182043a07

    View details for Web of Science ID 000287680200019

    View details for PubMedID 21364421

  • Acute Skeletal Injury Is Necessary for Human Adipose-Derived Stromal Cell-Mediated Calvarial Regeneration PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Nelson, E. R., Peng, M., Wan, D. C., Commons, G. W., Lee, M., Wu, B., Longaker, M. T. 2011; 127 (3): 1118-1129

    Abstract

    Studies have demonstrated that human adipose-derived stromal cells (ASCs) are able to repair acute calvarial injuries. The more clinically relevant repair of an established skeletal defect, however, has not been addressed. The authors sought to determine whether human ASCs could heal chronic (established) calvarial defects.Critical-sized (4 mm) mouse parietal defects were created. Human ASCs were engrafted either immediately postoperatively (acute defect) or 8 weeks following defect creation (established defect). Methods of analysis included microcomputer tomography scans, histology, and in situ hybridization. Finally, human ASCs were treated in vitro with platelet-rich plasma to simulate an acute wound environment; proliferation and osteogenic differentiation were assessed (alkaline phosphatase, alizarin red, and quantitative reverse transcriptase polymerase chain reaction).Nearly complete osseous healing was observed when calvarial defects were immediately engrafted with human ASCs. In contrast, when human ASCs were engrafted into established defects, little bone formation occurred. Histological analysis affirmed findings by microcomputer tomography, showing more robust staining for alkaline phosphatase and picrosirius red in an acute than in an established human ASC-engrafted defect. In situ hybridization and quantitative reverse transcriptase polymerase chain reaction showed an increase in bone morphogenetic protein (BMP) expression (BMP-2, BMP-4, and BMP-7) acutely following calvarial defect creation. Finally, in vitro treatment of human ASCs with platelet-rich plasma enhanced osteogenic differentiation and increased BMP-2 expression.Although human ASCs can be utilized to heal an acute mouse calvarial defect, they do not enhance healing of an established (or chronic) defect. Endogenous BMP signaling activated after injury may explain these differences in healing. Platelet-rich plasma enhances osteogenic differentiation of human ASCs in vitro and may prove a promising therapy for future skeletal tissue engineering efforts.

    View details for DOI 10.1097/PRS.0b013e318205f274

    View details for Web of Science ID 000287680200012

    View details for PubMedID 21364415

  • In brief: Regenerative medicine. Current problems in surgery Glotzbach, J. P., Wong, V. W., Gurtner, G. C., Longaker, M. T. 2011; 48 (3): 142-146

    View details for DOI 10.1067/j.cpsurg.2010.11.001

    View details for PubMedID 21295631

  • Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo STEM CELLS AND DEVELOPMENT James, A. W., Levi, B., Nelson, E. R., Peng, M., Commons, G. W., Lee, M., Wu, B., Longaker, M. T. 2011; 20 (3): 427-439

    Abstract

    Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)-polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT-polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze-thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze-thaw process in vitro (*P?

    View details for DOI 10.1089/scd.2010.0082

    View details for Web of Science ID 000287798300006

    View details for PubMedID 20536327

  • Regenerative Medicine CURRENT PROBLEMS IN SURGERY Glotzbach, J. P., Wong, V. W., Gurtner, G. C., Longaker, M. T. 2011; 48 (3): 148-212

    View details for DOI 10.1067/j.cpsurg.2010.11.002

    View details for Web of Science ID 000287543600002

    View details for PubMedID 21295632

  • Engineered Pullulan-Collagen Composite Dermal Hydrogels Improve Early Cutaneous Wound Healing TISSUE ENGINEERING PART A Wong, V. W., Rustad, K. C., Galvez, M. G., Neofyotou, E., Glotzbach, J. P., Januszyk, M., Major, M. R., Sorkin, M., Longaker, M. T., Rajadas, J., Gurtner, G. C. 2011; 17 (5-6): 631-644

    Abstract

    New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan-collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan-collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.

    View details for DOI 10.1089/ten.tea.2010.0298

    View details for Web of Science ID 000287801600005

    View details for PubMedID 20919949

  • Elastic Properties of Induced Pluripotent Stem Cells TISSUE ENGINEERING PART A Hammerick, K. E., Huang, Z., Sun, N., Lam, M. T., Prinz, F. B., Wu, J. C., Commons, G. W., Longaker, M. T. 2011; 17 (3-4): 495-502

    Abstract

    The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications, including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs), a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC), and also the gold standard human embryonic stem cell, we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness, and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly, cells exhibited a noticeable difference in stiffness. From least to most stiff, the order of cell stiffness was as follows: hASC-iPSC, human embryonic stem cell, fibroblast-iPSC, fibroblasts, and, lastly, as the stiffest cell, hASC. In comparing hASC-iPSCs to their origin cell, the hASC, the reprogrammed cell is significantly less stiff, indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence, material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state.

    View details for DOI 10.1089/ten.tea.2010.0211

    View details for Web of Science ID 000286661600023

    View details for PubMedID 20807017

  • Human Adipose-Derived Stromal Cells Stimulate Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts STEM CELLS AND DEVELOPMENT Levi, B., James, A. W., Nelson, E. R., Li, S., Peng, M., Commons, G. W., Lee, M., Wu, B., Longaker, M. T. 2011; 20 (2): 243-257

    Abstract

    Human adipose-derived stromal cells (hASCs) have the proven capacity to ossify skeletal defects. The mechanisms whereby hASCs stimulate bone repair are not fully understood. In this study, we examined the potential for hASCs to stimulate autogenous repair of a mouse calvarial defect. Immunofluoresence, osteogenic stains, and surface electron microscopy were used to demonstrate osteogenic differentiation of hASCs. hASCs were engrafted into 4 mm calvarial defects in athymic mice using an osteoconductive scaffold. Analysis included microcomputed tomography, histology, in situ hybridization, and quantitative real-time-polymerase chain reaction. Next, the in vitro interaction between hASCs and mouse calvarial osteoblasts (mOBs) was assessed by the conditioned medium and coculture assays. The medium was supplemented with Hedgehog signaling modifiers, including recombinant N-terminal Sonic hedgehog, smoothened agonist, and cyclopamine. Finally, cyclopamine was delivered in vivo to hASC-engrafted defects. Significant calvarial healing was observed among hASC-engrafted defects compared with control groups (no treatment or scaffold alone) (*P<0.05). hASCs showed evidence of stimulation of host mouse osteogenesis, including (1) increased expression of bone markers at the defect edge by in situ hybridization, and (2) increased host osteogenic gene expression by species-specific quantitative real-time polymerase chain reaction. Using the conditioned medium or coculture assays, hASCs stimulated mOB osteogenic differentiation, accompanied by Hedgehog signaling activation. N-terminal Sonic hedgehog or smoothened agonist replicated, while cyclopamine reversed, the pro-osteogenic effect of the conditioned medium on mOBs. Finally, cyclopamine injection arrested bone formation in vivo. hASCs heal critical-sized mouse calvarial defects, this is, at least in part, via stimulation of autogenous healing of the host defect. Our studies suggest that hASC-derived Hedgehog signaling may play a paracrine role in skeletal repair.

    View details for DOI 10.1089/scd.2010.0250

    View details for Web of Science ID 000286460000007

    View details for PubMedID 20698749

  • Locally Applied Vascular Endothelial Growth Factor A Increases the Osteogenic Healing Capacity of Human Adipose-Derived Stem Cells by Promoting Osteogenic and Endothelial Differentiation STEM CELLS Behr, B., Tang, C., Germann, G., Longaker, M. T., Quarto, N. 2011; 29 (2): 286-296

    Abstract

    Human adipose-derived stem cells (hASCs) are known for their capability to promote bone healing when applied to bone defects. For bone tissue regeneration, both sufficient angiogenesis and osteogenesis is desirable. Vascular endothelial growth factor A (VEGFA) has the potential to promote differentiation of common progenitor cells to both lineages. To test this hypothesis, the effects of VEGFA on hASCs during osteogenic differentiation were tested in vitro. In addition, hASCs were seeded in murine critical-sized calvarial defects locally treated with VEGFA. Our results suggest that VEGFA improves osteogenic differentiation in vitro as indicated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time polymerase chain reaction analysis. Moreover, local application of VEGFA to hASCs significantly improved healing of critical-sized calvarial defects in vivo. This repair was accompanied by a striking enhancement of angiogenesis. Both paracrine and, to a lesser degree, cell-autonomous effects of VEGFA-treated hASCs were accountable for angiogenesis. These data were confirmed by using CD31(-) /CD45(-) mouse ASCs(GFP+) cells. In summary, we demonstrated that VEGFA increased osteogenic differentiation of hASCS in vitro and in vivo, which was accompanied by an enhancement of angiogenesis. Additionally, we showed that during bone regeneration, the increase in angiogenesis of hASCs on treatment with VEGFA was attributable to both paracrine and cell-autonomous effects. Thus, locally applied VEGFA might prove to be a valuable growth factor that can mediate both osteogenesis and angiogenesis of multipotent hASCs in the context of bone regeneration.

    View details for DOI 10.1002/stem.581

    View details for Web of Science ID 000287698600012

    View details for PubMedID 21732486

  • Differential Expression of Sclerostin in Adult and Juvenile Mouse Calvariae PLASTIC AND RECONSTRUCTIVE SURGERY Kwan, M. D., Quarto, N., Gupta, D. M., Slater, B. J., Wan, D. C., Longaker, M. T. 2011; 127 (2): 595-602

    Abstract

    An understanding of the molecular mechanisms controlling bone formation is central to skeletal tissue engineering efforts. The observation that immature animals are able to heal calvarial defects while adult animals are not has proven to be a useful tool for examining these mechanisms. Thus, the authors compared expression of sclerostin, a bone inhibitor, between the calvariae of juvenile and adult mice.Parietal bone was harvested from juvenile (6-day-old; n = 20) and adult (60-day-old; n = 20) mice. Sclerostin transcript and protein levels were compared between the parietal bone of juvenile and adult mice using polymerase chain reaction, Western blotting, and immunohistochemistry. Finally, osteoblasts from the parietal bone of juvenile and adult mice were harvested and cultured under osteogenic differentiation conditions with and without recombinant sclerostin (200 ng/ml). Terminal osteogenic differentiation was assessed at 21 days with alizarin red staining.Polymerase chain reaction, Western blot analysis, and immunohistochemistry all confirmed greater expression of sclerostin in the parietal bone of adult mice when compared with that of juvenile mice. Osteoblasts, whether from juvenile or adult parietal bones, demonstrated reduced capacity for osteogenic differentiation when exposed to recombinant sclerostin.Given the role of sclerostin in inhibiting bone formation, the authors' findings suggest that differences in expression levels of sclerostin may play a role in the differential regenerative capacity of calvariae from juvenile and adult animals. These findings suggest it as a potential target to abrogate in future tissue engineering studies.

    View details for DOI 10.1097/PRS.0b013e3181fed60d

    View details for Web of Science ID 000286928100014

    View details for PubMedID 21285764

  • Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors NATURE PROTOCOLS Narsinh, K. H., Jia, F., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2011; 6 (1): 78-88

    Abstract

    Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ?4 weeks.

    View details for DOI 10.1038/nprot.2010.173

    View details for Web of Science ID 000285965000008

    View details for PubMedID 21212777

  • Salvage of the Crucified Chin PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Longaker, M. T., Allam, K. A., Perry, A., Kawamoto, H. K. 2011; 127 (1): 352-354

    View details for DOI 10.1097/PRS.0b013e3181fad3d6

    View details for Web of Science ID 000285992100048

    View details for PubMedID 21200230

  • Osteogenic differentiation of human multipotent mesenchymal stromal cells. Methods in molecular biology (Clifton, N.J.) Gupta, D. M., Panetta, N. J., Longaker, M. T. 2011; 698: 201-214

    Abstract

    A comprehensive knowledge of the molecular biology underlying osteogenic differentiation in a controlled, laboratory setting may promise optimization of future cell-based tissue engineering strategies for clinical problems. The scope of this review encompasses a discussion of the methodology utilized to perform such studies. Our laboratory routinely performs both in vitro and in vivo assays underlying osteogenic differentiation, and the widespread use of singular methodology across multiple investigators and institutions promises great advancements for the skeletal tissue engineering community.

    View details for DOI 10.1007/978-1-60761-999-4_16

    View details for PubMedID 21431521

  • Craniosynostosis of coronal suture in twist1 mice occurs through endochondral ossification recapitulating the physiological closure of posterior frontal suture. Frontiers in physiology Behr, B., Longaker, M. T., Quarto, N. 2011; 2: 37-?

    Abstract

    Craniosynostosis, the premature closure of cranial suture, is a pathologic condition that affects 1/2000 live births. Saethre-Chotzen syndrome is a genetic condition characterized by craniosynostosis. The Saethre-Chotzen syndrome, which is defined by loss-of-function mutations in the TWIST gene, is the second most prevalent craniosynostosis. Although much of the genetics and phenotypes in craniosynostosis syndromes is understood, less is known about the underlying ossification mechanism during suture closure. We have previously demonstrated that physiological closure of the posterior frontal suture occurs through endochondral ossification. Moreover, we revealed that antagonizing canonical Wnt-signaling in the sagittal suture leads to endochondral ossification of the suture mesenchyme and sagittal synostosis, presumably by inhibiting Twist1. Classic Saethre-Chotzen syndrome is characterized by coronal synostosis, and the haploinsufficient Twist1(+/-) mice represents a suitable model for studying this syndrome. Thus, we seeked to understand the underlying ossification process in coronal craniosynostosis in Twist1(+/-) mice. Our data indicate that coronal suture closure in Twist1(+/-) mice occurs between postnatal day 9 and 13 by endochondral ossification, as shown by histology, gene expression analysis, and immunohistochemistry. In conclusion, this study reveals that coronal craniosynostosis in Twist1(+/-) mice occurs through endochondral ossification. Moreover, it suggests that haploinsufficiency of Twist1 gene, a target of canonical Wnt-signaling, and inhibitor of chondrogenesis, mimics conditions of inactive canonical Wnt-signaling leading to craniosynostosis.

    View details for DOI 10.3389/fphys.2011.00037

    View details for PubMedID 21811467

  • The Role of Stem Cells in Cutaneous Wound Healing: What Do We Really Know? PLASTIC AND RECONSTRUCTIVE SURGERY Ko, S. H., Nauta, A., Wong, V., Glotzbach, J., Gurtner, G. C., Longaker, M. T. 2011; 127 (1): 10S-20S

    Abstract

    Wound repair is a complex process involving the orchestrated interaction of multiple growth factors, cytokines, chemokines, and cell types. Dysregulation of this process leads to problems such as excessive healing in the form of keloids and hypertrophic scars and chronic, nonhealing wounds. These issues have broad global implications. Stem cells offer enormous potential for enhancing tissue repair and regeneration following injury. The rapidly developing fields of stem cell biology and skin tissue engineering create translational opportunities for the development of novel stem cell-based wound-healing therapies.

    View details for DOI 10.1097/PRS.0b013e3181fbe2d8

    View details for Web of Science ID 000286122400003

    View details for PubMedID 21200267

  • Surgical Approaches to Create Murine Models of Human Wound Healing JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY Wong, V., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. 2011

    Abstract

    Wound repair is a complex biologic process which becomes abnormal in numerous disease states. Although in vitro models have been important in identifying critical repair pathways in specific cell populations, in vivo models are necessary to obtain a more comprehensive and pertinent understanding of human wound healing. The laboratory mouse has long been the most common animal research tool and numerous transgenic strains and models have been developed to help researchers study the molecular pathways involved in wound repair and regeneration. This paper aims to highlight common surgical mouse models of cutaneous disease and to provide investigators with a better understanding of the benefits and limitations of these models for translational applications.

    View details for DOI 10.1155/2011/969618

    View details for Web of Science ID 000286250000001

    View details for PubMedID 21151647

  • Diathermy or Surgical Scalpel for Abdominal Skin Incisions What Is the Impact On Clinical Practice? ANNALS OF SURGERY Longaker, M. T., Gurtner, G. C. 2011; 253 (1): 14-15

    View details for DOI 10.1097/SLA.0b013e318205718a

    View details for Web of Science ID 000286179100004

    View details for PubMedID 21294284

  • Deformational Plagiocephaly: A Look Into the Future JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Wan, D. C., Longaker, M. T., Habal, M. B. 2011; 22 (1): 3-5

    View details for DOI 10.1097/SCS.0b013e3181fb7ee5

    View details for Web of Science ID 000286195600002

    View details for PubMedID 21239916

  • Basic Science Review on Adipose Tissue for Clinicians PLASTIC AND RECONSTRUCTIVE SURGERY Brown, S. A., Levi, B., Lequex, C., Wong, V. W., Mojallal, A., Longaker, M. T. 2010; 126 (6): 1936-1946

    Abstract

    The recognition that fat contains stem cells has driven further examination into the potential uses of fat and adipose-derived stem cells in a wide number of clinical situations. New information about the harvesting, isolation, and subsequent differentiation properties of isolated adipose-derived stem cells has led to new research into novel tissue-engineered constructs and the transformation of adipose-derived stem cells to induced pluripotent stem cells. Clinically, use of fat grafts and adipose-derived stem cells worldwide and in the United States has dramatically increased in parallel to questions concerning the safety and efficacy of adipose-derived stem cell-based treatments. Currently, the U.S. Food and Drug Administration has not approved the use of isolated adipose-derived stem cells for medical indications.

    View details for DOI 10.1097/PRS.0b013e3181f44790

    View details for Web of Science ID 000284832400038

    View details for PubMedID 21124133

  • Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone PLOS ONE Li, S., Quarto, N., Longaker, M. T. 2010; 5 (11)

    Abstract

    As a culmination of efforts over the last years, our knowledge of the embryonic origins of the mammalian frontal and parietal cranial bones is unambiguous. Progenitor cells that subsequently give rise to frontal bone are of neural crest origin, while parietal bone progenitors arise from paraxial mesoderm. Given the unique qualities of neural crest cells and the clear delineation of the embryonic origins of the calvarial bones, we sought to determine whether mouse neural crest derived frontal bone differs in biology from mesoderm derived parietal bone.BrdU incorporation, immunoblotting and osteogenic differentiation assays were performed to investigate the proliferative rate and osteogenic potential of embryonic and postnatal osteoblasts derived from mouse frontal and parietal bones. Co-culture experiments and treatment with conditioned medium harvested from both types of osteoblasts were performed to investigate potential interactions between the two different tissue origin osteoblasts. Immunoblotting techniques were used to investigate the endogenous level of FGF-2 and the activation of three major FGF signaling pathways. Knockdown of FGF Receptor 1 (FgfR1) was employed to inactivate the FGF signaling.Our results demonstrated that striking differences in cell proliferation and osteogenic differentiation between the frontal and parietal bone can be detected already at embryonic stages. The greater proliferation rate, as well as osteogenic capacity of frontal bone derived osteoblasts, were paralleled by an elevated level of FGF-2 protein synthesis. Moreover, an enhanced activation of FGF-signaling pathways was observed in frontal bone derived osteoblasts. Finally, the greater osteogenic potential of frontal derived osteoblasts was dramatically impaired by knocking down FgfR1.Osteoblasts from mouse neural crest derived frontal bone displayed a greater proliferative and osteogenic potential and endogenous enhanced activation of FGF signaling compared to osteoblasts from mesoderm derived parietal bone. FGF signaling plays a key role in determining biological differences between the two types of osteoblasts.

    View details for DOI 10.1371/journal.pone.0014033

    View details for Web of Science ID 000284356900002

    View details for PubMedID 21124973

  • The basic science of vascular biology: implications for the practicing surgeon. Plastic and reconstructive surgery Glotzbach, J. P., Levi, B., Wong, V. W., Longaker, M. T., Gurtner, G. C. 2010; 126 (5): 1528-1538

    Abstract

    A thorough understanding of vascular biology will assist the reconstructive surgeon in both operative planning and development of novel surgical approaches to treat chronic wounds and tissue loss, and to optimize regenerative strategies for tissue reconstruction. In this review, several fundamental concepts of the basic science of vascular biology are discussed, with specific emphasis on the clinical implications most relevant to the reconstructive surgeon. Topics include the vascular physiology of tissue flaps and grafts, the principles of neovascularization including angiogenesis and vasculogenesis, and the basic concepts of bioengineering of vascularized tissue constructs for use in reconstruction. As basic science research increases our collective understanding of vascular physiology--specifically, in the areas of neovascularization and tissue engineering--reconstructive surgeons will be able to improve treatment of the sequelae of ischemic injuries, tissue loss, and chronic wounds.

    View details for DOI 10.1097/PRS.0b013e3181ef8ccf

    View details for PubMedID 21042110

  • The Basic Science of Vascular Biology: Implications for the Practicing Surgeon PLASTIC AND RECONSTRUCTIVE SURGERY Glotzbach, J. P., Levi, B., Wong, V. W., Longaker, M. T., Gurtner, G. C. 2010; 126 (5): 1527-1537
  • Scarless Fetal Wound Healing: A Basic Science Review PLASTIC AND RECONSTRUCTIVE SURGERY Larson, B. J., Longaker, M. T., Lorenz, H. P. 2010; 126 (4): 1172-1180

    Abstract

    Scar formation is a major medical problem that can have devastating consequences for patients. The adverse physiological and psychological effects of scars are broad, and there are currently no reliable treatments to prevent scarring. In contrast to adult wounds, early gestation fetal skin wounds repair rapidly and in the absence of scar formation. Despite extensive investigation, the exact mechanisms of scarless fetal wound healing remain largely unknown. For some time, it has been known that significant differences exist among the extracellular matrix, inflammatory response, cellular mediators, and gene expression profiles of fetal and postnatal wounds. These differences may have important implications in scarless wound repair.

    View details for DOI 10.1097/PRS.0b013e3181eae781

    View details for Web of Science ID 000282559100004

    View details for PubMedID 20885241

  • The Diverse Surgeons Initiative: An Effective Method for Increasing the Number of Under-represented Minorities in Academic Surgery JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS Butler, P. D., Britt, L. D., Green, M. L., Longaker, M. T., Geis, W. P., Franklin, M. E., Ruhalter, A., Fullum, T. M. 2010; 211 (4): 561-566
  • Opposite Spectrum of Activity of Canonical Wnt Signaling in the Osteogenic Context of Undifferentiated and Differentiated Mesenchymal Cells: Implications for Tissue Engineering TISSUE ENGINEERING PART A Quarto, N., Behr, B., Longaker, M. T. 2010; 16 (10): 3185-3197

    Abstract

    To delineate the competence window in which canonical wingless (Wnt)-signaling can either inhibit or promote osteogenic differentiation, we have analyzed cells with different status, specifically undifferentiated mesenchymal cells, such as adipose-derived stem cells and embryonic calvarial mesenchymal cells, and differentiated mesenchymal cells such as juvenile immature calvarial osteoblasts and adult calvarial osteoblasts. Our analysis indicated that undifferentiated mesenchymal cells and juvenile calvarial osteoblasts are endowed with higher levels of endogenous canonical Wnt signaling compared to fully differentiated adult calvarial osteoblasts, and that different levels of activation inversely correlated with expression levels of several Wnt antagonists. We have observed that activation of canonical Wnt signaling may elicit opposite biological activity in the context of osteogenic differentiation depending on the status of cell, the threshold levels of its activation, and Wnt ligands concentration. The results presented in this study indicate that treatment with Wnt3 and/or expression of constitutively activated β-catenin inhibits osteogenic differentiation of undifferentiated mesenchymal cells, whereas expression of dominant negative transcription factor 4 (Tcf4) and/or secreted frizzled related protein 1 treatment enhances their osteogenic differentiation. Wnt3a treatment also inhibits osteogenesis in juvenile calvarial osteoblasts in a dose-dependent fashion. Conversely, Wnt3a treatment strongly induces osteogenesis in mature calvarial osteoblasts in a dose-dependent manner. Importantly, in vitro data correlated with in vivo results showing that Wnt3a treatment of calvarial defects, created in juvenile mice, promotes calvarial healing and bone regeneration only at low doses, whereas high doses of Wnt3a impairs tissue regeneration. In contrast, high doses of Wnt3a enhance bony tissue regeneration and calvarial healing in adult mice. Therefore, the knowledge of both endogenous activity of canonical Wnt signaling and appropriate concentrations of Wnt3a treatment may lead to significant improvement for bony tissue engineering, as well as for the efficient implement of adipose-derived stem cells in bone regeneration. Indeed, this study has important potential implications for tissue engineering, specifically for repair of juvenile bone defects.

    View details for DOI 10.1089/ten.tea.2010.0133

    View details for Web of Science ID 000282361100017

    View details for PubMedID 20590472

  • Stem Cells PLASTIC AND RECONSTRUCTIVE SURGERY Behr, B., Ko, S. H., Wong, V. W., Gurtner, G. C., Longaker, M. T. 2010; 126 (4): 1163-1171

    Abstract

    Stem cells are self-renewing cells capable of differentiating into multiple cell lines and are classified according to their origin and their ability to differentiate. Enormous potential exists in use of stem cells for regenerative medicine. To produce effective stem cell-based treatments for a range of diseases, an improved understanding of stem cell biology and better control over stem cell fate are necessary. In addition, the barriers to clinical translation, such as potential oncologic properties of stem cells, need to be addressed. With renewed government support and continued refinement of current stem cell methodologies, the future of stem cell research is exciting and promises to provide novel reconstructive options for patients and surgeons limited by traditional paradigms.

    View details for DOI 10.1097/PRS.0b013e3181ea42bb

    View details for Web of Science ID 000282559100003

    View details for PubMedID 20555302

  • Discussion: Hes1 is required for the development of craniofacial structures derived from ectomesenchymal neural crest cells. journal of craniofacial surgery Levi, B., Brugmann, S., Longaker, M. T. 2010; 21 (5): 1450-1451

    View details for DOI 10.1097/SCS.0b013e3181ecc54b

    View details for PubMedID 20818250

  • Tissue Engineering in Plastic Surgery: A Review PLASTIC AND RECONSTRUCTIVE SURGERY Wong, V. W., Rustad, K. C., Longaker, M. T., Gurtner, G. C. 2010; 126 (3): 858-868

    Abstract

    Novel tissue- and organ-engineering strategies are needed to address the growing need for replacement biological parts. Collective progress in stem cell technology, biomaterials, engineering, and molecular medicine has advanced the state of regenerative medicine, yet many hurdles to clinical translation remain. Plastic surgeons are in an ideal position to capitalize on emerging technologies and will be at the forefront of transitioning basic science research into the clinical reconstructive arena. This review highlights fundamental principles of bioengineering, recent progress in tissue-specific engineering, and future directions for this exciting and rapidly evolving area of medicine.

    View details for DOI 10.1097/PRS.0b013e3181e3b3a3

    View details for Web of Science ID 000281606700016

    View details for PubMedID 20811219

  • Paracrine Interaction between Adipose-Derived Stromal Cells and Cranial Suture-Derived Mesenchymal Cells PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Levi, B., Commons, G. W., Glotzbach, J., Longaker, M. T. 2010; 126 (3): 806-821

    Abstract

    Adipose-derived stromal cells are a potential cell source for the successful healing of skeletal defects. In this study, the authors sought to investigate the potential for cranial suture-derived mesenchymal cells to promote the osteogenic differentiation of adipose-derived stromal cells. Various reports have previously examined the unique in vitro attributes of suture-derived mesenchymal cells; this study sought to extend those findings.Suture-derived mesenchymal cells were isolated from wild-type mice (n = 30) from both fusing posterofrontal and patent sagittal sutures. Cells were placed in Transwell inserts with human adipose-derived stromal cells (n = 5 patients) with osteogenic differentiation medium with or without recombinant Noggin (10 to 400 ng/ml). Specific gene expression of osteogenic markers and Hedgehog pathway were assayed; standard osteogenic assays (alkaline phosphatase and alizarin red staining) were performed. All assays were performed in triplicate.Both posterofrontal and sagittal suture-derived mesenchymal cells induced osteogenic differentiation of adipose-derived stromal cells (p < 0.05). Posterofrontal suture-derived mesenchymal cells induced adipose-derived stromal cell osteogenesis to a greater degree than sagittal suture-derived mesenchymal cells (p < 0.05). This was accompanied by an increase in bone morphogenetic protein expression (p < 0.05). Finally, recombinant Noggin mitigated the pro-osteogenic effects of co-culture accompanied by a reduction in Hedgehog signaling (p < 0.05).Suture-derived mesenchymal cells secrete paracrine factors that induce osteogenic differentiation of multipotent stromal cells (human adipose-derived stromal cells). Cells derived from the fusing posterofrontal suture do this to a significantly greater degree than cells from the patent sagittal suture. Enhanced bone morphogenetic protein and Hedgehog signaling may underlie this paracrine effect.

    View details for DOI 10.1097/PRS.0b013e3181e5f81a

    View details for Web of Science ID 000281606700010

    View details for PubMedID 20811214

  • Hes1 Is Required for the Development of Craniofacial Structures Derived From Ectomesenchymal Neural Crest Cells DISCUSSION JOURNAL OF CRANIOFACIAL SURGERY Levi, B., Brugmann, S., Longaker, M. T. 2010; 21 (5): 1450-1451
  • So You Want to Be an Innovator? PLASTIC AND RECONSTRUCTIVE SURGERY Rohrich, R. J., Rosen, J., Longaker, M. T. 2010; 126 (3): 1107-1109

    View details for DOI 10.1097/PRS.0b013e3181e3b854

    View details for Web of Science ID 000281606700045

    View details for PubMedID 20811242

  • Depot-Specific Variation in the Osteogenic and Adipogenic Potential of Human Adipose-Derived Stromal Cells PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Glotzbach, J. P., Wan, D. C., Commons, G. W., Longaker, M. T. 2010; 126 (3): 822-834

    Abstract

    Adipose-derived stromal cells hold promise for use in tissue regeneration. However, multiple facets of their biology remain unclear. The authors examined the variations in osteogenesis and adipogenesis in adipose-derived stromal cells between subcutaneous fat depots and potential molecular causes.Adipose-derived stromal cells were isolated from human patients from subcutaneous fat depots, including arm, flank, thigh, and abdomen (n = 5 patients). Osteogenic and adipogenic differentiation was performed (alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction). Co-cultures were established to assess the paracrine effect of human adipose-derived stromal cells on mouse osteoblasts. Finally, HOX gene expression was analyzed by quantitative real-time polymerase chain reaction.Subcutaneous fat depots retain markedly different osteogenic and adipogenic potentials. Osteogenesis was most robust in adipose-derived stromal cells from the flank and thigh, as compared with those from the arm and abdomen (p < 0.05 by all markers examined). This was accompanied by elevations of BMP4 and BMPR1B (p < 0.05 by all markers examined). The osteogenic advantage of cells from the flank and thigh was again observed when analyzing the paracrine effects of these cells. Conversely, those cells isolated from the flank had a lesser ability to undergo adipogenic differentiation. Adipose-associated HOX genes were less expressed in flank-derived adipose-derived stromal cells.Variations exist between fat depots in terms of adipose-derived stromal cell osteogenic and adipogenic differentiation. Differences in HOX expression and bone morphogenetic protein signaling may underlie these observations. This study indicates that the choice of fat depot derivation of adipose-derived stromal cells may be an important one for future efforts in tissue engineering.

    View details for DOI 10.1097/PRS.0b013e3181e5f892

    View details for Web of Science ID 000281606700011

    View details for PubMedID 20811215

  • Differential activation of canonical Wnt signaling determines cranial sutures fate: A novel mechanism for sagittal suture craniosynostosis DEVELOPMENTAL BIOLOGY Behr, B., Longaker, M. T., Quarto, N. 2010; 344 (2): 922-940

    Abstract

    Premature closure of cranial sutures, which serve as growth centers for the skull vault, result in craniosynostosis. In the mouse posterior frontal (PF) suture closes by endochondral ossification, whereas sagittal (SAG) remain patent life time, although both are neural crest tissue derived. We therefore, investigated why cranial sutures of same tissue origin adopt a different fate. We demonstrated that closure of the PF suture is tightly regulated by canonical Wnt signaling, whereas patency of the SAG suture is achieved by constantly activated canonical Wnt signaling. Importantly, the fate of PF and SAG sutures can be reversed by manipulating Wnt signaling. Continuous activation of canonical Wnt signaling in the PF suture inhibits endochondral ossification and therefore, suture closure, In contrast, inhibition of canonical Wnt signaling in the SAG suture, upon treatment with Wnt antagonists results in endochondral ossification and suture closure. Thus, inhibition of canonical Wnt signaling in the SAG suture phenocopies craniosynostosis. Moreover, mice haploinsufficient for Twist1, a target gene of canonical Wnt signaling which inhibits chondrogenesis, have sagittal craniosynostosis. We propose that regulation of canonical Wnt signaling is of crucial importance during the physiological patterning of PF and SAG sutures. Importantly, dysregulation of this pathway may lead to craniosynostosis.

    View details for DOI 10.1016/j.ydbio.2010.06.009

    View details for Web of Science ID 000280787100034

    View details for PubMedID 20547147

  • Divergent Modulation of Adipose-Derived Stromal Cell Differentiation by TGF-beta 1 Based on Species of Derivation PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Xu, Y., Commons, G. W., Longaker, M. T. 2010; 126 (2): 412-425

    Abstract

    Adipose-derived stromal cells hold promise for skeletal tissue engineering. However, various studies have observed that adipose-derived stromal cells differ significantly in their biology depending on species of derivation. In the following study, the authors sought to determine the species-specific response of adipose-derived stromal cells to recombinant TGF-beta1 (rTGF-beta1).Adipose-derived stromal cells were derived from mouse and human sources. Recombinant TGF-beta1 was added to culture medium (2.5 to 10 ng/ml); proliferation and osteogenic and adipogenic differentiation were assessed by standardized parameters, including cell counting, alkaline phosphatase, alizarin red, oil red O staining, and quantitative real-time polymerase chain reaction.Recombinant TGF-beta1 was found to significantly repress cellular proliferation in both mouse and human adipose-derived stromal cells (p < 0.01). Recombinant TGF-beta1 was found to significantly repress osteogenic differentiation in mouse adipose-derived stromal cells. In contrast, osteogenic differentiation of human adipose-derived stromal cells proceeded unimpeded in either the presence or the absence of rTGF-beta1. Interestingly, rTGF-beta1 induced expression of a number of osteogenic genes in human adipose-derived stromal cells, including BMP2 and BMP4.The authors' results further detail an important facet in which mouse and human adipose-derived stromal cells differ. Mouse adipose-derived stromal cell osteogenesis is completely inhibited by rTGF-beta1, whereas human adipose-derived stromal cell osteogenesis progresses in the presence of rTGF-beta1. These data highlight the importance of species of derivation in basic adipose-derived stromal cell biology. Future studies will examine in more detail the species-specific differences among adipose-derived stromal cell populations.

    View details for DOI 10.1097/PRS.0b013e3181df64dc

    View details for Web of Science ID 000280143800009

    View details for PubMedID 20679827

  • Different endogenous threshold levels of Fibroblast Growth Factor-ligands determine the healing potential of frontal and parietal bones BONE Behr, B., Panetta, N. J., Longaker, M. T., Quarto, N. 2010; 47 (2): 281-294

    Abstract

    In the skull vault, neural crest derived frontal bones have an increased healing capacity and higher expression levels of Fibroblast Growth Factor-ligands as compared to mesoderm-derived parietal bones. Thus, we asked whether Fibroblast Growth Factor-ligands are responsible for the superior healing potential of frontal bones. Parietal defects in juvenile and adult mice treated with Fibroblast Growth Factor-2, -9 and -18 showed increased bone regeneration, comparable to frontal defects. Immunohistochemistry revealed increased recruitment of osteoprogenitors and activation of FGF-signaling pathways in FGF-treated parietal defects. Conversely, calvarial defects in Fgf-9(+/-) and Fgf-18(+/-) mice showed impaired calvarial healing which could be rescued by exogenous Fibroblast Growth Factor-ligands. Moreover, by utilizing Wnt1Cre/R26R mice, the migration and contribution of dura mater and pericranium cells to calvarial healing could be demonstrated. Taken together our results demonstrated that different endogenous threshold levels of Fibroblast Growth Factor-ligands in frontal and parietal bones have a profound impact on calvarial regeneration. The present study thereby opens new avenues for translational medicine.

    View details for DOI 10.1016/j.bone.2010.05.008

    View details for Web of Science ID 000280449300015

    View details for PubMedID 20472108

  • Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A James, A. W., Leucht, P., Levi, B., Carre, A. L., Xu, Y., Helms, J. A., Longaker, M. T. 2010; 16 (8): 2605-2616

    Abstract

    Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed that Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, and Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgehog (Shh-N) significantly increased in vitro osteogenic differentiation in mouse ASCs, by all markers examined (*p < 0.01). Concomitantly, Shh-N abrogated adipogenic differentiation, by all markers examined (*p < 0.01). Conversely, blockade of endogenous Hedgehog signaling, with the Hedgehog antagonist cyclopamine, enhanced adipogenesis at the expense of osteogenesis. We next translated these results to a mouse model of appendicular skeletal regeneration. Using quantitative real-time polymerase chain reaction and in situ hybridization, we found that skeletal injury (a monocortical 1 mm defect in the tibia) results in a localized increase in Hedgehog signaling. Moreover, grafting of ASCs treated with Shh-N resulted in significantly increased bone regeneration within the defect site. In conclusion, Hedgehog signaling enhances the osteogenic differentiation of mouse ASCs, at the expense of adipogenesis. These data suggest that Hedgehog signaling directs the lineage differentiation of mesodermal stem cells and represents a promising strategy for skeletal tissue regeneration.

    View details for DOI 10.1089/ten.tea.2010.0048

    View details for Web of Science ID 000280648700018

    View details for PubMedID 20367246

  • Origin Matters: Differences in Embryonic Tissue Origin and Wnt Signaling Determine the Osteogenic Potential and Healing Capacity of Frontal and Parietal Calvarial Bones JOURNAL OF BONE AND MINERAL RESEARCH Quarto, N., Wan, D. C., Kwan, M. D., Panetta, N. J., Li, S., Longaker, M. T. 2010; 25 (7): 1680-1694

    Abstract

    Calvarial bones arise from two embryonic tissues, namely, the neural crest and the mesoderm. In this study we have addressed the important question of whether disparate embryonic tissue origins impart variable osteogenic potential and regenerative capacity to calvarial bones, as well as what the underlying molecular mechanism(s). Thus, by performing in vitro and in vivo studies, we have investigated whether differences exist between neural crest-derived frontal and paraxial mesodermal-derived parietal bone. Of interest, our data indicate that calvarial bone osteoblasts of neural crest origin have superior potential for osteogenic differentiation. Furthermore, neural crest-derived frontal bone displays a superior capacity to undergo osseous healing compared with calvarial bone of paraxial mesoderm origin. Our study identified both in vitro and in vivo enhanced endogenous canonical Wnt signaling in frontal bone compared with parietal bone. In addition, we demonstrate that constitutive activation of canonical Wnt signaling in paraxial mesodermal-derived parietal osteoblasts mimics the osteogenic potential of frontal osteoblasts, whereas knockdown of canonical Wnt signaling dramatically impairs the greater osteogenic potential of neural crest-derived frontal osteoblasts. Moreover, fibroblast growth factor 2 (FGF-2) treatment induces phosphorylation of GSK-3beta and increases the nuclear levels of beta-catenin in osteoblasts, suggesting that enhanced activation of Wnt signaling might be mediated by FGF. Taken together, our data provide compelling evidence that indeed embryonic tissue origin makes a difference and that active canonical Wnt signaling plays a major role in contributing to the superior intrinsic osteogenic potential and tissue regeneration observed in neural crest-derived frontal bone.

    View details for DOI 10.1359/jbmr.091116

    View details for Web of Science ID 000280395900023

    View details for PubMedID 19929441

  • Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271 NATURE Boiko, A. D., Razorenova, O. V., van de Rijn, M., Swetter, S. M., Johnson, D. L., Ly, D. P., Butler, P. D., Yang, G. P., Joshua, B., Kaplan, M. J., Longaker, M. T., Weissman, I. L. 2010; 466 (7302): 133-U155

    Abstract

    The question of whether tumorigenic cancer stem cells exist in human melanomas has arisen in the last few years. Here we show that in melanomas, tumour stem cells (MTSCs, for melanoma tumour stem cells) can be isolated prospectively as a highly enriched CD271(+) MTSC population using a process that maximizes viable cell transplantation. The tumours sampled in this study were taken from a broad spectrum of sites and stages. High-viability cells isolated by fluorescence-activated cell sorting and re-suspended in a matrigel vehicle were implanted into T-, B- and natural-killer-deficient Rag2(-/-)gammac(-/-) mice. The CD271(+) subset of cells was the tumour-initiating population in 90% (nine out of ten) of melanomas tested. Transplantation of isolated CD271(+) melanoma cells into engrafted human skin or bone in Rag2(-/-)gammac(-/-) mice resulted in melanoma; however, melanoma did not develop after transplantation of isolated CD271(-) cells. We also show that in mice, tumours derived from transplanted human CD271(+) melanoma cells were capable of metastatsis in vivo. CD271(+) melanoma cells lacked expression of TYR, MART1 and MAGE in 86%, 69% and 68% of melanoma patients, respectively, which helps to explain why T-cell therapies directed at these antigens usually result in only temporary tumour shrinkage.

    View details for DOI 10.1038/nature09161

    View details for Web of Science ID 000279343800049

    View details for PubMedID 20596026

  • Regulation of Human Adipose-Derived Stromal Cell Osteogenic Differentiation by Insulin-Like Growth Factor-1 and Platelet-Derived Growth Factor-alpha PLASTIC AND RECONSTRUCTIVE SURGERY Levi, B., James, A. W., Wan, D. C., Glotzbach, J. P., Commons, G. W., Longaker, M. T. 2010; 126 (1): 41-52

    Abstract

    Human adipose-derived stromal cells possess a great potential for tissue engineering purposes. The authors' laboratory is interested in harnessing human adipose-derived stromal cells for skeletal tissue regeneration and identifying those factors that enhance human adipose-derived stromal cell osteogenic differentiation. The authors hypothesized that insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF) would stimulate human adipose-derived stromal cell osteogenesis and that IGF would stimulate adipogenesis.Adipose-derived stromal cells were harvested from human lipoaspirate. Previously, a microarray analysis examined gene expression throughout osteogenic differentiation. In a candidate fashion, the authors added recombinant IGF-1 and PDGF-alpha individually and in combination. Osteogenesis and adipogenesis were assessed by alkaline phosphatase, alizarin red, and oil red O staining, and quantitative real-time polymerase chain reaction (RUNX2, ALP, OCN, IGF1, PPARG, LPL, AP2, and GCP1). Finally, intersection between IGF and PDGF signaling pathways was evaluated.IGF-1 was observed to increase osteogenic differentiation by all markers (p < 0.01). However, PDGF-alpha when added alone primarily did not affect osteogenic markers. PDGF-alpha positively regulated transcription of IGF1. Addition of PDGF-alpha in combination with or before IGF-1 enhanced osteogenesis more than either alone. IGF-1 increased whereas PDGF-alpha diminished human adipose-derived stromal cell adipogenesis.IGF signaling significantly increased osteogenesis in human adipose-derived stromal cells and may be used for tissue-engineering purposes. The combination of PDGF and IGF may be more beneficial than either alone in driving adipose-derived stromal cell osteogenesis. Future in vivo applications will focus on the combination of adipose-derived stromal cells, biomimetic scaffolds, and recombinant IGF.

    View details for DOI 10.1097/PRS.0b013e3181da8858

    View details for Web of Science ID 000279097500006

    View details for PubMedID 20220555

  • Strategies for organ level tissue engineering ORGANOGENESIS Rustad, K. C., Sorkin, M., Levi, B., Longaker, M. T., Gurtner, G. C. 2010; 6 (3): 151-157

    Abstract

    The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue-engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs-biomaterials, cells and bioactive molecules-and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to "scale up" every aspect of the research and development process.

    View details for DOI 10.4161/org.6.3.12139

    View details for Web of Science ID 000290266200003

    View details for PubMedID 21197216

  • Elucidating Mechanisms of Osteogenesis in Human Adipose-Derived Stromal Cells via Microarray Analysis JOURNAL OF CRANIOFACIAL SURGERY Lee, J., Gupta, D., Panetta, N. J., Levi, B., James, A. W., Wan, D., Commons, G. W., Longaker, M. T. 2010; 21 (4): 1136-1141

    Abstract

    The osteogenic potential of human adipose-derived stromal cells (hASCs), the ease of cell procurement, and the shortcomings of conventional skeletal reconstruction call for further analysis of the molecular mechanisms governing hASC osteogenic differentiation. We have examined the expression profile of the human transcriptome during osteogenic differentiation of ASCs using microarray. Subsequently, we analyzed those genes related to osteogenesis that have not been previously studied about hASCs. We have preliminarily assessed the role of IGFBP3, TGF-B3, TNC, CTGF, DKK-1, and PDGFRB in hASC osteogenic differentiation.We compared the expression profile of undifferentiated hASCs to that of hASCs treated with osteogenic differentiation medium for 1, 3, or 7 days using the Human Exonic Evidence-Based Oligonucleotide chip. Genes significantly overexpress or underexpressed were validated with quantitative reverse transcription-polymerase chain reaction. The osteogenic capability of ASCs was verified by Alizarin Red staining.IGFBP3, TGF-B3, TNC, CTGF, and PDGFRB were all upregulated in early osteogenesis, and TGF-B3, TNC, and PDGFRB were upregulated in late osteogenesis by microarray and quantitative reverse transcription analysis. In contrast, DKK-1 was downregulated in early and late osteogenesis. Alizarin Red staining showed a significant increase in mineralization in hASCs, even after 1 day in osteogenic differentiation medium.Factors that commit hASCs to an osteogenic pathway remain largely unknown. We have described 6 genes that play key roles in hASC osteogenic differentiation. We plan to further exploit these data via in vitro treatment of hASCs with these soluble cytokines and in vivo translation using a nude mouse calvarial defect model.

    View details for DOI 10.1097/SCS.0b013e3181e488d6

    View details for Web of Science ID 000280149100044

    View details for PubMedID 20613589

  • Fgf-9 is required for angiogenesis and osteogenesis in long bone repair PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Behr, B., Leucht, P., Longaker, M. T., Quarto, N. 2010; 107 (26): 11853-11858

    Abstract

    Bone healing requires a complex interaction of growth factors that establishes an environment for efficient bone regeneration. Among these, FGFs have been considered important for intrinsic bone-healing capacity. In this study, we analyzed the role of Fgf-9 in long bone repair. One-millimeter unicortical defects were created in tibias of Fgf-9(+/-) and wild-type mice. Histomorphometry revealed that half-dose gene of Fgf-9 markedly reduced bone regeneration as compared with wild-type. Both immunohistochemistry and RT-PCR analysis revealed markedly decreased levels of proliferating cell nuclear antigen (PCNA), Runt-related transcription factor 2 (Runx2), osteocalcin, Vega-a, and platelet endothelial cell adhesion molecule 1 (PECAM-1) in Fgf-9(+/-) defects. muCT angiography indicated dramatic impairment of neovascularization in Fgf-9(+/-) mice as compared with controls. Treatment with FGF-9 protein promoted angiogenesis and successfully rescued the healing capacity of Fgf-9(+/-) mice. Importantly, although other pro-osteogenic factors [Fgf-2, Fgf-18, and bone morphogenic protein 2 (Bmp-2)] still were present in Fgf-9(+/-) mice, they could not compensate for the haploinsufficiency of the Fgf-9 gene. Therefore, endogenous Fgf-9 seems to play an important role in long bone repair. Taken together our data suggest a unique role for Fgf-9 in bone healing, presumably by initiating angiogenesis through Vegf-a. Moreover, this study further supports the embryonic phenotype previously observed in the developing limb, thus promoting the concept that healing processes in adult organisms may recapitulate embryonic skeletal development.

    View details for DOI 10.1073/pnas.1003317107

    View details for Web of Science ID 000279332300037

    View details for PubMedID 20547837

  • Connective Tissue Growth Factor in Regulation of RhoA Mediated Cytoskeletal Tension Associated Osteogenesis of Mouse Adipose-Derived Stromal Cells PLOS ONE Xu, Y., Wagner, D. R., Bekerman, E., Chiou, M., James, A. W., Carter, D., Longaker, M. T. 2010; 5 (6)

    Abstract

    Cytoskeletal tension is an intracellular mechanism through which cells convert a mechanical signal into a biochemical response, including production of cytokines and activation of various signaling pathways.Adipose-derived stromal cells (ASCs) were allowed to spread into large cells by seeding them at a low-density (1,250 cells/cm(2)), which was observed to induce osteogenesis. Conversely, ASCs seeded at a high-density (25,000 cells/cm(2)) featured small cells that promoted adipogenesis. RhoA and actin filaments were altered by changes in cell size. Blocking actin polymerization by Cytochalasin D influenced cytoskeletal tension and differentiation of ASCs. To understand the potential regulatory mechanisms leading to actin cytoskeletal tension, cDNA microarray was performed on large and small ASCs. Connective tissue growth factor (CTGF) was identified as a major regulator of osteogenesis associated with RhoA mediated cytoskeletal tension. Subsequently, knock-down of CTGF by siRNA in ASCs inhibited this osteogenesis.We conclude that CTGF is important in the regulation of cytoskeletal tension mediated ASC osteogenic differentiation.

    View details for DOI 10.1371/journal.pone.0011279

    View details for Web of Science ID 000279135400023

    View details for PubMedID 20585662

  • In vitro effects of direct current electric fields on adipose-derived stromal cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Hammerick, K. E., Longaker, M. T., Prinz, F. B. 2010; 397 (1): 12-17

    Abstract

    Endogenous electric fields play an important role in embryogenesis, regeneration, and wound repair and previous studies have shown that many populations of cells, leukocytes, fibroblasts, epithelial cells, and endothelial cells, exhibit directed migration in response to electric fields. As regenerative therapies continue to explore ways to control mesenchymal progenitor cells to recreate desirable tissues, it is increasingly necessary to characterize the vast nature of biological responses imposed by physical phenomena. Murine adipose-derived stromal cells (mASCs) migrated toward the cathode in direct current (DC) fields of physiologic strength and show a dose dependence of migration rate to stronger fields. Electric fields also caused mASCs to orient perpendicularly to the field vector and elicited a transient increase in cytosolic calcium. Additionally, their galvanotactic response appears to share classic chemotactic signaling pathways that are involved in the migration of other cell types. Galvanotaxis is one predominant result of electric fields on mASCs and it may be exploited to engineer adult stem cell concentrations and locations within implanted grafts or toward sites of wound repair.

    View details for DOI 10.1016/j.bbrc.2010.05.003

    View details for Web of Science ID 000279292800003

    View details for PubMedID 20452327

  • Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects PLOS ONE Levi, B., James, A. W., Nelson, E. R., Vistnes, D., Wu, B., Lee, M., Gupta, A., Longaker, M. T. 2010; 5 (6)

    Abstract

    Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect.Human ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo.Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

    View details for DOI 10.1371/journal.pone.0011177

    View details for Web of Science ID 000278886300019

    View details for PubMedID 20567510

  • Bone Regeneration and Repair CURRENT STEM CELL RESEARCH & THERAPY Panetta, N. J., Gupta, D. M., Longaker, M. T. 2010; 5 (2): 122-128

    Abstract

    In the face of mounting clinical demand, and armed with reconstructive techniques that are technically challenging and frequently result in suboptimal patient outcomes, increasing focus is being placed on tissue engineering and regenerative medicine as a potential source of novel skeletal reconstructive approaches. Specifically, evidence is accumulating that highlights the promise of osteoprogenitor cell-based reconstructive strategies to meet the needs of an expanding patient population. Historically, the study of cell and molecular biology guiding physiologic and pathologic skeletal development, as well as endogenous bone regeneration following injury, has provided a wealth of information that lends insight toward potential parallel processes that may regulate the osteogenic differentiation of progenitor cells. Multiple progenitor cell populations are now known to possess a capacity to undergo robust osteogenic differentiation in the presence of appropriate environmental cues (hESC, BMSC, ASC, etc.) Recent investigations have put forth multiple advantages of ASC relative to BMSC. Of note, ASC exist in relative abundance, lack the need for in vitro expansion prior to utilization, and can be harvested with relative ease and reduced donor morbidity. Collectively, these factors, paired with promising in vitro and in vivo observations that speak toward the substantial osteogenic potential of ASC, have spurred enthusiasm to pursue the application of ASC in the maturation of skeletal tissue engineering applications. Yet, elucidating what structural and functional properties of scaffolds designed for ASC-mediated skeletal tissue engineering applications (porosity, pore size, composition, mechanical stability, degradation kinetics, etc.), as well as evolving our understanding and capacity to deliver spatiotemporally specific pro-osteogenic targeted molecular manipulation to progenitor cells, remain important hurdles to clear. The scope of this review encompasses the current state of ongoing investigations along these fronts, as well as what future direction will be critical to the transition of cell-based skeletal tissue engineering strategies to the bedside.

    View details for Web of Science ID 000208262700006

    View details for PubMedID 19941457

  • Retinoic Acid Enhances Osteogenesis in Cranial Suture-Derived Mesenchymal Cells: Potential Mechanisms of Retinoid-Induced Craniosynostosis PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Levi, B., Xu, Y., Carre, A. L., Longaker, M. T. 2010; 125 (5): 1352-1361

    Abstract

    In utero retinoid exposure results in numerous craniofacial malformations, including craniosynostosis. Although many malformations associated with retinoic acid syndrome are associated with neural crest defects, the specific mechanisms of retinoid-induced craniosynostosis remain unclear. The authors used the culture of mouse cranial suture-derived mesenchymal cells to probe the potential cellular mechanisms of this teratogen to better elucidate mechanisms of retinoid-induced suture fusion.Genes associated with retinoid signaling were assayed in fusing (posterofrontal) and patent (sagittal, coronal) sutures by quantitative real-time polymerase chain reaction. Cultures of mouse suture-derived mesenchymal cells from the posterofrontal suture were established from 4-day-old mice. Cells were cultured with all-trans retinoic acid (1 and 5 muM). Proliferation, osteogenic differentiation, and specific gene expression were assessed.Mouse sutures were found to express genes necessary for retinoic acid synthesis, binding, and signal transduction, demonstrated by quantitative real-time polymerase chain reaction (Raldh1, Raldh2, Raldh3, and Rbp4). These genes were not found to be differentially expressed in fusing as compared with patent cranial sutures in vivo. Addition of retinoic acid enhanced the osteogenic differentiation of suture-derived mesenchymal cells in vitro, including up-regulation of alkaline phosphatase activity and Runx2 expression. Contemporaneously, cellular proliferation was repressed, as shown by proliferative cell nuclear antigen expression. The pro-osteogenic effect of retinoic acid was accompanied by increased gene expression of several hedgehog and bone morphogenetic protein ligands.Retinoic acid represses proliferation and enhances osteogenic differentiation of suture-derived mesenchymal cells. These in vitro data suggest that retinoid exposure may lead to premature cranial suture fusion by means of enhanced osteogenesis and hedgehog and bone morphogenetic protein signaling.

    View details for DOI 10.1097/PRS.0b013e3181d62980

    View details for Web of Science ID 000276886600007

    View details for PubMedID 20134361

  • Highlights of the proceedings from the 13th International Congress of the International Society of Craniofacial Surgery: ISCFS 2009. journal of craniofacial surgery Bradley, J. P., Warren, S., Longaker, M. T. 2010; 21 (3): 944-946

    View details for DOI 10.1097/SCS.0b013e3181d88125

    View details for PubMedID 20485093

  • The SNaP System: Biomechanical and Animal Model Testing of a Novel Ultraportable Negative-Pressure Wound Therapy System PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Hu, D., Eichstadt, S., Gupta, D. M., Pinto, M., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2010; 125 (5): 1362-1371

    Abstract

    Negative-pressure wound therapy is traditionally achieved by attaching an electrically powered pump to a sealed wound bed and applying subatmospheric pressure by means of gauze or foam. The Smart Negative Pressure (SNaP) System (Spiracur, Inc., Sunnyvale, Calif.) is a novel ultraportable negative-pressure wound therapy system that does not require an electrically powered pump.Negative pressure produced by the SNaP System, and a powered pump, the wound vacuum-assisted closure advanced-therapy system (Kinetic Concepts, Inc., San Antonio, Texas), were compared in vitro using bench-top pressure sensor testing and microstrain and stress testing with pressure-sensitive film and micro-computed tomographic scan analysis. In addition, to test in vivo efficacy, 10 rats underwent miniaturized SNaP (mSNaP) device placement on open wounds. Subject rats were randomized to a system activation group (approximately -125 mmHg) or a control group (atmospheric pressure). Wound measurements and histologic data were collected for analysis.Bench measurement revealed nearly identical negative-pressure delivery and mechanical strain deformation patterns between both systems. Wounds treated with the mSNaP System healed faster, with decreased wound size by postoperative day 7 (51 percent versus 12 percent reduction; p < 0.05) and had more rapid complete reepithelialization (21 days versus 32 days; p < 0.05). The mSNaP device also induced robust granulation tissue formation.The SNaP System and an existing electrically powered negative-pressure wound therapy system have similar biomechanical properties and functional wound-healing benefits. The potential clinical efficacy of the SNaP device for the treatment of wounds is supported.

    View details for DOI 10.1097/PRS.0b013e3181d62b25

    View details for Web of Science ID 000276886600008

    View details for PubMedID 20440156

  • Commentary. Aesthetic surgery journal Levi, B., Ko, S. H., Longaker, M. T. 2010; 30 (3): 387-389

    View details for DOI 10.1177/1090820X10374102

    View details for PubMedID 20601561

  • Cyclophilin C-Associated Protein/Mac-2 Binding Protein Colocalizes With Calnexin and Regulates the Expression of Tissue Transglutaminase JOURNAL OF CELLULAR PHYSIOLOGY Kong, W., Lin, B. W., Li, S., Longaker, M. T., Lorenz, H. P. 2010; 223 (1): 151-157

    Abstract

    Cyclophilin C-associated protein (CyCAP) or Mac-2 binding protein has been identified as a binding protein for cyclophilin C in mice and for Mac-2 (galectin-3) in human, suggesting its multiple binding activity to proteins. In the present study, using specific anti-rat-CyCAP antibody, we found that CyCAP colocalizes with calnexin at the location near the nuclear envelope, however CyCAP does not have colocalization with calreticulin. In senescent fibroblasts and interferon-gamma (IFNgamma) treated fibroblasts, both calnexin and CyCAP form larger polymers and are released from the endoplasmic reticulum (ER) through the cellular membrane to the extracellular area. Immunoprecipitation studies further confirm that the release of calnexin is through binding to CyCAP. Further, we found that tissue transglutaminase (tTG) protein is decreased, however not at the RNA level, in CyCAP null fibroblasts, which suggests that CyCAP is involved in tTG post-translational modification. Our data give novel evidence that CyCAP regulates the post-translational modification of tTG through its colocalization with calnexin in ER.

    View details for DOI 10.1002/jcp.22020

    View details for Web of Science ID 000275589300017

    View details for PubMedID 20049854

  • A nonviral minicircle vector for deriving human iPS cells NATURE METHODS Jia, F., Wilson, K. D., Sun, N., Gupta, D. M., Huang, M., Li, Z., Panetta, N. J., Chen, Z. Y., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2010; 7 (3): 197-U46

    Abstract

    Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.

    View details for DOI 10.1038/NMETH.1426

    View details for Web of Science ID 000275058200018

    View details for PubMedID 20139967

  • Pulsed Direct Current Electric Fields Enhance Osteogenesis in Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Hammerick, K. E., James, A. W., Huang, Z., Prinz, F. B., Longaker, M. T. 2010; 16 (3): 917-931

    Abstract

    Adipose-derived stromal cells (ASCs) constitute a promising source of cells for regenerative medicine applications. Previous studies of osteogenic potential in ASCs have focused on chemicals, growth factors, and mechanical stimuli. Citing the demonstrated role electric fields play in enhancing healing in bone fractures and defects, we investigated the ability of pulsed direct current electric fields to drive osteogenic differentiation in mouse ASCs. Employing 50 Hz direct current electric fields in concert with and without osteogenic factors, we demonstrated increased early osteoblast-specific markers. We were also able to establish that commonly reported artifacts of electric field stimulation are not the primary mediators of the observed effects. The electric fields caused marked changes in the cytoskeleton. We used atomic force microscopy-based force spectroscopy to record an increase in the cytoskeletal tension after treatment with electric fields. We abolished the increased cytoskeletal stresses with the rho-associated protein kinase inhibitor, Y27632, and did not see any decrease in osteogenic gene expression, suggesting that the pro-osteogenic effects of the electric fields are not transduced via cytoskeletal tension. Electric fields may show promise as candidate enhancers of osteogenesis of ASCs and may be incorporated into cell-based strategies for skeletal regeneration.

    View details for DOI 10.1089/ten.tea.2009.0267

    View details for Web of Science ID 000275041500016

    View details for PubMedID 19824802

  • Human iPS cell-based therapy Considerations before clinical applications CELL CYCLE Sun, N., Longaker, M. T., Wu, J. C. 2010; 9 (5): 880-885

    Abstract

    Generation of induced pluripotent stem (iPS) cells has revolutionized the field of regenerative medicine. With the exponential increase in iPS cell research in the past three years, human iPS cells have been derived with different technologies and from various cell types. From a translational perspective, however, a number of issues must be addressed before safe and high quality patient-specific iPS cells can be derived for clinical applications. In addition, iPS cell-based therapies also need to be thoroughly evaluated in pre-clinical animal models before they can be applied to human subjects.

    View details for Web of Science ID 000276307700017

    View details for PubMedID 20160515

  • Human Adipose-Derived Stromal Cells Respond to and Elaborate Bone Morphogenetic Protein-2 during In Vitro Osteogenic Differentiation PLASTIC AND RECONSTRUCTIVE SURGERY Panetta, N. J., Gupta, D. M., Lee, J. K., Wan, D. C., Commons, G. W., Longaker, M. T. 2010; 125 (2): 483-493

    Abstract

    Interest in the potential application of adipose-derived stromal cells in cell-mediated tissue engineering of bone and other mesenchymal-derived tissues is growing. This study aimed to investigate the hypothesis that human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein (BMP) 2, which could represent an important target of molecular manipulation to enhance the osteogenic potential of human adipose-derived stromal cells.Human adipose-derived stromal cells were differentiated for 10 days toward the osteogenic lineage in osteogenic differentiation media alone or supplemented with recombinant human BMP2 (rhBMP2). Alizarin red staining was quantified by spectrophotometry. Gene expression analyses were performed using quantitative real-time polymerase chain reaction. BMP2 levels in conditioned media were titered by enzyme-linked immunosorbent assay daily during osteogenic differentiation. Human adipose-derived stromal cells were cultured in complete or partially (50 percent) changed osteogenic differentiation media, or unchanged osteogenic differentiation media, to assay for pro-osteogenic secreted factors. In addition, human adipose-derived stromal cells were cultured in osteogenic differentiation media supplemented with BMP2/BMP4-neutralizing antibody.Exogenous rhBMP2 significantly augmented the in vitro osteogenic potential of human adipose-derived stromal cells in a dose-dependent fashion, and significantly increased transcript levels of RUNX2 and osteocalcin. BMP2, BMP4, BMPR1B, and SMAD1/5 expression was significantly increased during differentiation. Enzyme-linked immunosorbent assay demonstrated significantly increased BMP2 elaboration during differentiation. Culture in conditioned osteogenic differentiation media led to significantly increased matrix mineralization. Mineralization was significantly decreased when osteogenic differentiation media was supplemented with a BMP2/BMP4-neutralizing antibody.These data strongly support that BMP signaling is dynamic and important during normal in vitro osteogenic differentiation of human adipose-derived stromal cells. Thus, BMP2 may be used to enhance the osteogenic differentiation of human adipose-derived stromal cells for bone tissue engineering. Future studies will examine the effect of rhBMP2 on osteogenic differentiation of human adipose-derived stromal cells in vivo.

    View details for DOI 10.1097/PRS.0b013e3181c82d75

    View details for Web of Science ID 000274741700008

    View details for PubMedID 20124834

  • Addressing the paucity of underrepresented minorities in academic surgery: can the "Rooney Rule" be applied to academic surgery? AMERICAN JOURNAL OF SURGERY Butler, P. D., Longaker, M. T., Britt, L. D. 2010; 199 (2): 255-262
  • Interaction of Wingless Protein (Wnt), Transforming Growth Factor-beta 1, and Hyaluronan Production in Fetal and Postnatal Fibroblasts PLASTIC AND RECONSTRUCTIVE SURGERY Carre, A. L., James, A. W., Macleod, L., Kong, W., Kawai, K., Longaker, M. T., Lorenz, H. P. 2010; 125 (1): 74-88

    Abstract

    Mammalian fetal skin injury heals scarlessly. The intrinsic differences between embryonic and adult fibroblasts that underlie this observation are poorly understood. Several studies have linked Wnt proteins with skin morphogenesis. The authors' study aimed to establish a correlation between beta-catenin-dependent (canonical) Wnt protein, transforming growth factor (TGF)-beta1, and the expression of hyaluronan synthesis enzymes during scarless versus scarring wound healing.Wnt signaling was quantified after 1.5-mm skin wounds were created in BAT-gal fetal (e16.5) and postnatal (p1) mice. Canonical Wnt signals were localized by X-gal staining and quantified with quantitative real-time polymerase chain reaction. Primary embryonic and postnatal mouse dermal fibroblasts were treated with recombinant Wnt3a or TGF-beta1. Proliferation was assayed by bromodeoxyuridine incorporation. Gene expression of enzymes that regulate hyaluronan production and turnover was examined by quantitative real-time polymerase chain reaction (hyaluronan synthases or HAS1-3, hyaluronadase-2), as well as other target genes for Wnt and TGF-beta (Axin2, TGF-beta1, TGF-beta3, type 1 collagen, proliferating cell nuclear antigen).Canonical Wnt signaling increased following wounding in postnatal, but not fetal, mice. In vitro, rmWnt3a increased postnatal fibroblast proliferation but not in embryonic cells. Both Wnt3a and TGF-beta1 induced HAS2 and HAS3 gene expression in embryonic fibroblasts, while HAS1 and Hyal2 were induced in postnatal fibroblasts. Finally, rmWnt3a significantly increased type I collagen expression, particularly in postnatal fibroblasts, and influenced expression of TGF-beta isoforms.Increased canonical Wnt signaling occurs during postnatal but not fetal cutaneous wound repair. Fetal and postnatal fibroblasts have a disparate response to rmWnt3a in vitro. rmWnt3a affects postnatal fibroblasts in a similar fashion to rhTGF-beta1, a known profibrotic cytokine.

    View details for DOI 10.1097/PRS.0b013e3181c495d1

    View details for Web of Science ID 000273417000011

    View details for PubMedID 20048602

  • Meta Analysis of the Effectiveness of Surgical Scalpel or Diathermy in Making Abdominal Skin Incisions. Annals of surgery Longaker, M. T., Gurtner, G. C. 2010

    View details for PubMedID 21135686

  • Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure CELLS TISSUES ORGANS Sahar, D. E., Behr, B., Fong, K. D., Longaker, M. T., Quarto, N. 2010; 191 (5): 401-413

    Abstract

    Cranial suture development involves coordinated expression of multiple genes and tissue contribution from neural crest cells and paraxial mesoderm for timely sutural morphogenesis. Transcription factors, growth factors, and neural crest determinant genes play critical roles in calvarial growth ensuring normal development of the underlying brain. In vitro studies have implicated cell-cell adhesion molecules as a driving force behind suture closure. We performed cDNA microarray to study differential expression of adhesion molecules during the timing of suture closure in a mouse model where only the posterior frontal (PF) suture closes. Our results indicate increased expression of E-cadherin during the period of PF suture closure. Quantitative RT-PCR analysis of E- and N-cadherin in PF closing suture revealed a biphasic expression of N-cadherin, the first phase coinciding with cellular condensation preceding chondrogenesis followed by a second phase coinciding with E-cadherin co-expression and suture closure. Furthermore, expression analysis of the N-cadherin and E-cadherin transcriptional repressors Wnt7a and Snail indicate a specific temporal regulation of these genes, suggesting their potential role as regulators of both E- and N-cadherin during the PF suture development and closure. Finally, given the in vitro evidence of fibroblast growth factor (FGF)-2 as a potential regulator of E- and N-cadherin we investigated the expression of E-cadherin during PF suture closure in Fgf-2 deficient mice. In contrast to in vitrodata previously reported, E-cadherin expression is normal in these animals, and PF suture closure occurs properly, probably due to potential redundancy of FGF ligands ensuring normal temporal expression of E-cadherin and PF suture closure.

    View details for DOI 10.1159/000272318

    View details for Web of Science ID 000276748700007

    View details for PubMedID 20051668

  • Inhibition of Histone Deacetylase Activity in Reduced Oxygen Environment Enhances the Osteogenesis of Mouse Adipose-Derived Stromal Cells TISSUE ENGINEERING PART A Xu, Y., Hammerick, K. E., James, A. W., Carre, A. L., Leucht, P., Giaccia, A. J., Longaker, M. T. 2009; 15 (12): 3697-3707

    Abstract

    Recent studies suggest that oxygen tension has a great impact on the osteogenic differentiation capacity of mesenchymal cells derived from adipose tissue: reduced oxygen impedes osteogenesis. We have found that expansion of mouse adipose-derived stromal cells (mASCs) in reduced oxygen tension (10%) results in increased cell proliferation along with induction of histone deacetylase (HDAC) activity. In this study, we utilized two HDAC inhibitors (HDACi), sodium butyrate (NaB) and valproic acid (VPA), and studied their effects on mASCs expanded in various oxygen tensions (21%, 10%, and 1% O(2)). Significant growth inhibition was observed with NaB or VPA treatment in each oxygen tension. Osteogenesis was enhanced by treatment with NaB or VPA, particularly in reduced oxygen tensions (10% and 1% O(2)). Conversely, adipogenesis was decreased with treatments of NaB or VPA at all oxygen tensions. Finally, NaB- or VPA-treated, reduced oxygen tension-exposed (1% O(2)) ASCs were grafted into surgically created mouse tibial defects and resulted in significantly increased bone regeneration. In conclusion, HDACi significantly promote the osteogenic differentiation of mASCs exposed to reduced oxygen tension; HDACi may hold promise for future clinical applications of ASCs for skeletal regeneration.

    View details for DOI 10.1089/ten.tea.2009.0213

    View details for Web of Science ID 000272528400003

    View details for PubMedID 19505250

  • TGF-beta 1 RNA Interference in Mouse Primary Dura Cell Culture: Downstream Effects on TGF Receptors, FGF-2, and FGF-R1 mRNA Levels PLASTIC AND RECONSTRUCTIVE SURGERY Ko, S. H., Behr, B., Longaker, M. T. 2009; 124 (5): 1474-1476
  • Human skin wounds: A major and snowballing threat to public health and the economy WOUND REPAIR AND REGENERATION Sen, C. K., Gordillo, G. M., Roy, S., Kirsner, R., Lambert, L., Hunt, T. K., Gottrup, F., Gurtner, G. C., Longaker, M. T. 2009; 17 (6): 763-771

    Abstract

    ABSTRACT In the United States, chronic wounds affect 6.5 million patients. An estimated excess of US$25 billion is spent annually on treatment of chronic wounds and the burden is rapidly growing due to increasing health care costs, an aging population and a sharp rise in the incidence of diabetes and obesity worldwide. The annual wound care products market is projected to reach $15.3 billion by 2010. Chronic wounds are rarely seen in individuals who are otherwise healthy. In fact, chronic wound patients frequently suffer from "highly branded" diseases such as diabetes and obesity. This seems to have overshadowed the significance of wounds per se as a major health problem. For example, NIH's Research Portfolio Online Reporting Tool (RePORT; http://report.nih.gov/), directed at providing access to estimates of funding for various disease conditions does list several rare diseases but does not list wounds. Forty million inpatient surgical procedures were performed in the United States in 2000, followed closely by 31.5 million outpatient surgeries. The need for post-surgical wound care is sharply on the rise. Emergency wound care in an acute setting has major significance not only in a war setting but also in homeland preparedness against natural disasters as well as against terrorism attacks. An additional burden of wound healing is the problem of skin scarring, a $12 billion annual market. The immense economic and social impact of wounds in our society calls for allocation of a higher level of attention and resources to understand biological mechanisms underlying cutaneous wound complications.

    View details for DOI 10.1111/j.1524-475X.2009.00543.x

    View details for Web of Science ID 000271314900001

    View details for PubMedID 19903300

  • Discussion. TGF-beta1 RNA interference in mouse primary dura cell culture: downstream effects on TGF receptors, FGF-2, and FGF-R1 mRNA levels. Plastic and reconstructive surgery Ko, S. H., Behr, B., Longaker, M. T. 2009; 124 (5): 1474-1476

    View details for DOI 10.1097/PRS.0b013e3181b989de

    View details for PubMedID 20009833

  • From Bedside to Bench and Back Again: Technology Innovation in Plastic Surgery PLASTIC AND RECONSTRUCTIVE SURGERY Gurtner, G. C., Rohrich, R. J., Longaker, M. T. 2009; 124 (4): 1355-1356

    View details for DOI 10.1097/PRS.0b013e3181b8901a

    View details for Web of Science ID 000270332300042

    View details for PubMedID 19935323

  • Estrogen/Estrogen Receptor Alpha Signaling in Mouse Posterofrontal Cranial Suture Fusion PLOS ONE James, A. W., Theologis, A. A., Brugmann, S. A., Xu, Y., Carre, A. L., Leucht, P., Hamilton, K., Korach, K. S., Longaker, M. T. 2009; 4 (9)

    Abstract

    While premature suture fusion, or craniosynostosis, is a relatively common condition, the cause is often unknown. Estrogens are associated with growth plate fusion of endochondral bones. In the following study, we explore the previously unknown significance of estrogen/estrogen receptor signaling in cranial suture biology.Firstly, estrogen receptor (ER) expression was examined in physiologically fusing (posterofrontal) and patent (sagittal) mouse cranial sutures by quantitative RT-PCR. Next, the cranial suture phenotype of ER alpha and ER beta knockout (alphaERKO, betaERKO) mice was studied. Subsequently, mouse suture-derived mesenchymal cells (SMCs) were isolated; the effects of 17-beta estradiol or the estrogen antagonist Fulvestrant on gene expression, osteogenic and chondrogenic differentiation were examined in vitro. Finally, in vivo experiments were performed in which Fulvestrant was administered subcutaneously to the mouse calvaria. Results showed that increased ERalpha but not ERbeta transcript abundance temporally coincided with posterofrontal suture fusion. The alphaERKO but not betaERKO mouse exhibited delayed posterofrontal suture fusion. In vitro, addition of 17-beta estradiol enhanced both osteogenic and chondrogenic differentiation in suture-derived mesenchymal cells, effects reversible by Fulvestrant. Finally, in vivo application of Fulvestrant significantly diminished calvarial osteogenesis, inhibiting suture fusion.Estrogen signaling through ERalpha but not ERbeta is associated with and necessary for normal mouse posterofrontal suture fusion. In vitro studies suggest that estrogens may play a role in osteoblast and/or chondrocyte differentiation within the cranial suture complex.

    View details for DOI 10.1371/journal.pone.0007120

    View details for Web of Science ID 000270154400013

    View details for PubMedID 19771170

  • Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sun, N., Panetta, N. J., Gupta, D. M., Wilson, K. D., Lee, A., Jia, F., Hu, S., Cherry, A. M., Robbins, R. C., Longaker, M. T., Wu, J. C. 2009; 106 (37): 15720-15725

    Abstract

    Ectopic expression of transcription factors can reprogram somatic cells to a pluripotent state. However, most of the studies used skin fibroblasts as the starting population for reprogramming, which usually take weeks for expansion from a single biopsy. We show here that induced pluripotent stem (iPS) cells can be generated from adult human adipose stem cells (hASCs) freshly isolated from patients. Furthermore, iPS cells can be readily derived from adult hASCs in a feeder-free condition, thereby eliminating potential variability caused by using feeder cells. hASCs can be safely and readily isolated from adult humans in large quantities without extended time for expansion, are easy to maintain in culture, and therefore represent an ideal autologous source of cells for generating individual-specific iPS cells.

    View details for DOI 10.1073/pnas.0908450106

    View details for Web of Science ID 000269806600040

    View details for PubMedID 19805220

  • Bone Tissue Engineering Scaffolds of Today and Tomorrow DISCUSSION JOURNAL OF CRANIOFACIAL SURGERY Panetta, N. J., Gupta, D. M., Longaker, M. T. 2009; 20 (5): 1531-1532

    View details for Web of Science ID 000270369000047

    View details for PubMedID 19816291

  • Tissue Harvest by Means of Suction-Assisted or Third-Generation Ultrasound-Assisted Lipoaspiration Has No Effect on Osteogenic Potential of Human Adipose-Derived Stromal Cells PLASTIC AND RECONSTRUCTIVE SURGERY Panetta, N. J., Gupta, D. M., Kwan, M. D., Wan, D. C., Commons, G. W., Longaker, M. T. 2009; 124 (1): 65-73

    Abstract

    Human adipose-derived stromal cells readily undergo osteogenic differentiation in vitro and in vivo. Thus, interest in their potential role in skeletal tissue engineering continues to escalate. Very little is known regarding the effects that energy delivered by means of third-generation ultrasound-assisted lipoaspiration may have on the osteogenic potential of these cells. The authors investigated whether differences in adipose-derived stromal cell yield, and the in vitro proliferation and osteogenic potential of these cells obtained by suction-assisted lipoaspiration or third-generation ultrasound-assisted lipoaspiration, exist.Adipose-derived stromal cells were harvested from lipoaspiration specimens of patients undergoing elective suction-assisted lipoaspiration and third-generation ultrasound-assisted lipoaspiration. Harvested cells were seeded to evaluate proliferative capacity and in vitro osteogenic potential. Alkaline phosphatase and alizarin red staining were performed to evaluate early and terminal osteogenic differentiation, respectively. Quantitative real-time polymerase chain reaction analysis was used to examine osteogenic gene expression patterns of RUNX2/CFBA1 (early differentiation) and osteocalcin (late differentiation).No significant differences in the proliferative capacity (n = 3), alkaline phosphatase staining (n = 3), or extracellular matrix mineralization (n = 3) of suction-assisted lipoaspiration- or third-generation ultrasound-assisted lipoaspiration-derived cells were appreciated. Transcript levels of markers of early and terminal osteogenic differentiation were not significantly different (n = 3).These findings suggest that exposure of adipose-derived stromal cells to ultrasound energy during tissue harvest by means of third-generation ultrasound-assisted lipoaspiration does not impart a negative consequence toward their proliferative capacity or osteogenic potential. Thus, the cells harvested using third-generation ultrasound-assisted lipoaspiration are comparable to those obtained by means of suction-assisted lipoaspiration for use in the study of osteogenic differentiation and skeletal tissue engineering.

    View details for DOI 10.1097/PRS.0b013e3181ab10cd

    View details for Web of Science ID 000267895000011

    View details for PubMedID 19568046

  • The Use of Polymer Scaffolds in Skeletal Tissue Engineering Applications JOURNAL OF CRANIOFACIAL SURGERY Gupta, D. M., Panetta, N. J., Longaker, M. T. 2009; 20 (3): 860-861

    View details for Web of Science ID 000266295400035

    View details for PubMedID 19461326

  • Ethnic Diversity Remains Scarce in Academic Plastic and Reconstructive Surgery PLASTIC AND RECONSTRUCTIVE SURGERY Butler, P. D., Britt, L. D., Longaker, M. T. 2009; 123 (5): 1618-1627

    Abstract

    Plastic surgery has been dedicated to advancing academic surgery in education, research, innovation, and patient care. Thus, as U.S. health care disparities persist, it would be befitting for plastic surgery to assume the lead in alleviating these disparities. As part of a multifaceted approach to ameliorate health care disparities, increasing diversity in the health care workforce will be imperative. Investigating the demographics of the U.S. plastic surgery residents and faculty can bring attention to a deficit that, if corrected, could benefit the field and improve the entire health care system.Medical students, plastic surgery residents/fellows, and plastic surgery faculty demographic information from 1966 to 2006 was analyzed from the Association of American Medical Colleges' data files.Caucasians encompass 68.7 percent of U.S. plastic surgery residents/fellows, while Asian-, African-, and Latino-Americans encompass 20.9, 3.7, and 6.2 percent, respectively. Caucasians comprise 74.9 percent of academic plastic surgeons, while Asian-, African-, and Latino-Americans comprise 10.9, 1.4, and 3.6 percent, respectively. Caucasians constitute 82.0 percent of tenured full professors, while Asian-, African-, and Latino-Americans constitute 4.9, 1.6, and 4.9 percent, respectively. In 2004, African-Americans and Latino-Americans comprised 3.6 percent and 5.7 percent of all U.S. plastic surgeons, but only 1.5 percent and 4.9 percent of plastic academicians, respectively.Over the last 40 years, plastic surgery has been ineffective in adequately increasing the number of minority residents and faculty. Expanding the number of minority academic plastic surgeons could establish a health care environment more accommodating to minority patients, increase studies highlighting minority health needs, and provide additional role models and mentors.

    View details for DOI 10.1097/PRS.0b013e3181a07610

    View details for Web of Science ID 000266067900029

    View details for PubMedID 19407636

  • Untitled AESTHETIC SURGERY JOURNAL Gupta, D. M., Panetta, N. J., Longaker, M. T. 2009; 29 (2): 147-149
  • Craniofacial Autologous Fat Transfer JOURNAL OF CRANIOFACIAL SURGERY Wan, D. C., Lim, A. T., Longaker, M. T. 2009; 20 (2): 273-274

    View details for DOI 10.1097/SCS.0b013e31819921d3

    View details for Web of Science ID 000264570300003

    View details for PubMedID 19305242

  • Perspectives in Cell-Based Skeletal Tissue JOURNAL OF CRANIOFACIAL SURGERY Panetta, N. J., Gupta, D. M., Longaker, M. T. 2009; 20 (2): 347-348

    View details for Web of Science ID 000264570300016

    View details for PubMedID 19305243

  • Cell Permeant Peptide Analogues of the Small Heat Shock Protein, HSP20, Reduce TGF-beta 1-Induced CTGF Expression in Keloid Fibroblasts JOURNAL OF INVESTIGATIVE DERMATOLOGY Lopes, L. B., Furnish, E. J., Komalavilas, P., Flynn, C. R., Ashby, P., Hansen, A., Ly, D. P., Yang, G. P., Longaker, M. T., Panitch, A., Brophy, C. M. 2009; 129 (3): 590-598

    Abstract

    A growing body of evidence suggests the involvement of connective tissue growth factor (CTGF) in the development and maintenance of fibrosis and excessive scarring. As the expression of this protein requires an intact actin cytoskeleton, disruption of the cytoskeleton represents an attractive strategy to decrease CTGF expression and, consequently, excessive scarring. The small heat-shock-related protein (HSP20), when phosphorylated by cyclic nucleotide signaling cascades, displaces phospho-cofilin from the 14-3-3 scaffolding protein leading to activation of cofilin as an actin-depolymerizing protein. In the present study, we evaluated the effect of AZX100, a phosphopeptide analogue of HSP20, on transforming growth factor-beta-1 (TGF-beta1)-induced CTGF and collagen expression in human keloid fibroblasts. We also examined the effect of AZX100 on scar formation in vivo in dermal wounds in a Siberian hamster model. AZX100 decreased the expression of CTGF and type I collagen induced by TGF-beta1, endothelin, and lysophosphatidic acid. Treatment with AZX100 decreased stress fiber formation and altered the morphology of human dermal keloid fibroblasts. In vivo, AZX100 significantly improved collagen organization in a Siberian hamster scarring model. Taken together, these results suggest the potential use of AZX100 as a strategy to prevent excessive scarring and fibrotic disorders.

    View details for DOI 10.1038/jid.2008.264

    View details for Web of Science ID 000263569500011

    View details for PubMedID 18787533

  • Mesenchymal cells for skeletal tissue engineering PANMINERVA MEDICA Panetta, N. J., Gupta, D. M., Quarto, N., Longaker, M. T. 2009; 51 (1): 25-41

    Abstract

    Today, surgical intervention remains the mainstay of treatment to intervene upon a multitude of skeletal deficits and defects attributable to congenital malformations, oncologic resection, pathologic degenerative bone destruction, and post-traumatic loss. Despite this significant demand, the tools with which surgeons remain equipped are plagued with a surfeit of inadequacies, often resulting in less than ideal patient outcomes. The failings of current techniques largely arise secondary to their inability to produce a regenerate which closely resembles lost tissue. As such, focus has shifted to the potential of mesenchymal stem cell (MSC)-based skeletal tissue engineering. The successful development of such techniques would represent a paradigm shift from current approaches, carrying with it the potential to regenerate tissues which mimic the form and function of endogenous bone. Lessons learned from investigations probing the endogenous regenerative capacity of skeletal tissues have provided direction to early studies investigating the osteogenic potential of MSC. Additionally, increasing attention is being turned to the role of targeted molecular manipulations in augmenting MSC osteogenesis, as well as the development of an ideal scaffold ''vehicle'' with which to deliver progenitor cells. The following discussion presents the authors' current working knowledge regarding these critical aspects of MSC application in cell-based skeletal tissue engineering strategies, as well as provides insight towards what future steps must be taken to make their clinical translation a reality.

    View details for Web of Science ID 000266771000004

    View details for PubMedID 19352307

  • Aging and Diabetes Impair the Neovascular Potential of Adipose-Derived Stromal Cells PLASTIC AND RECONSTRUCTIVE SURGERY El-Ftesi, S., Chang, E. I., Longaker, M. T., Gurtner, G. C. 2009; 123 (2): 475-485

    Abstract

    Aging and diabetes are major risk factors for poor wound healing and tissue regeneration that reflect an impaired ability to respond to ischemic insults. The authors explored the intrinsic neovascular potential of adipose-derived stromal cells in the setting of advanced age and in type 1 and type 2 diabetes.Adipose-derived stromal cells isolated from young, aged, streptozotocin-induced, and db/db diabetic mice were exposed to normoxia and hypoxia in vitro. Vascular endothelial growth factor (VEGF) expression, proliferation, and tubulization were measured. Conditioned media harvested from adipose-derived stromal cell cultures were assessed for their ability to stimulate human umbilical vein endothelial cell proliferation (n = 3 and n = 3).Young adipose-derived stromal cells demonstrated significantly higher levels of VEGF production, proliferation, and tubulogenesis than those derived from aged, streptozotocin-induced, and db/db mice in both normoxia and hypoxia. Although aged and diabetic adipose-derived stromal cells retained the ability to up-regulate VEGF secretion, proliferation, and tubulogenesis in response to hypoxia, the response was blunted compared with young controls. Conditioned media derived from these cells cultured in normoxia in vitro also had a significantly greater ability to increase human umbilical vein endothelial cell proliferation compared with media harvested from aged, streptozotocin-induced, and db/db adipose-derived stromal cells. This effect was magnified in conditioned media harvested from hypoxic adipose-derived stromal cell cultures.This study demonstrates that aging and type 1 and type 2 diabetes impair intrinsic adipose-derived stromal cell function; however, these cells may still be a suitable source of angiogenic cells that can potentially improve neovascularization of ischemic tissues.

    View details for DOI 10.1097/PRS.0b013e3181954d08

    View details for Web of Science ID 000265669200006

    View details for PubMedID 19182604

  • The Role of Regional Posterior Frontal Dura Mater in the Overlying Suture Morphology PLASTIC AND RECONSTRUCTIVE SURGERY Slater, B. J., Kwan, M. D., Gupta, D. M., Lee, J. K., Longaker, M. T. 2009; 123 (2): 463-469

    Abstract

    Craniosynostosis, the premature fusion of one or more cranial sutures, is a common developmental disorder resulting in morphologic and functional consequences. The rat model is useful for studying pathologic and normal suture fusion because the posterior frontal suture undergoes fusion but the remaining sutures remain patent. The authors investigated the influence of regional posterior frontal dura mater on the overlying suture morphology and fate.In 8-day-old Sprague-Dawley rats, an 8-mm calvarial disk was excised without disrupting the underlying dura mater (n = 22) and flipped so that the previously ectocranial aspect was adjacent to the dura mater. The animals were humanely killed after 5, 7, 9, 11, and 28 days, and the posterior frontal sutures were analyzed histologically. A comparison was made to control animals in which the disk was excised and then placed back into its anatomical position (n = 5). Immunohistochemistry of the transforming growth factor (TGF)-beta isoforms was performed to investigate their differential, temporal, and spatial expression.Posterior frontal suture fusion occurred on the side adjacent to the dura mater (previously patent ectocranial aspect) in an anterior-to-posterior direction, similar to that in the control group. There was specific expression of the TGF-beta isoforms in the dura mater and suture mesenchyme adjacent to the dura mater.Regional dura mater plays an important role in suture morphology, and the posterior frontal-associated dura mater possesses potent, pro-osteogenic signals that influence the overlying suture fate. The differential expression pattern of TGF-beta signaling from the dura mater further supports the regional paracrine effect of the dura mater.

    View details for DOI 10.1097/PRS.0b013e3181954d21

    View details for Web of Science ID 000265669200004

    View details for PubMedID 19182602

  • Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development PLOS ONE Slater, B. J., Liu, K. J., Kwan, M. D., Quarto, N., Longaker, M. T. 2009; 4 (1)

    Abstract

    The tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied.We examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (microCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13.A better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.

    View details for DOI 10.1371/journal.pone.0003914

    View details for Web of Science ID 000265481900001

    View details for PubMedID 19156194

  • Ex vivo Model of Cranial Suture Morphogenesis and Fate CELLS TISSUES ORGANS Slater, B. J., Lenton, K. A., James, A., Longaker, M. T. 2009; 190 (6): 336-346

    Abstract

    Craniosynostosis, the premature fusion of cranial sutures, is a common congenital defect. In vivo models for studying cranial suture biology impose inherent restrictions on tissue accessibility and manipulation. The present study was performed to investigate the utility of the renal capsule assay in overcoming these limitations and providing a reproducible model system for studying cranial suture morphogenesis and fate.The posterior frontal suture, which fuses physiologically, and the coronal and sagittal sutures, which remain patent, were dissected from postnatal and embryonic mouse calvaria and placed under the renal capsule of syngeneic recipient mice (n = 72 in total). Sutures were harvested from 1-14 days after transplantation for histological and morphometric analysis. Suture transplants were compared with nonmanipulated sutures at equivalent developmental stages. The derivation of cells associated with the growing transplants was analyzed using beta-actin-GFP (green fluorescent protein) transgenic mice.Sutures transplanted under the renal capsule maintained normal suture morphology and fate with the posterior frontal suture fusing and the coronal and sagittal sutures remaining patent. In posterior frontal suture transplants, the fusion process mimicked in vivo suture fusion with a delay of 1-2 days. In comparison to in vivo suture complexes, transplant thickness and trabeculation were significantly increased. In addition, we found that osteoblasts within the growing transplant were derived from the transplant itself rather than the host.The renal capsule supports the growth of cranial sutures. In this system transplanted sutures recapitulate the anatomical development and fate (fusion or patency) of cranial sutures in vivo. This model system will facilitate controlled ex vivo manipulations of both embryonic and postnatal sutures.

    View details for DOI 10.1159/000228157

    View details for Web of Science ID 000271819700004

    View details for PubMedID 19590164

  • Differential FGF Ligands and FGF Receptors Expression Pattern in Frontal and Parietal Calvarial Bones CELLS TISSUES ORGANS Quarto, N., Behr, B., Li, S., Longaker, M. T. 2009; 190 (3): 158-169

    Abstract

    The mammalian skull vault consists mainly of 5 flat bones, the paired frontals and parietals, and the unpaired interparietal. All of these bones are formed by intramembranous ossification within a layer of mesenchyme, the skeletogenic membrane, located between the dermal mesenchyme and the meninges surrounding the brain. While the frontal bones are of neural crest in origin, the parietal bones arise from mesoderm. The present study is a characterization of frontal and parietal bones at their molecular level, aiming to highlight distinct differences between the neural crest-derived frontal and mesodermal-derived parietal bone. We performed a detailed comparative gene expression profile of FGF ligands and their receptors known to play crucial role in skeletogenesis. This analysis revealed that a differential expression pattern of the major FGF osteogenic molecules and their receptors exists between the neural crest-derived frontal bone and the paraxial mesoderm-derived parietal bone. Particularly, the expression of ligands such as Fgf-2, Fgf-9 and Fgf-18 was upregulated in frontal bone on embryonic day 17.5, postnatal day 1 and postnatal day 60 mice. Frontal bone also elaborated higher levels of Fgf receptor 1, 2 and 3 transcripts versus parietal bone. Taken together, these data suggest that the frontal bone is a domain with higher FGF-signaling competence than parietal bone.

    View details for DOI 10.1159/000202789

    View details for Web of Science ID 000268865700004

    View details for PubMedID 19218784

  • Topical Lineage-Negative Progenitor-Cell Therapy for Diabetic Wounds (Invited Discussion) PLASTIC AND RECONSTRUCTIVE SURGERY Gurtner, G. C., Longaker, M. T. 2009; 123 (1): 421-423

    View details for DOI 10.1097/PRS.0b013e318194d2b8

    View details for Web of Science ID 000262317700054

    View details for PubMedID 19116584

  • Re: Chen et al. Expression of Inducible Nitric Oxide Synthase and Vascular Endothelial Growth Factor in Ameloblastoma. J Craniofac Surg 2009;20;171-175 DISCUSSION JOURNAL OF CRANIOFACIAL SURGERY Slater, B. J., Kwan, M. D., Longaker, M. T. 2009; 20 (1): 176-177
  • FETAL SKIN WOUND HEALING ADVANCES IN CLINICAL CHEMISTRY VOL 48 Buchanan, E. P., Longaker, M. T., Lorenz, H. P. 2009; 48: 137-161

    Abstract

    The developing fetus has the ability to heal wounds by regenerating normal epidermis and dermis with restoration of the extracellular matrix (ECM) architecture, strength, and function. In contrast, adult wounds heal with fibrosis and scar. Scar tissue remains weaker than normal skin with an altered ECM composition. Despite extensive investigation, the mechanism of fetal wound healing remains largely unknown. We do know that early in gestation, fetal skin is developing at a rapid pace and the ECM is a loose network facilitating cellular migration. Wounding in this unique environment triggers a complex cascade of tightly controlled events culminating in a scarless wound phenotype of fine reticular collagen and abundant hyaluronic acid. Comparison between postnatal and fetal wound healing has revealed differences in inflammatory response, cellular mediators, cytokines, growth factors, and ECM modulators. Investigation into cell signaling pathways and transcription factors has demonstrated differences in secondary messenger phosphorylation patterns and homeobox gene expression. Further research may reveal novel genes essential to scarless repair that can be manipulated in the adult wound and thus ameliorate scar.

    View details for DOI 10.1016/S0065-2423(09)48006-5

    View details for Web of Science ID 000270339800006

    View details for PubMedID 19803418

  • 5-Fluorouracil Treatment of Problematic Scars Discussion PLASTIC AND RECONSTRUCTIVE SURGERY Yang, G. P., Longaker, M. T. 2009; 123 (1): 149-151
  • IFATS Collection: Adipose Stromal Cells Adopt a Proangiogenic Phenotype Under the Influence of Hypoxia STEM CELLS Thangarajah, H., Vial, I. N., Chang, E., El-Ftesi, S., Januszyk, M., Chang, E. I., Paterno, J., Neofytou, E., Longaker, M. T., Gurtner, G. C. 2009; 27 (1): 266-274

    Abstract

    Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell-derived factor (SDF)-1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF-1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis.

    View details for DOI 10.1634/stemcells.2008-0276

    View details for Web of Science ID 000263032400030

    View details for PubMedID 18974212

  • Differential Effects of TGF-beta 1 and TGF-beta 3 on Chondrogenesis in Posterofrontal Cranial Suture-Derived Mesenchymal Cells In Vitro PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Xu, Y., Lee, J. K., Wang, R., Longaker, M. T. 2009; 123 (1): 31-43

    Abstract

    Transforming growth factor (TGF)-beta1 has been associated with cranial suture fusion, whereas TGF-beta3 has been associated with suture patency. The mouse posterofrontal suture, analogous to the human metopic suture, fuses through endochondral ossification.TGF-beta1 and TGF-beta3 expression in the posterofrontal suture was examined by immunohistochemistry. Next, the authors established cultures of suture-derived mesenchymal cells from the posterofrontal suture and examined the cellular responses to TGF-beta1 and TGF-beta3. Proliferation in response to TGF-beta isoforms was examined by bromodeoxyuridine incorporation. High-density micromass culture of posterofrontal mesenchymal cells was used to study the effect of TGF-beta1 and TGF-beta3 on chondrogenic differentiation.TGF-beta1 but not TGF-beta3 protein was highly expressed in chondrocytes within the posterofrontal suture. Significant increases in posterofrontal cell proliferation were observed with TGF-beta3 but not TGF-beta1. TGF-beta1 led to significant increases in chondrogenic-specific gene expression (including Sox9, Col II, Aggrecan, and Col X) as compared with moderate effects of TGF-beta3. TGF-beta1 increased cellular adhesion molecule expression (N-cadherin and fibronectin) and promoted cellular condensation, whereas TGF-beta3 increased cellular proliferation (PCNA expression). Finally, TGF-beta1 and, to a lesser extent, TGF-beta3 induced the expression of fibroblast growth factors (FGF-2 and FGF-18).TGF-beta1 and TGF-beta3 exhibit marked differences in their effects on chondrogenesis in posterfrontal suture-derived mesenchymal cells, influencing different stages of chondrogenic differentiation. TGF-beta3 significantly increased cellular proliferation, whereas TGF-beta1 induced precartilage condensation, promoting chondrocyte differentiation.

    View details for DOI 10.1097/PRS.0b013e3181904c19

    View details for Web of Science ID 000262317700006

    View details for PubMedID 19116522

  • Where Do We Find the Best Evidence? Discussion PLASTIC AND RECONSTRUCTIVE SURGERY Butler, P. D., Longaker, M. T. 2008; 122 (6): 1950-1951
  • Gene expression analysis of Dupuytren's disease: the role of TGF-beta2. The Journal of hand surgery, European volume Zhang, A. Y., Fong, K. D., Pham, H., Nacamuli, R. P., Longaker, M. T., Chang, J. 2008; 33 (6): 783-790

    Abstract

    Dupuytren's disease is characterised by nodular fibroblastic proliferation of the palmar fascia leading to contracture of the hand. Transforming growth factor beta (TGF-beta) is thought to play a role in its pathogenesis. We performed a cDNA microarray analysis of Dupuytren's diseased cord tissue with an emphasis on TGF-beta isoforms. Normal-appearing transverse ligament of the palmar fascia from adjacent to the diseased cord and palmar fascia from patients undergoing carpal tunnel release were used as controls. TGF-beta gene expression was confirmed by quantitative real-time polymerase chain reaction. Over 20 unique genes were found to be significantly up-regulated, including several previously reported genes. A dominant increase in TGF-beta2 expression was seen in the cord tissue, whereas TGF-beta1 and TGF-beta3 were found not to be significantly up-regulated. Quantitative real-time polymerase chain reaction confirmed these findings. This gene expression profile allows for further experiments that may eventually lead to gene therapy to block the development and progression of Dupuytren's disease clinically.

    View details for DOI 10.1177/1753193408091352

    View details for PubMedID 18694919

  • Transforming Growth Factor-beta 1 Stimulates Chondrogenic Differentiation of Posterofrontal Suture-Derived Mesenchymal Cells In Vitro PLASTIC AND RECONSTRUCTIVE SURGERY Xu, Y., James, A. W., Longaker, M. T. 2008; 122 (6): 1649-1659

    Abstract

    Evidence from animal studies has associated transforming growth factor (TGF)-beta signaling with both normal and premature cranial suture fusion. However, the mechanisms whereby this pleiotropic cytokine mediates suture fusion remain uncertain. The authors established cultures of suture-derived mesenchymal cells from normally fusing (posterofrontal) and patent (sagittal) sutures and examined the in vitro effects of TGF-beta1 on these distinct cell populations.Skulls were harvested from 80 5-day-old mice. Posterofrontal and sagittal sutures were dissected, and cultures of suture-derived mesenchymal cells were established. The mitogenic, osteogenic, and chondrogenic effects of recombinant TGF-beta1 were then assessed on posterofrontal and sagittal suture-derived mesenchymal cells (1 to 10 ng/ml). Quantitative real-time polymerase chain reaction was used to examine the effects of TGF-beta1 on gene expression.TGF-beta1 significantly decreased proliferation of both posterofrontal and sagittal suture-derived mesenchymal cells, by bromodeoxyuridine incorporation assays (n = 6). TGF-beta1 also inhibited osteogenesis in both suture-derived mesenchymal cells determined by alkaline phosphatase activity and mineralization (n = 3 for all assays). During chondrogenic differentiation, TGF-beta1 markedly increased expression of chondrocyte-specific gene markers in posterofrontal suture-derived mesenchymal cells (Sox9, Col II, Aggrecan, and Col X) (p

    View details for DOI 10.1097/PRS.0b013e31818cbf44

    View details for Web of Science ID 000263721500006

    View details for PubMedID 19050517

  • GENE EXPRESSION ANALYSIS OF DUPUYTREN'S DISEASE: THE ROLE OF TGF-beta 2 JOURNAL OF HAND SURGERY-EUROPEAN VOLUME Zhang, A. Y., Fong, K. D., Pham, H., Nacamuli, R. P., Longaker, M. T., Chang, J. 2008; 33E (6): 783-790
  • Differential expression of specific FGF ligands and receptor isoforms during osteogenic differentiation of mouse Adipose-derived Stem Cells (mASCs) recapitulates the in vivo osteogenic pattern GENE Quarto, N., Longaker, M. T. 2008; 424 (1-2): 130-140

    Abstract

    The ability of Adipose-derived Stem Cells (ASCs) to differentiate into various tissues in vitro and in vivo, a function known as "stem cell plasticity", makes them an appealing cell source for tissue engineering. Our laboratory is particularly focused on the potential role of adipose tissue as a readily available postnatal source of osteoprogenitor. Fibroblast growth factors (FGF) and their receptors (FGFR) are important regulators of osteogenesis. The goal of this study was to elucidate how changes in temporal expression patterns of individual components of the fibroblast growth factor (FGF) signaling axis correlate with osteogenic differentiation of mASCs. Our results indicate that FGF ligand genes, such as Fgf-2, -4, -8, and -18, displayed a differential and dynamic profile during mouse ASC (mASC) osteogenesis. Fgf-2 transcript was down-regulated, while Fgf-18 transcript level was strongly up-regulated. Interestingly, a drift in the ratio of different FGF-2 protein forms, with translation favoring the HMWFGF-2 forms, occurred during osteogenic differentiation, whereas, the expression of LMWFGF-2 form was down-regulated. This finding shares similarity with a previous study suggesting that preferential expression of the HMWFGF-2 forms is associated with a more osteogenic differentiated state of calvarial osteoblast. Moreover, a differential expression of Fgf Receptor 1 and 2 resembling that previously found in in vivo osteogenic study was observed. Thus, mASCs undergoing osteogenesis recapitulate the in vivo osteogenic differentiation expression pattern of FGF ligands and receptors of calvarial mesenchymal cells during their own osteogenic differentiation. Indeed, this observation further validates ASCs as a suitable resource for skeletal tissue engineering.

    View details for DOI 10.1016/j.gene.2008.07.029

    View details for Web of Science ID 000260563800018

    View details for PubMedID 18718860

  • Plastic Surgery and the Society of University Surgeons: An Expanding Relationship PLASTIC AND RECONSTRUCTIVE SURGERY Longaker, M. T. 2008; 122 (5): 1581-1582

    View details for DOI 10.1097/PRS.0b013e31818824b0

    View details for Web of Science ID 000261138700032

    View details for PubMedID 18971743

  • Major Deficit in the Number of Underrepresented Minority Academic Surgeons Persists ANNALS OF SURGERY Butler, P. D., Longaker, M. T., Britt, L. D. 2008; 248 (5): 704-711

    Abstract

    Eliminating health care disparities in the United States will require a multifaceted approach that will include increasing diversity in the health care workforce. Historically, the field of medicine, and particularly surgery, has had an incumbent that grossly misrepresents the patient population. Delineating the exact demographics of the U.S. surgical residents and faculty could provide outstanding information, yielding insight into a possible deficit that, if rectified by the medical education system, could change the face of surgery and the entire health care system.Demographic information regarding medical students, surgical residents, and surgical faculty was retrieved and analyzed from the Association of American Medical Colleges data files dating back to 1966.Whites comprise 64.4% of U.S. surgical residents, whereas Asian Americans, African Americans, and Latino Americans comprise 17.2%, 4.7%, and 5.1%, respectively. Whites comprise 74.1%, of academic surgeons, whereas Asian Americans, African Americans, and Latino Americans comprise 10.8%, 2.9%, and 3.6%, respectively. African Americans and Latino Americans comprise 5.4% and 4.8% of all U.S. surgeons, but only 2.9% and 3.6% of the academic surgeons, respectively. Whites comprise 85.7% of tenured surgical professors, whereas Asian Americans, African Americans, and Latino Americans comprise 4.9%, 1.8%, and 2.7%, respectively.Academic surgery is exceedingly deficient of minority residents, junior faculty, and professors. Correcting this misrepresentation would facilitate establishing a more culturally and ethnically sensitive health care environment for patients who otherwise would not seek care. Additionally, with more minority academic surgeons, there will likely be a commensurate increase in investigative studies highlighting minority specific health care needs and provide additional role models and mentors for future minority surgeons.

    View details for DOI 10.1097/SLA.0b013e31817f2c30

    View details for Web of Science ID 000260726200003

    View details for PubMedID 18948795

  • Optimization of Flexor Tendon Tissue Engineering With a Cyclic Strain Bioreactor JOURNAL OF HAND SURGERY-AMERICAN VOLUME Riboh, J., Chong, A. K., Pham, H., Longaker, M., Jacobs, C., Chang, J. 2008; 33A (8): 1388-1396

    Abstract

    Mechanical manipulation of cultured tendon cells can enhance cell proliferation and matrix production. This study aims to determine the bioreactor strain patterns (amplitude, frequency, and on/off ratio) that favor cellular proliferation, promote collagen production, and maintain morphology in candidate cell lines cultured for flexor tendon tissue engineering, including multipotent stromal cells.We studied epitenon tenocytes (Es), sheath fibroblasts (Ss), bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived stem cells (ASCs). We examined the effects of 3 patterns of cyclic uniaxial strain on cell proliferation, collagen I production, and cell morphology.Adipoderived stem cells (33% adhesion) and Ss (29%) adhered more strongly to bioreactor membranes than did Es (15%) and BMSCs (7%), p=.04. Continuous cyclic strain (CCS, 8%, 1 Hz) inhibited cell proliferation (p=.01) and increased per-cell collagen production (p=.04) in all cell types. Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/5 hours off) increased proliferation in ASCs (p=.06) and Ss (p=.04). Intermittent cyclic strain (4%, 0.1 Hz, 1 hour on/2 hours off) increased total collagen production by 25% in ASCs (p=.004) and 20% in Ss (p=.05). Cyclic strain resulted in cell alignment perpendicular to the strain axis, cytoskeletal alignment, and nuclear elongation. These morphological characteristics are similar to those of tenocytes.These results demonstrate that intermittent cyclic strain can increase cell proliferation, promote collagen I production, and maintain tenocyte morphology in vitro. Use of a cell bioreactor might accelerate the in vitro stage of tendon tissue engineering.

    View details for DOI 10.1016/j.jhsa.2008.04.019

    View details for Web of Science ID 000260049100021

    View details for PubMedID 18929207

  • Global age-dependent differences in gene expression in response to calvarial injuryd JOURNAL OF CRANIOFACIAL SURGERY Wan, D. C., Kwan, M. D., Gupta, D. M., Wang, Z., Slater, B. J., Panetta, N. J., Morrell, N. T., Longaker, M. T. 2008; 19 (5): 1292-1301

    Abstract

    Children less than 2 years of age are capable of healing large calvarial defects, whereas adults have been found to lack this endogenous ability. In this study, we used microarray analysis to compare genomewide expression patterns during active regeneration after injury with calvaria in skeletally immature and mature mice. Parietal bone defects were created in 6-day-old (juvenile) and 60-day-old (adult) mice using a 4-mm trephine bit (n = 20 mice per age group). The calvarial disc was removed, leaving the underlying dura mater intact. Two weeks after injury, the region of regeneration with the underlying dura mater was harvested, and RNA was extracted for microarray analysis. The 25 most differentially upregulated genes in juvenile regenerates compared with adults were listed, as well as selected bone-related genes. In addition, QRT-PCR confirmation of specific genes was performed for validation. Juvenile regenerates expressed significantly greater amounts of BMP-2, -4, -7, as well as FGF-2 and its receptor FGFR-1. Various other growth factors were also noted to be upregulated, including IGF-2 and Ptn. This corresponded with the increased expression of markers for osteogenic differentiation of Sparc and Oc. Markers of osteoclast activity, Acp5, Ctsk, and Mmp2, were noted to be greater in juvenile regenerates compared with adults. The observation of Mmp14 upregulation, however, highlights the importance of balanced osteoclast-mediated bone resorption for ultimate healing. The 2 most differentially regulated genes, transthyretin (Ttr) and prostaglandin D2 synthase (Ptgds), highlight the potential role of retinoic acid signaling and the prostaglandin axis on skeletal regeneration. These findings underscore the multitude of biomolecular mechanisms at play, allowing juvenile calvaria to heal after injury. The identification of various growth factors and cytokines involved also suggests novel therapeutic strategies for tissue-engineering purposes.

    View details for Web of Science ID 000259503400015

    View details for PubMedID 18812854

  • Presidential address: leadership, teamwork, and SUS brand extension SURGERY Longaker, M. T. 2008; 144 (2): 109-118

    View details for DOI 10.1016/j.surg.2008.03.002

    View details for Web of Science ID 000258308400001

    View details for PubMedID 18656615

  • Hepatic injury and the kinetics of bone marrow-derived hepatocyte transgene expression JOURNAL OF PEDIATRIC SURGERY Perryman, S. V., Jenkins, D. D., Streetz, K. L., Longaker, M. T., Sylvester, K. G. 2008; 43 (8): 1511-1519

    Abstract

    Numerous congenital and acquired liver diseases could benefit from a successful hepatic cell therapy strategy. Hepatotypic cells derived from bone marrow have been recognized during liver injury, repair, and regeneration. To study this phenomenon, we compared the effect of several modes of experimental hepatic injury on hepatotypic protein expression in a mouse model after bone marrow transplantation.Male mice transgenic for the liver-specific protein human alpha-1 antitrypsin (hAAT) were used as bone marrow donors. Syngeneic wild-type recipient mice were subjected to 1 of 3 hepatic injuries: (1) sublethal irradiation, (2) injection of a hepatotoxic adenoviral construct, and (3) administration of a hepatotoxic diet. Bone marrow-derived hepatotypic (BMdH) transgene expression was determined by serial serum enzyme-linked immunosorbent assay for hAAT.In both acute injury models, hAAT expression was detected as early as 1 week, whereas the control group never elicited hAAT expression. The adenovirus-treated group demonstrated transient hAAT level expression lasting up to 2 weeks postinjury, whereas the irradiated group maintained persistent hAAT expression through 4 months. In the chronic injury (hepatotoxin) model, hAAT expression persisted and was noted to increase over time to 200 to 300 ng/mL.Irradiation favors long-term establishment of BMdH transgene expression, and chronic injury further promotes this phenomenon.

    View details for DOI 10.1016/j.jpedsurg.2007.12.047

    View details for Web of Science ID 000258592500016

    View details for PubMedID 18675644

  • Fluid shear stress magnitude, duration, and total applied load regulate gene expression and nitric oxide production in primary calvarial osteoblast cultures PLASTIC AND RECONSTRUCTIVE SURGERY Gonzalez, O., Fong, K. D., Trindade, M. C., Warren, S. M., Longaker, M. T., Smith, R. L. 2008; 122 (2): 419-428

    Abstract

    Successful bone engineering requires an understanding of the effects of mechanical stress on osteoblast differentiation. Therefore, we examined the effects of varying magnitude and duration of fluid shear stress on factors associated with osteoblastic differentiation.Using a cone viscometer, primary neonatal rat calvarial osteoblasts were exposed to continuous fluid shear stress at varying doses: 0.21, 0.43, and 0.85 Pa for varying time periods. Gene expression was analyzed using Northern blots and nitric oxide production was quantified with the colorimetric Griess reaction.Fluid shear stress stimulated comparable transient increases in TGF-beta1 and TGF-beta3 expression by 3 hours. TGF-beta1 expression returned to baseline by 12 hours at all shear doses. In contrast, TGF-beta3 expression decreased by 22 percent and 47 percent at 12 hours in response to 0.43 Pa and 0.85 Pa, respectively. Osteopontin and Msx-2 expression patterns were consistent with a more differentiated phenotype at all shear levels. The maximum level of shear stress increased nitric oxide production 2.5-fold at 12 hours and 6.0-fold at 24 hours.These data demonstrate differential regulation of TGF-beta1 and TGF-beta3 isoforms with fluid shear stress. Furthermore, because osteopontin and Msx-2 changes were consistent with progressive differentiation at all levels of shear stress, dosage appears to be less important than the presence of an effective physical stimulus. Lastly, nitric oxide does not appear to be the primary regulator of early transcriptional changes found in this study.

    View details for DOI 10.1097/PRS.0b013e31817d5ff1

    View details for Web of Science ID 000258136900011

    View details for PubMedID 18626357

  • Microarray analysis of the role of regional dura mater in cranial suture fate PLASTIC AND RECONSTRUCTIVE SURGERY Kwan, M. D., Wan, D. C., Wang, Z., Gupta, D. M., Slater, B. J., Longaker, M. T. 2008; 122 (2): 389-399

    Abstract

    Craniosynostosis, the premature fusion of cranial sutures, results in serious neurologic and morphologic abnormalities when left untreated. Surgical excision of the fused sutures and remodeling of the skull remains the standard therapy. Development of novel, minimally invasive therapies for craniosynostosis will undoubtedly be dependent on a more thorough understanding of the molecular mechanisms underlying this abnormality. Significant evidence suggests the influence of regional dura mater on the behavior of the overlying suture complex. The mouse model has been instrumental in investigating this observation because of the natural juxtaposition of the posterior frontal suture, which fuses early in life, with the other cranial sutures, which remain patent.The authors used microarray analysis to compare genomic changes in the dura mater underlying the posterior frontal and sagittal sutures of mice. Suture-associated dura mater was harvested from mice before (postnatal day 5), during (postnatal day 10), and after (postnatal day 20) posterior frontal suture fusion (n = 20 mice for each of the three time points).Microarray results confirmed differential regulation of genes involved in paracrine signaling, extracellular matrix, and bone remodeling between the dura mater underlying the fusing posterior frontal suture and the patent sagittal suture.These data confirm global differences in gene expression between regional dura mater underlying fusing and patent sutures. These results provide further insight into potential molecular mechanisms that may play a role in cranial suture biology.

    View details for DOI 10.1097/PRS.0b013e31817d6244

    View details for Web of Science ID 000258136900008

    View details for PubMedID 18626354

  • Regenerative medicine: The next frontier TRANSPLANTATION Kwan, M. D., Longaker, M. T. 2008; 86 (2): 206-207

    View details for DOI 10.1097/TP.0b013e31817f17b2

    View details for Web of Science ID 000257887500006

    View details for PubMedID 18645480

  • Proliferation, osteogenic differentiation, and FGF-2 modulation of posterofrontal/sagittal suture-derived mesenchymal cells in vitro PLASTIC AND RECONSTRUCTIVE SURGERY James, A. W., Xu, Y., Wang, R., Longaker, M. T. 2008; 122 (1): 53-63

    Abstract

    Fibroblast growth factor (FGF) signaling is of central importance in premature cranial suture fusion. In the murine skull, the posterofrontal suture normally fuses in early postnatal life, whereas the adjacent sagittal suture remains patent. The authors used a recently developed isolation technique for in vitro culture of suture-derived mesenchymal cells to examine the effects of FGF-2 on proliferation and differentiation of posterofrontal and sagittal suture-derived mesenchymal cells.Skulls were harvested from 40 mice (5-day-old). Posterofrontal and sagittal sutures were dissected, separating sutural mesenchymal tissue from dura mater and pericranium, and cultured. After cell migration from the explant and subculture, differences in proliferation and osteogenic differentiation of these distinct populations were studied. The mitogenic and osteogenic effects of recombinant FGF-2 were then assessed. FGF-2 regulation of gene expression was evaluated.Suture-derived mesenchymal cells isolated from the posterofrontal suture demonstrated significantly higher proliferation rates and a robust mitogenic response to FGF-2 as compared with suture-derived mesenchymal cells isolated from the sagittal suture. Interestingly, posterofrontal suture-derived mesenchymal cells retained a higher in vitro osteogenic potential, as shown by alkaline phosphatase activity and bone nodule formation. FGF-2 significantly diminished osteogenesis in both suture-derived mesenchymal cell populations. Subsequently, Ob-cadherin and Sox9 were found to be differentially expressed in posterofrontal versus sagittal suture-derived mesenchymal cells and dynamically regulated by FGF-2.In vitro osteogenesis of suture-derived mesenchymal cells recapitulates in vivo posterofrontal and sagittal sutural fates. Posterofrontal rather than sagittal suture-derived mesenchymal cells are more responsive to FGF-2 in vitro, in terms of both mitogenesis and osteogenesis.

    View details for DOI 10.1097/PRS.0b013e31817747b5

    View details for Web of Science ID 000257104300006

    View details for PubMedID 18594386

  • Mesenchymal cells for skeletal tissue engineering EXPERT OPINION ON BIOLOGICAL THERAPY Slater, B. J., Kwan, M. D., Gupta, D. M., Panetta, N. J., Longaker, M. 2008; 8 (7): 885-893

    Abstract

    Skeletal defects represent a significant socioeconomic burden to the US healthcare system. Current options for reconstructing osseous deficits have shortcomings.To review the use of mesenchymal stem cells for skeletal tissue engineering.We focused on the application of mesenchymal cells in skeletal regeneration, optimization of this technique, tropic effects of multipotent mesenchymal cells, and future directions.A number of cell-based modalities have been investigated. We have been interested in the role of adipose-derived stromal cells in bone regeneration and understanding the mechanisms behind osteogenic differentiation of progenitor cells and acceleration of this process. Future clinical applications of multipotent mesenchymal cells will depend on better understanding of the molecular signaling involved in osteogenic differentiation and maintaining pluripotency.

    View details for DOI 10.1517/14712590802120981

    View details for Web of Science ID 000257408000004

    View details for PubMedID 18549320

  • Dissecting the influence of regional dura mater on cranial suture biology PLASTIC AND RECONSTRUCTIVE SURGERY Slater, B. J., Kwan, M. D., Gupta, D. M., Amasha, R. R., Wan, D. C., Longaker, M. T. 2008; 122 (1): 77-84

    Abstract

    Craniosynostosis is a relatively common developmental disorder that leads to a number of serious consequences. Previous studies have shown the influence of dura mater on the overlying cranial suture. This study was conducted to determine the role of regional dura mater versus the intrinsic nature of the suture in directing the overlying suture's fate.The authors examined the effect of regional dura mater on the fate and morphology of the posterofrontal and coronal sutures. In 8-day-old Sprague-Dawley rats, calvarial disks, consisting of the posterofrontal and coronal sutures, were excised and placed in one of three positions: (1) native position (control group), (2) rotated 45 degrees, or (3) rotated 90 degrees (n = 5 animals per group). The animals were euthanized 1 month postoperatively, and the sutures were analyzed histologically.The control group revealed normal suture morphology (n = 5). In the 45-degree rotation group, which placed the posterofrontal and coronal sutures over non-suture-associated dura mater, the posterofrontal sutures fused with thin morphology, and the coronal sutures remained patent (n = 5). In the 90-degree rotation group, the posterofrontal sutures, which were positioned over coronal suture-associated dura mater, were found to be fused with thinner morphology. The coronal sutures of the 90-degree rotation group, which were placed over posterofrontal suture-associated dura mater, remained patent but had acquired a posterofrontal-like morphology (n = 5).This study further elucidates variations in the biology of dura mater, depending on its location. Furthermore, these results illustrate the interplay between regional dura mater and the inherent characteristics of the suture complex in determining suture biology.

    View details for DOI 10.1097/PRS.0b013e318177478c

    View details for Web of Science ID 000257104300008

    View details for PubMedID 18594389

  • Advances in science and technology: Impact on craniofacial surgery JOURNAL OF CRANIOFACIAL SURGERY Kwan, M. D., Longaker, M. T. 2008; 19 (4): 1136-1139

    View details for Web of Science ID 000257921300049

    View details for PubMedID 18650748

  • Molecular mechanisms of FGF-2 inhibitory activity in the osteogenic context of mouse adipose-derived stem cells (mASCs) BONE Quarto, N., Wan, D. C., Longaker, M. T. 2008; 42 (6): 1040-1052

    Abstract

    Adipose-derived adult stem cells (ASCs), like their bone-marrow derived counterparts, possess the ability to differentiate down osteogenic, chondrogenic, adipogenic, and myogenic pathways. For bone differentiation of mouse ASCs (mASCs), retinoic-acid mediated upregulation of BMPR-IB has been found to be necessary. Interestingly, our previous work has also shown Fibroblast Growth Factor-2 (FGF-2) to strongly inhibit this osteogenic differentiation, even in the presence of retinoic acid. In this report, we investigated the molecular mechanisms underlying FGF-2 mediated osteogenic inhibition, demonstrating that addition of exogenous FGF-2 to mASCs antagonizes upregulation of BMPR-IB gene expression in response to retinoic acid. In addition, constitutive expression of BMPR-IB, but not BMPR-IA or BMPR-II, was found to counteract the inhibitory effects of FGF-2. Finally, p53(-/-) mASCs and human ASCs, both of which express high levels of endogenous BMPR-IB, underwent normal osteogenic differentiation even in the presence of FGF-2. Collectively, our data therefore indicate that FGF-2 antagonizes the response of mASCs to retinoic acid and also suggest that threshold levels of BMPR-IB may play a crucial role both in counteracting the inhibitory role of FGF-2 and in promoting osteogenic differentiation of ASCs in the absence of retinoic acid. Moreover, the present study also indicates that differences exist between mouse and human ASCs in relationship to FGF-2 activity in the osteogenic context.

    View details for DOI 10.1016/j.bone.2008.01.026

    View details for Web of Science ID 000256330000006

    View details for PubMedID 18420480

  • Wound repair and regeneration NATURE Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. 2008; 453 (7193): 314-321

    Abstract

    The repair of wounds is one of the most complex biological processes that occur during human life. After an injury, multiple biological pathways immediately become activated and are synchronized to respond. In human adults, the wound repair process commonly leads to a non-functioning mass of fibrotic tissue known as a scar. By contrast, early in gestation, injured fetal tissues can be completely recreated, without fibrosis, in a process resembling regeneration. Some organisms, however, retain the ability to regenerate tissue throughout adult life. Knowledge gained from studying such organisms might help to unlock latent regenerative pathways in humans, which would change medical practice as much as the introduction of antibiotics did in the twentieth century.

    View details for DOI 10.1038/nature07039

    View details for Web of Science ID 000255868400038

    View details for PubMedID 18480812

  • Tissue engineering in cleft palate and other congenital malformations PEDIATRIC RESEARCH Panetta, N. J., Gupta, D. M., Slater, B. J., Kwan, M. D., Liu, K. J., Longaker, M. T. 2008; 63 (5): 545-551

    Abstract

    Contributions from multidisciplinary investigations have focused attention on the potential of tissue engineering to yield novel therapeutics. Congenital malformations, including cleft palate, craniosynostosis, and craniofacial skeletal hypoplasias represent excellent targets for the implementation of tissue engineering applications secondary to the technically challenging nature and inherent inadequacies of current reconstructive interventions. Apropos to the search for answers to these clinical conundrums, studies have focused on elucidating the molecular signals driving the biologic activity of the aforementioned maladies. These investigations have highlighted multiple signaling pathways, including Wnt, fibroblast growth factor, transforming growth factor-beta, and bone morphogenetic proteins, that have been found to play critical roles in guided tissue development. Furthermore, a comprehensive knowledge of these pathways will be of utmost importance to the optimization of future cell-based tissue engineering strategies. The scope of this review encompasses a discussion of the molecular biology involved in the development of cleft palate and craniosynostosis. In addition, we include a discussion of craniofacial distraction osteogenesis and how its applied forces influence cell signaling to guide endogenous bone regeneration. Finally, this review discusses the future role of cell-based tissue engineering in the treatment of congenital malformations.

    View details for Web of Science ID 000255311900014

    View details for PubMedID 18427300

  • Increased rate of hair regrowth in mice with constitutive overexpression of Del 1 JOURNAL OF SURGICAL RESEARCH Hsu, G. P., Mathy, J. A., Wang, Z., Xia, W., Sakamoto, G., Kundu, R., Longaker, M. T., Quertermous, T., Yang, G. P. 2008; 146 (1): 73-80

    Abstract

    Developmental endothelial locus (Del)1 is a secreted extracellular matrix-associated protein that stimulates angiogenesis through integrin binding and is implicated in vasculogenesis. We hypothesized that increased expression of an angiogenic factor would lead to enhanced wound healing.Transgenic mice had Del1 cloned behind a keratin 14 promoter (K14-Del1) to drive constitutive expression in basal keratinocytes. Transgenic animals and wild-type litter mates underwent excisional wounding or depilation, and tissues were harvested at various time points. Wound healing and hair regrowth were assessed by photography, histology, and immunohistochemistry. For injection experiments, purified Del1 protein was injected in the flanks of wild-type mice with carrier on the contralateral flank as a control. Del1 expression during hair development was performed using transgenic mice with a LacZ cassette introduced downstream from the native promoter.K14-Del1 animals appeared normal and healed excisional wounds normally but demonstrated an increased rate of hair regrowth after wound healing. Using depilation experiments to specifically address hair follicle growth, we found increased hair regrowth was independent of wounding. This was confirmed by injection of purified Del1 protein. During normal hair anagenesis, Del1 is expressed in the root of the hair follicle.Constitutive expression of Del1 in skin does not affect skin vascularity or improve wound healing. Surprisingly, we found the primary effect of constitutive Del1 expression in the basal keratinocytes was increased hair growth following induction of anagenesis. During normal hair anagenesis, we see expression of Del1 in the root of the hair follicle suggesting it may function there to stimulate hair growth.

    View details for DOI 10.1016/j.jss.2007.02.024

    View details for Web of Science ID 000254798700011

    View details for PubMedID 17764695

  • Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium ANNALS OF BIOMEDICAL ENGINEERING Wagner, D. R., Lindsey, D. P., Li, K. W., Tummala, P., Chandran, S. E., Smith, R. L., Longaker, M. T., Carter, D. R., Beaupre, G. S. 2008; 36 (5): 813-820

    Abstract

    This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

    View details for DOI 10.1007/s10439-008-9448-5

    View details for Web of Science ID 000254755800013

    View details for PubMedID 18266109

  • Silva et al: Repair of cranial bone defects with calcium phosphate ceramic implant or autogenous bone graft. journal of craniofacial surgery Amasha, R. R., Kwan, M. D., Longaker, M. T. 2008; 19 (3): 675-677

    View details for DOI 10.1097/SCS.0b013e31815d063b

    View details for PubMedID 18520382

  • Blood-derived small Dot cells reduce scar in wound healing EXPERIMENTAL CELL RESEARCH Kong, W., Li, S., Longaker, M. T., Lorenz, H. P. 2008; 314 (7): 1529-1539

    Abstract

    Wounds in fetal skin heal without scar, however the mechanism is unknown. We identified a novel group of E-cadherin positive cells in the blood of fetal and adult mice and named them "Dot cells". The percentage of Dot cells in E16.5 fetal mice blood is more than twenty times higher compared to adult blood. Dot cells also express integrin beta1, CD184, CD34, CD13low and Sca1low, but not CD45, CD44, and CD117. Dot cells have a tiny dot shape between 1 and 7 microm diameters with fast proliferation in vitro. Most of the Dot cells remain positive for E-cadherin and integrin beta1 after one month in culture. Transplantation of Dot cells to adult mice heals skin wounds with less scar due to reduced smooth muscle actin and collagen expression in the repair tissue. Tracking GFP-positive Dot cells demonstrates that Dot cells migrate to wounds and differentiate into dermal cells, which also express strongly to FGF-2, and later lose their GFP expression. Our results indicate that Dot cells are a group of previously unidentified cells that have strong wound healing effect. The mechanism of scarless wound healing in fetal skin is due to the presence of a large number of Dot cells.

    View details for DOI 10.1016/j.yexcr.2008.01.022

    View details for Web of Science ID 000254798600010

    View details for PubMedID 18295204

  • Cell-based therapies for skeletal regenerative medicine HUMAN MOLECULAR GENETICS Kwan, M. D., Slater, B. J., Wan, D. C., Longaker, M. T. 2008; 17: R93-R98

    Abstract

    Skeletal deficits represent a substantial biomedical burden on the US healthcare system. Current strategies for reconstructing bony defects are fraught with inadequacies. Cell-based therapies for skeletal regeneration offer a paradigm shift that may provide alternative solutions. Substantial work has identified a host of cellular sources that possess the potential for osteogenic differentiation. Significant efforts have been devoted toward characterizing the role of postnatal cellular sources that are relatively abundant and easily accessible. Among these, the potential of using adipose-derived stromal cells for skeletal regeneration has garnered much interest. Integral to these efforts directed at characterizing cellular sources are studies that seek to understand the factors that initiate and regulate osteogenic differentiation of progenitor cells. Specifically, focus has been directed on elucidating the role of bone morphogenetic protein and fibroblast growth factor signaling in regulating osteogenic differentiation of osteoprogenitor cells. Concurrent studies in the field of scaffold design have also helped to advance the potential for cell-based therapies.

    View details for DOI 10.1093/hmg/ddn071

    View details for Web of Science ID 000258261600015

    View details for PubMedID 18632703

  • Current progress in keloid research and treatment JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS Butler, P. D., Longaker, M. T., Yang, G. P. 2008; 206 (4): 731-741
  • Cranial Sutures: A Brief Review PLASTIC AND RECONSTRUCTIVE SURGERY Slater, B. J., Lenton, K. A., Kwan, M. D., Gupta, D. M., Wan, D. C., Longaker, M. T. 2008; 121 (4): 170E-178E

    Abstract

    Craniosynostosis, or the premature fusion of one or more cranial sutures, is a relatively common congenital defect that causes a number of morphologic and functional abnormalities. With advances in genetics and molecular biology, research of craniosynostosis has progressed from describing gross abnormalities to understanding the molecular interactions that underlie these cranial deformities. Animal models have been extremely valuable in improving our comprehension of human craniofacial morphogenesis, primarily by human genetic linkage analysis and the development of knock-out animals. This article provides a brief review of perisutural tissue interactions, embryonic origins, signaling molecules and their receptors, and transcription factors in maintaining the delicate balance between proliferation and differentiation of cells within the suture complex that determines suture fate. Finally, this article discusses the potential implications for developing novel therapies for craniosynostosis.

    View details for DOI 10.1097/01.prs.0000304441.99483.97

    View details for Web of Science ID 000207666900003

    View details for PubMedID 18349596

  • Use of organotypic coculture to study keloid biology AMERICAN JOURNAL OF SURGERY Butler, P. D., Ly, D. P., Longaker, M. T., Yang, G. P. 2008; 195 (2): 144-148

    Abstract

    Keloids are pathologic scars afflicting a large segment of our population and for which there is no definitive therapy. The lack of an animal model for keloid formation has hampered study. We developed an in vitro organotypic skin model to simulate normal keloid biology, which may allow us to study keloid formation without an animal model.Normal (NFs) and keloid (KFs) human fibroblasts were cultured in a collagen matrix to create a 3-dimensional dermal structure. Normal human keratinocytes (NKs) were cultured as a second layer on top and exposed to an air-fluid interface to allow differentiation into a mature keratinocyte layer. The organotypic skin was maintained for 28 days in Dulbecco's modified eagle medium with 10% fetal calf serum. Samples were collected, processed, sectioned, stained with hematoxylin and eosin, and then measured for qualitative analysis. alpha-smooth-muscle actin was also evaluated by immunoblotting.KF/NK organotypic skin showed increased collagen deposition, based on significantly denser collagen staining, with increased dermal thickness compared with NF/NK organotypic skin. We saw increased contracture in the KF/NK construct, and this correlated with increased organization of alpha-smooth-muscle actin fibers in the dermal layer of KF/NK organotypic skin compared with NF/NK skin.We have shown that coculture of KFs with keloid keratinocytes leads to an increased collagen production and dermal contracture compared with NFs and NKs, consistent with known keloid behavior. Given the lack of an animal model, we believe that organotypic skin culture can serve as a surrogate to study keloid formation.

    View details for DOI 10.1016/j.amjsurg.2007.10.003

    View details for Web of Science ID 000252598400002

    View details for PubMedID 18070722

  • Periosteal biaxial residual strains correlate with bone specific growth rates in chick embryos COMPUTER METHODS IN BIOMECHANICS AND BIOMEDICAL ENGINEERING Chen, J. C., Zhao, B., Longaker, M. T., Helms, J. A., Carter, D. R. 2008; 11 (5): 453-461

    Abstract

    It has been proposed that periosteal residual tensile strains influence periosteal bone apposition and endochondral ossification. The role of bone growth rates on the development of residual strains is not well known. This study examined the relationships between specific growth rate and residual strains in chick tibiotarsi. We measured length and circumference during embryonic days 11-20 using microCT. Bones grew faster in length, with longitudinal and circumferential specific growth rates decreasing from 17 to 9% and 14 to 8% per day, respectively. To calculate residual strains, opening dimensions of incisions through the periosteum were analysed using finite element techniques. Results indicate that Poisson's ratio for an isotropic material model is between 0 and 0.04. For the model with Poisson's ratio 0.03, longitudinal and circumferential residual strains decreased from 46.2 to 29.3% and 10.6 to 3.9%, respectively, during embryonic days 14-20. Specific growth rates and residual strains were positively correlated (p<0.05).

    View details for DOI 10.1080/10255840802129817

    View details for Web of Science ID 000260457900004

    View details for PubMedID 18608339

  • Confocal laser scanning microscopic analysis of collagen scaffolding patterns in cranial sutures JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Walder, B., Dec, W., Longaker, M. T., Ting, K. 2008; 19 (1): 198-203

    Abstract

    Although recent studies indicate that regional dura mater influences the fate of the overlying cranial suture, little is known about the assembly of extracellular matrix (ECM) molecules within the patent and fusing murine cranial suture complexes. Confocal laser scanning microscopy was used to study ECM assembly within patent and fusing cranial suture complexes. Coronal sections (20 microm thick) of patent sagittal (SAG) and fusing posterior frontal (PF) sutures from postnatal 8-, 14-, and 18-day-old Sprague-Dawley rats were scanned in 0.5-microm increments, and images were collected consecutively to create a z-series for three-dimensional reconstruction. Spatial and temporal collagen arrangements were compared between SAG and PF sutures by measuring interfiber distance, fiber thickness, and total collagen surface area at each time point. We demonstrate that on day 8 (before the onset of suture fusion), collagen bundles are randomly arranged in both the SAG and PF sutures. By day 14 (midfusion period), there was a statistically significant reduction in total collagen surface area (80.5% versus 67.4%; P < 0.05) as the collagen bundles were organized into orthogonal lattices along the anterior and endocranial margins of the PF suture. Furthermore, new bone matrix deposition was observed along the edges of these organized collagen bundles. In contrast, collagen within the SAG suture remained randomly arranged and unossified. By day 18 (late fusion period), the PF suture was completely fused except for the posterior-ectocranial portion. This patent section of the PF suture contained a highly organized mineralizing orthogonal collagen lattice. The total collagen surface area in the day-18 PF suture continued to decline compared with the day-8 PF suture (80.5% versus 55.6%; P < 0.05). In the day-18 SAG suture, the collagen bundles remained randomly arranged, and the total surface area did not change. The same analysis was performed in a human pathologic fusing and patent suture. Similar results were observed. The total collagen surface area significantly decreased in the pathologic fusing human suture compared with the patent suture (92.8% versus 60.6%; P < 0.05). Moreover, the pathologically fusing suture contained a highly organized mineralizing orthogonal collagen lattice. This is the first analysis of collagen patterns in patent and fusing cranial sutures.

    View details for Web of Science ID 000252619900033

    View details for PubMedID 18216689

  • Applications of an athymic nude mouse model of nonhealing critical-sized calvarial defects JOURNAL OF CRANIOFACIAL SURGERY Gupta, D. M., Kwan, M. D., Slater, B. J., Wan, D. C., Longaker, M. T. 2008; 19 (1): 192-197

    Abstract

    Calvarial bone defects are a common clinical scenario in craniofacial surgery. Numerous approaches are used to reconstruct skull defects, and each possesses its own inherent disadvantages. This fact underscores the opportunity to develop a novel method to repair osseous defects in craniofacial surgery. Recent literature strongly suggests that cell-based therapies in the form of regenerative medicine may be a developing paradigm in reconstructive surgery. Although numerous studies have probed osteoprogenitor cells from mice, few have explored the biology of human cells in the setting of osteogenesis in an equally rigorous manner. This study proposes a nude mouse model of critical-sized calvarial defects to study the in vivo biology of human osteoprogenitor cells. Critical-sized 4.0-mm calvarial defects were created in nude mice (n = 15) with a custom trephine drill bit outfitted to a dental drill handpiece. During the craniotomy, the dura mater was spared from injury. Gross inspection, routine histology, and micro-computed tomographic scanning were performed at 2, 4, 8, and 16 weeks postoperatively. There was no calvarial healing in any of the animals by 16 weeks. The dura mater remained intact in all subjects. Gross, histologic, and radiographic assays confirmed these findings. Although several studies have implanted human osteoprogenitor cells in vivo in various animal models, few have documented the appropriate controls or conditions necessary to support the potential to translate benchtop findings into clinical applications. We propose in this study that the nude mouse critical-sized calvarial defect model will be valuable with increasing investigations with human osteoprogenitor cells.

    View details for Web of Science ID 000252619900032

    View details for PubMedID 18216688

  • Current treatment of craniosynostosis and future therapeutic directions. Frontiers of oral biology Wan, D. C., Kwan, M. D., Lorenz, H. P., Longaker, M. T. 2008; 12: 209-230

    Abstract

    Normal craniofacial development is contingent upon coordinated growth between the brain and overlying calvaria. Craniosynostosis, the premature fusion of one or more cranial sutures, perturbs this natural framework, resulting in dramatic dysmorphology of the skull and face along with a multitude of associated functional abnormalities. Traditional approaches to the treatment of craniosynostosis have employed complex surgical remodeling of the skull vault and facial deformities all aimed at increasing the amount of intracranial volume and restoring a more normal craniofacial appearance. Significant morbidity and mortality, however, have plagued these procedures, driving dramatic evolution in our approach towards the treatment of pathologically fused sutures. Recent clinical and genetic studies have identified multiple forms of human craniosynostosis, each associated with mutations within various cytokine signaling pathways. Knowledge garnered from these investigations bear promise for the future development of alternative strategies to enhance or perhaps even replace contemporary approaches for the treatment of craniosynostosis.

    View details for DOI 10.1159/0000115043

    View details for PubMedID 18391503

  • Dura mater-derived FGF-2 mediates mitogenic signaling in calvarial osteoblasts AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Li, S., Quarto, N., Longaker, M. T. 2007; 293 (6): C1834-C1842

    Abstract

    Although dura mater tissue is believed to have an important role in calvarial reossification in many in vivo studies, few studies have shown the direct effect of dura mater cells on osteoblasts. In addition, no reports have yet identified the potential factor(s) responsible for various biological activities exerted by dura mater on calvarial reossification (e.g., cell proliferation). In this study, we tested the effect of dura mater on calvarial-derived osteoblasts by performing both heterotypic coculture and by culturing osteoblast cells with conditioned media harvested from dura mater cells of juvenile (3-day-old) and adult (30-day-old) mice. The results presented here demonstrate that cellular proliferation of juvenile osteoblast cells was significantly increased by juvenile dura mater either in the coculture system or when dura mater cell-conditioned medium was applied to the osteoblast cells. Moreover, high levels of FGF-2 protein were detected in juvenile dura mater cells and their conditioned medium. In contrast, low levels of FGF-2 protein were detected in adult dura mater cells, whereas FGF-2 protein was not detectable in their conditioned medium. Abrogation of the mitogenic effect induced by juvenile dura mater cell-conditioned medium was achieved by introducing a neutralizing anti-FGF-2 antibody, thus indicating that FGF-2 may be responsible for the mitogenic effect of the juvenile dura mater. Moreover, data obtained by exploring the three major FGF-2 signaling pathways further reinforced the idea that FGF-2 might be an important paracrine signaling factor in vivo supplied by the underlying dura mater to the overlying calvarial osteoblasts.

    View details for DOI 10.1152/ajpcell.00135.2007

    View details for Web of Science ID 000251562200013

    View details for PubMedID 17913846

  • In vitro expansion of adipose-derived adult stromal cells in hypoxia enhances early chondrogenesis TISSUE ENGINEERING Xu, Y., Malladi, P., Chiou, M., Bekerman, E., Giaccia, A. J., Longaker, M. T. 2007; 13 (12): 2981-2993

    Abstract

    Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.

    View details for DOI 10.1089/ten.2007.0050

    View details for Web of Science ID 000251788400018

    View details for PubMedID 17916040

  • Molecular and cellular characterization of mouse calvarial osteoblasts derived from neural crest and paraxial mesoderm PLASTIC AND RECONSTRUCTIVE SURGERY Xu, Y., Malladi, P., Zhou, D., Longaker, M. T. 2007; 120 (7): 1783-1795

    Abstract

    Cranial skeletogenic mesenchyme is derived from two distinct embryonic sources: mesoderm and cranial neural crest. Previous studies have focused on molecular and cellular differences of juvenile and adult osteoblasts.To further understand the features of mouse-derived juvenile osteoblasts, the authors separated calvarial osteoblasts by their developmental origins: frontal bone-derived osteoblasts from cranial neural crest, and parietal bone-derived osteoblasts from paraxial mesoderm. Cells were harvested from a total of 120 mice.Interestingly, the authors observed distinct morphologies and proliferation potential of the two populations of osteoblasts. Osteogenic genes such as alkaline phosphatase, osteopontin, collagen I, and Wnt5a, which was recently identified as playing a role in skeletogenesis, were abundantly expressed in parietal bone-derived osteoblasts versus frontal bone-derived osteoblasts. In addition, fibroblast growth factor (FGF) receptor 2, and FGF-18 were more highly expressed in the parietal bone-derived osteoblasts, suggesting a more differentiated phenotype. In contrast, FGF-2, and adhesion molecules osteoblast cadherins and bone morphogenetic protein receptor IB, the bone tissue-specific type receptor were overexpressed in frontal bone-derived osteoblasts compared with parietal bone-derived osteoblasts.The authors observed that although neural crest-derived osteoblasts represented a population of less differentiated, faster growing cells, they formed bone nodules more rapidly than parietal bone-derived osteoblasts. This in vitro study suggests that embryonic tissue derivations influence postnatal in vitro calvarial osteoblast cell biology.

    View details for DOI 10.1097/01.prs.0000279491.48283.51

    View details for Web of Science ID 000251668400005

    View details for PubMedID 18090740

  • Increased CCN2 transcription in keloid fibroblasts requires cooperativity between AP-1 and SMAD binding sites ANNALS OF SURGERY Xia, W., Kong, W., Wang, Z., Phan, T., Lim, I. J., Longaker, M. T., Yang, G. P. 2007; 246 (5): 886-895

    Abstract

    We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth.Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal.Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers.Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun.We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.

    View details for DOI 10.1097/SLA.0b013e318070d54f

    View details for Web of Science ID 000250773400028

    View details for PubMedID 17968183

  • Mechanical load initiates hypertrophic scar formation through decreased cellular apoptosis FASEB JOURNAL Aarabi, S., Bhatt, K. A., Shi, Y., Paterno, J., Chang, E. I., Loh, S. A., Holmes, J. W., Longaker, M. T., Yee, H., Gurtner, G. C. 2007; 21 (12): 3250-3261

    Abstract

    Hypertrophic scars occur following cutaneous wounding and result in severe functional and esthetic defects. The pathophysiology of this process remains unknown. Here, we demonstrate for the first time that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. The resulting scars are histopathologically identical to human hypertrophic scars and persist for more than six months following a brief (one-week) period of augmented mechanical stress during the proliferative phase of wound healing. Resulting scars are structurally identical to human hypertrophic scars and showed dramatic increases in volume (20-fold) and cellular density (20-fold). The increased cellularity is accompanied by a four-fold decrease in cellular apoptosis and increased activation of the prosurvival marker Akt. To clarify the importance of apoptosis in hypertrophic scar formation, we examine the effects of mechanical loading on cutaneous wounds of animals with altered pathways of cellular apoptosis. In p53-null mice, with down-regulated cellular apoptosis, we observe significantly greater scar hypertrophy and cellular density. Conversely, scar hypertrophy and cellular density are significantly reduced in proapoptotic BclII-null mice. We conclude that mechanical loading early in the proliferative phase of wound healing produces hypertrophic scars by inhibiting cellular apoptosis through an Akt-dependent mechanism.

    View details for DOI 10.1096/fj.07-8218com

    View details for Web of Science ID 000249781600025

    View details for PubMedID 17504973

  • Noggin suppression enhances in vitro osteogenesis and accelerates in vivo bone formation JOURNAL OF BIOLOGICAL CHEMISTRY Wan, D. C., Pomerantz, J. H., Brunet, L. J., Kim, J., Chou, Y., Wu, B. M., Harland, R., Blau, H. M., Longaker, M. T. 2007; 282 (36): 26450-26459

    Abstract

    Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.

    View details for DOI 10.1074/jbc.M703282200

    View details for Web of Science ID 000249239600050

    View details for PubMedID 17609215

  • Hypertrophic scar formation following burns and trauma: New approaches to treatment PLOS MEDICINE Aarabi, S., Longaker, M. T., Gurtner, G. C. 2007; 4 (9): 1464-1470

    View details for DOI 10.1371/journal.pmed.0040234

    View details for Web of Science ID 000249768100011

    View details for PubMedID 17803351

  • Live imaging of Smad2/3 signaling in mouse skin wound healing WOUND REPAIR AND REGENERATION Chong, A. K., Satterwhite, T., Pham, H. M., Costa, M. A., Luo, J., Longaker, M. T., Wyss-Coray, T., Chang, J. 2007; 15 (5): 762-766

    Abstract

    Biophotonics and real-time imaging are novel technologies that can greatly enhance the study of complex biological processes. We applied this technology in a transgenic mouse with a luciferase reporter gene fused to a transforming growth factor-beta (TGF-beta) responsive Smad2/3-binding element to study bioluminescence after skin wounding. Two dorsal midline excisional skin wounds were made using a biopsy punch. One wound was randomized to suture closure and the other allowed to heal by secondary intention (n=8 each wound). Bioluminescence was measured at fixed time points following surgery. Phospho-Smad2/3 immunohistochemistry was performed to localize expression in skin wound samples. In vivo bioluminescence increased following skin wounding. Peak activity occurred on day 17 and was fourfold that of baseline (p<0.05). Subgroup analysis of primary and secondary healing showed that primarily sutured wounds had peak activities earlier than those with secondary healing, although this did not reach statistical significance. Intense phospho-Smad2/3 staining was found in the hair follicles. In vivo bioluminescence tracks Smad2/3-dependent TGF-beta signaling in the in vivo wound healing process. Our findings suggest that signaling increases after wound healing, which contrasts with other studies that show raised TGF-beta signaling in the initial days following wounding.

    View details for DOI 10.1111/j.1524-475X.2007.00299.x

    View details for Web of Science ID 000249846800019

    View details for PubMedID 17971023

  • In vitro analysis of transforming growth factor-beta 1 inhibition in novel Transgenic SBE-luciferase mice ANNALS OF PLASTIC SURGERY Satterwhite, T. S., Chong, A. K., Luo, J., Pham, H., Costa, M., Longaker, M. T., Wyss-Coray, T., Chang, J. 2007; 59 (2): 207-213

    Abstract

    Transforming growth factor beta1 (TGF-beta1) expression correlates with scarring. A novel transgenic mouse model with a Smad2/3-responsive luciferase reporter construct (SBE-luc) has been developed. We hypothesized that bioluminescence in SBE-luc dermal fibroblasts could be measured to assess TGF-beta1 inhibition.Cultured dermal fibroblasts from SBE-luc mice were treated simultaneously with TGF-beta1 and increasing doses of either neutralizing antibody to TGF-beta (NA-TGFbeta) or SB-431542, a novel TGF-beta receptor kinase inhibitor. Fibroblasts were measured for luciferase activity. SBE-luc fibroblasts underwent Western blot analysis for collagen type I production.TGF-beta1 produced maximal luciferase activity in SBE-luc fibroblasts at 0.1 ng/mL (P < 0.05). NA-TGFbeta and SB-431542 inhibited luciferase activity in a dose-dependent fashion, with complete inhibition achieved by 0.1 microg/mL and 1 microM, respectively (P < 0.05). NA-TGFbeta and SB-431542 inhibited collagen type I production.Our in vitro results provide validation for further in vivo real-time imaging studies using the SBE-luc mouse as a novel wound-healing model.

    View details for DOI 10.1097/01.sap.0000252732.25168.34

    View details for Web of Science ID 000248363400017

    View details for PubMedID 17667417

  • Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells (ASC) using an in vitro three-dimensional micromass culture system BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Xu, Y., Balooch, G., Chiou, M., Bekerman, E., Ritchie, R. O., Longaker, M. T. 2007; 359 (2): 311-316

    Abstract

    Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.

    View details for DOI 10.1016/j.bbrc.2007.05.098

    View details for Web of Science ID 000247494400020

    View details for PubMedID 17543281

  • Refining retinoic acid stimulation for osteogenic differentiation of murine adipose-derived adult stromal cells TISSUE ENGINEERING Wan, D. C., Siedhoff, M. T., Kwan, M. D., Nacamuli, R. P., Wu, B. M., Longaker, M. T. 2007; 13 (7): 1623-1631

    Abstract

    Murine adipose-derived adult stromal cells (ADAS) seeded onto appropriate scaffolds and pre-incubated with retinoic acid have been shown to generate in vivo bone rapidly. Prompt resorption ensues, however, as a result of osteoclastogenesis, likely secondary to retinoic acid carryover. In this study, we determined the effects of abbreviated retinoic acid exposure on ADAS osteogenic differentiation. Histological staining and gene expression analysis revealed that longer retinoic acid exposure resulted in better in vitro bone differentiation. However, significant osteogenesis was observed in ADAS after just 15 days of retinoic acid supplementation, suggesting that continual culture with retinoic acid is unnecessary for initiation of the osteogenic program. This was confirmed using ADAS pre-incubated in monolayer with an abbreviated 15 days of retinoic acid exposure before implantation into critical-sized calvarial defects. Similar rates of regeneration were observed between ADAS exposed to for 15 days or for a full 25-day course of retinoic acid before defect repair. Furthermore, by limiting retinoic acid exposure to ADAS in monolayer without scaffold, accelerated bone formation was observed without concomitant osteoclastic resorption. These data suggest that skeletal regeneration may be improved by modulating retinoic acid exposure before implantation, markedly accelerating the repair of bone defects using ADAS.

    View details for DOI 10.1089/ten.2006.0283

    View details for Web of Science ID 000248035500022

    View details for PubMedID 17518707

  • Accelerated bone repair after plasma laser corticotomies ANNALS OF SURGERY Leucht, P., Lam, K., Kim, J., Mackanos, M. A., Simanovskii, D. M., Longaker, M. T., Contag, C. H., Schwettman, H. A., Helms, J. A. 2007; 246 (1): 140-150

    Abstract

    To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal.Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing.Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling.Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery.In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.

    View details for DOI 10.1097/01.sla.0000258559.07435.b3

    View details for Web of Science ID 000247672300022

    View details for PubMedID 17592303

  • Hypoxia inducible factor-1 alpha deficiency affects chondrogenesis of adipose-derived adult stromal cells TISSUE ENGINEERING Malladi, P., Xu, Y., Chiou, M., Giaccia, A. J., Longaker, M. T. 2007; 13 (6): 1159-1171

    Abstract

    Increased cartilage-related disease, poor regeneration of cartilage tissue, and limited treatment options have led to intense research in tissue engineering of cartilage. Adipose-derived adult stromal cells (ADAS) are a promising cell source for skeletal tissue engineering; understanding ADAS cellular signaling and chondrogenesis will advance cell-based therapies in cartilage repair. Chondrocytes are unique-they are continuously challenged by a hypoxic microenvironment. Hypoxia inducible factor-1-alpha (HIF-1alpha), a critical mediator of a cell's response to hypoxia, plays a significant role in chondrocyte survival, growth arrest, and differentiation. By using an established in vitro 3-dimensional micromass system, we investigated the role of HIF-1alpha in chondrogenesis. Targeted deletion of HIF-1alpha in ADAS substantially inhibited the chondrogenic pathway specifically. In marked contrast, deletion of HIF-1alpha did not affect osteogenic differentiation but enhanced adipogenic differentiation. This study demonstrates the critical and specific interplay between HIF-1alpha and chondrogenesis in vitro.

    View details for DOI 10.1089/ten.2006.0265

    View details for Web of Science ID 000247237600003

    View details for PubMedID 17518738

  • Proposition 71 and CIRM - assessing the return on investment NATURE BIOTECHNOLOGY Longaker, M. T., Baker, L. C., Greely, H. T. 2007; 25 (5): 513-521

    Abstract

    Given that Californian voters authorized state coffers to sell $3 billion in bonds to fund the California Institute for Regenerative Medicine (CIRM) with the expectation of health and financial benefits, what benchmarks should be used to measure the initiative's success?

    View details for Web of Science ID 000246369400014

    View details for PubMedID 17483831

  • Keratinocytes modulate fetal and postnatal fibroblast transforming growth factor-beta and Smad expression in co-culture PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Yun, R., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2007; 119 (5): 1440-1445

    Abstract

    The mechanism of fetal scarless wound repair is poorly understood but is thought to involve unique characteristics and behavior patterns of the fetal dermal fibroblast. The authors hypothesized that keratinocytes may differentially modulate expression of key growth factors expressed during wound healing in fetal and postnatal fibroblasts.Murine E17 fetal (n = 12 animals) and newborn (n = 8 animals) fibroblasts were grown in isolation and co-culture with newborn keratinocytes (n = 12 animals). Quantitative real-time polymerase chain reaction was performed for transforming growth factor (TGF)-beta isoform, receptor, and signaling molecule (Smad) gene expression in each group under both conditions.At baseline, fetal fibroblasts have 1.8-fold greater TGF-beta3 expression than postnatal fibroblasts (p < 0.01). Keratinocytes induce a further increase of TGF-beta3 expression (p < 0.01) but decreased TGF-beta1, TGF-beta2, TGF-beta receptor (R)-I, and TGF-betaR-II expression in fetal fibroblasts. Keratinocytes also induce an increase in TGF-beta3 (p < 0.01) and a decrease TGF-beta2, TGF-betaR-I, and TGF-betaR-II expression in postnatal fibroblasts; however, TGF-beta1 expression is unchanged. Fetal fibroblasts have lower baseline expression of Smad3 and Smad4 than postnatal fibroblasts (p < 0.05). Keratinocytes decrease Smad3 and increase Smad7 expression in both fetal and postnatal fibroblasts (p < 0.01). In contrast, keratinocytes decrease Smad2 only in fetal fibroblasts (p < 0.05).Keratinocytes have an overall antifibrotic influence on both fetal and postnatal fibroblasts in co-culture conditions. These data further characterize intrinsic differences between fetal and postnatal fibroblasts.

    View details for DOI 10.1097/01.prs.0000256049.53562.39

    View details for Web of Science ID 000245711700007

    View details for PubMedID 17415238

  • Chemical rescue of cleft palate and midline defects in conditional GSK-3 beta mice NATURE Liu, K. J., Arron, J. R., Stankunas, K., Crabtree, G. R., Longaker, M. T. 2007; 446 (7131): 79-82

    Abstract

    Glycogen synthase kinase-3beta (GSK-3beta) has integral roles in a variety of biological processes, including development, diabetes, and the progression of Alzheimer's disease. As such, a thorough understanding of GSK-3beta function will have a broad impact on human biology and therapeutics. Because GSK-3beta interacts with many different pathways, its specific developmental roles remain unclear. We have discovered a genetic requirement for GSK-3beta in midline development. Homozygous null mice display cleft palate, incomplete fusion of the ribs at the midline and bifid sternum as well as delayed sternal ossification. Using a chemically regulated allele of GSK-3beta (ref. 6), we have defined requirements for GSK-3beta activity during discrete temporal windows in palatogenesis and skeletogenesis. The rapamycin-dependent allele of GSK-3beta produces GSK-3beta fused to a tag, FRB* (FKBP/rapamycin binding), resulting in a rapidly destabilized chimaeric protein. In the absence of drug, GSK-3beta(FRB)*(/FRB)* mutants appear phenotypically identical to GSK-3beta-/- mutants. In the presence of drug, GSK-3betaFRB* is rapidly stabilized, restoring protein levels and activity. Using this system, mutant phenotypes were rescued by restoring endogenous GSK-3beta activity during two distinct periods in gestation. This technology provides a powerful tool for defining windows of protein function during development.

    View details for DOI 10.1038/nature05557

    View details for Web of Science ID 000244525600041

    View details for PubMedID 17293880

  • Transforming growth factor-beta, smad, and collagen expression patterns in fetal and adult keratinocytes PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Faudoa, R., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2007; 119 (3): 852-857

    Abstract

    The transforming growth factor (TGF)-beta family regulates cellular proliferation, differentiation, and migration. To better define the influence of keratinocyte-derived TGF-beta during development and repair, the authors examined the TGF-beta isoform, receptor, signal messenger Smad, and collagen type I expression in fetal and postnatal keratinocytes.Sprague-Dawley rat keratinocytes were isolated in primary culture from fetal E17 (n = 6), newborn (n = 4), and 6-week-old adults (n = 4). Under serum-free conditions, quantitative polymerase chain reaction was performed for TGF-beta1, TGF-beta2, and TGF-beta3 ligands; TGF-beta receptor 1 (RI) and TGF-beta receptor 2 (RII); Smad4 and Smad7; and collagen type I expression.Total TGF-beta isoform expression increased 1.7-fold from E17 to newborn (p < 0.05) and adult (p < 0.01) ages. TGF-beta1 expression was 25-fold greater than TGF-beta2 and 10-fold greater than TGF-beta3 in fetal keratinocytes (p < 0.01 for each). The expression of TGF-beta1 was fivefold greater compared with TGF-beta2 and TGF-beta3 in newborn and adult keratinocytes (p < 0.01). TGF-beta-RI expression increased more than twofold (p < 0.01), whereas TGF-beta-RII expression increased by 25 percent (p < 0.01) from E17 to adult age. Smad4 increased more than twofold (p < 0.01), whereas Smad7 did not change appreciably. Collagen type I expression increased over 100-fold from E17 to adult (p < 0.005).The TGF-beta system and collagen type I have increased expression with increasing gestational age in keratinocytes. This suggests an increased profibrotic TGF-beta response and collagen type I production in keratinocytes during skin differentiation at ages associated with scarring.

    View details for DOI 10.1097/01.prs.0000255541.39993.66

    View details for Web of Science ID 000244438700011

    View details for PubMedID 17312487

  • Re: Differential effects of FGFR2 mutation in ophthalniologic findings in Apert syndrome. Discussion JOURNAL OF CRANIOFACIAL SURGERY Kwan, M. D., Wan, D. C., Lorenz, H. P., Longaker, M. T. 2007; 18 (2): 459-460

    View details for Web of Science ID 000245424400039

    View details for PubMedID 17414305

  • Isolation and characterization of posterofrontal/sagittal suture mesenchymal cells in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Xu, Y., Malladi, P., Chiou, M., Longaker, M. T. 2007; 119 (3): 819-829

    Abstract

    Craniosynostosis, the premature fusion of cranial sutures, affects one in 2500 children. In the mouse, the posterofrontal suture is programed to fuse postnatally, but the adjacent sagittal suture remains patent throughout life. To study the cellular process of suture fusion, the authors isolated and studied suture-derived mesenchymal cells.Skulls were harvested from 80 mice (2 to 5 days old), and posterofrontal and sagittal sutures were dissected meticulously. Suture mesenchymal tissue was separated from the underlying dura mater and overlying pericranium and cultured in growth media. After the cells migrated from the explant tissues, the morphologies of the two cell populations were studied carefully, and quantitative real-time polymerase chain reaction was performed to evaluate gene expression.Both posterofrontal and sagittal cells exhibited highly heterogeneous morphologies, and the posterofrontal cells migrated faster than the sagittal cells. Accordingly, growth factors such as transforming growth factor-beta1 and fibroblast growth factor (FGF)-2 were expressed significantly more highly in posterofrontal compared with sagittal suture mesenchymal cells. In contrast, FGF receptor 2 and FGF-18 were expressed significantly more in sagittal than in posterofrontal suture cells. Importantly, bone morphogenic protein-3, the only osteogenic inhibitor in the bone morphogenic protein family, and noggin, a bone morphogenic protein antagonist, were expressed significantly more in sagittal than in posterofrontal suture cells, suggesting a possible mechanism of suture patency.To the authors' knowledge, this is the first analysis of mouse suture-derived mesenchymal cells. The authors conclude that isolation of suture-derived mesenchymal cells will provide a useful in vitro system with which to study the mechanisms underlying suture biology.

    View details for DOI 10.1097/01.prs.0000255540.91987.a0

    View details for Web of Science ID 000244438700007

    View details for PubMedID 17312483

  • Progress and potential for regenerative medicine ANNUAL REVIEW OF MEDICINE Gurtner, G. C., Callaghan, M. J., Longaker, M. T. 2007; 58: 299-312

    Abstract

    Regenerative medicine focuses on new therapies to replace or restore lost, damaged, or aging cells in the human body to restore function. This goal is being realized by collaborative efforts in nonmammalian and human development, stem cell biology, genetics, materials science, bioengineering, and tissue engineering. At present, understanding existing reparative processes in humans and exploring the latent ability to regenerate tissue remains the focus in this field. This review covers recent work in limb regeneration, fetal wound healing, stem cell biology, somatic nuclear transfer, and tissue engineering as a foundation for developing new clinical therapies to augment and stimulate human regeneration.

    View details for DOI 10.1146/annurev.med.58.085405.095329

    View details for Web of Science ID 000244461500020

    View details for PubMedID 17076602

  • Cyclophilin C-associated protein is up-regulated during wound healing JOURNAL OF CELLULAR PHYSIOLOGY Kong, W., Li, S., Longaker, M. T., Lorenz, H. P. 2007; 210 (1): 153-160

    Abstract

    Cyclophilin C-associated protein (CyCAP) is identified from macrophages. It locates in intracellular, membrane bound and extracellular, suggesting it has an important role, however both of its regulation and function have not been elucidated. The expression of CyCAP in skin and during wound healing is also unknown. We demonstrate that CyCAP is expressed in both dermal fibroblasts and keratinocytes. In the dermis, the majority of CyCAP protein is located intracellular in a filamentous protein form while a lesser amount is in the extracellular matrix (ECM). CyCAP gene and protein expression is increased 1 day after skin wound healing in both fetal and adult rats and remains elevated level up to 1 week in adult rats. Immunohistochemistry studies demonstrate that the increased CyCAP expression locates mainly to inflammatory cells, including macrophages, monocytes and lymphocytes during wound healing. Interferon-gamma increases CyCAP gene and protein expression in cultured rat fibroblasts. We also found that wound healing is slower and less collagen is expressed in skin of CyCAP null mice. These data are the first observations of CyCAP expression in skin and during wound repair. Our data indicates that CyCAP is regulated by IFNgamma and may function on immune defense in macrophages, lymphocytes, dermal fibroblasts and keratinocytes during wound healing.

    View details for DOI 10.1002/jcp.20830

    View details for Web of Science ID 000242568200016

    View details for PubMedID 16998803

  • Epithelial-mesenchymal transition occurs after epidermal development in mouse skin EXPERIMENTAL CELL RESEARCH Kong, W., Li, S., Liu, C., Bari, A. S., Longaker, M. T., Lorenz, H. P. 2006; 312 (19): 3959-3968

    Abstract

    In the present study, we studied epithelial-mesenchymal transition (EMT) with fetal and postnatal serial skin sections. E-cadherin, occludin and zonula occludens 1 (ZO-1)-expressing cells appear in the dermal area from E18.5 to postnatal day 9 (P9), with highest expression from P2 to P5. The co-expression of mesenchymal marker alpha-smooth muscle (alpha-SMA), fibronectin and vimentin with E-cadherin in these dermal cells was further examined. Almost no dermal cells express alpha-SMA before P0. From P2 to P6, cells expressing both E-cadherin and alpha-SMA appear in the dermis. In contrast, fibronectin-releasing cells were detected in the dermis as early as on E15.5, although on P5, some dermal cells was found weakly expressing both fibronectin and E-cadherin, most cells strongly expressing fibronectin did not express E-cadherin. Vimentin was mainly expressed in both endothelial and blood-derived cells and did not show co-expression with E-cadherin. Confocal microscopy studies further found that during EMT, E-cadherin appears intracellularly, while the expression of alpha-SMA starts from the membrane area and moves to the cytosol of the cells. Our data are the first in vivo evidence that EMT occurs during mouse skin development. Dermal cells are derived from EMT and other origins, including blood, during skin development.

    View details for DOI 10.1016/j.yexcr.2006.08.029

    View details for Web of Science ID 000241790000023

    View details for PubMedID 17027753

  • Mannose-6-phosphate, an inhibitor of transforming growth factor-beta, improves range of motion after flexor tendon repair JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME Bates, S. J., Morrow, E., Zhang, A. Y., Pham, H., Longaker, M. T., Chang, J. 2006; 88A (11): 2465-2472

    Abstract

    Adhesion formation between the flexor tendon and its surrounding fibro-osseous sheath results in a decreased postoperative range of motion in the hand. Transforming growth factor-beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. In this study, the effects of two natural inhibitors of TGF-beta, decorin and mannose-6-phosphate, were investigated in vitro and in vivo.In the in vitro investigation, primary cell cultures from rabbit flexor tendon sheath, epitenon, and endotenon were established and each was supplemented with TGF-beta along with increasing doses of decorin or mannose-6-phosphate. Collagen-I production was measured with enzyme-linked immunosorbent assay (ELISA). For the in vivo study, rabbit zone-II flexor tendons were transected and then immediately repaired. Single intraoperative graded doses of decorin, mannose-6-phosphate, or phosphate-buffered saline solution (control) were added to the repair sites, and the forepaws were tested for the range of motion and repair strength at eight weeks postoperatively.Decorin and mannose-6-phosphate both reduced TGF-beta upregulated collagen production. Intraoperative application of low-dose mannose-6-phosphate significantly improved the range of motion of the operatively treated digits. The effect on breaking strength of the tendon repair was inconclusive.Mannose-6-phosphate is effective in reducing TGF-beta upregulated collagen production in an in vitro model. This finding correlated with our in vivo finding that a single intraoperative dose of mannose-6-phosphate improved the postoperative range of motion.

    View details for DOI 10.2106/JBJS.E.00143

    View details for Web of Science ID 000241769800020

  • Innovation in surgery - A historical perspective ANNALS OF SURGERY Riskin, D. J., T Longaker, M., Gertner, M., Krummel, T. M. 2006; 244 (5): 686-693

    Abstract

    To describe the field of surgical innovation from a historical perspective, applying new findings from research in technology innovation.While surgical innovation has a rich tradition, as a field of study it is embryonic. Only a handful of academic centers of surgical innovation exist, all of which have arisen within the last 5 years. To this point, the field has not been well defined, nor have future options to promote surgical innovation been thoroughly explored. It is clear that surgical innovation is fundamental to surgical progress and has significant health policy implications. A process of systematically evaluating and promoting innovation in surgery may be critical in the evolving practice of medicine.A review of the academic literature in technology innovation was undertaken. Articles and books were identified through technical, medical, and business sources. Luminaries in surgical innovation were interviewed to develop further relevance to surgical history. The concepts in technology innovation were then applied to innovation in surgery, using the historical example of surgical endoscopy as a representative area, which encompasses millennia of learning and spans multiple specialties of care.The history of surgery is comprised largely of individual, widely respected surgeon innovators. While respecting individual accomplishments, surgeons as a group have at times hindered critical innovation to the detriment of our profession and patients. As a clinical discipline, surgery relies on a tradition of research and attracting the brightest young minds. Innovation in surgery to date has been impressive, but inconsistently supported.A body of knowledge on technology innovation has been developed over the last decade but has largely not been applied to surgery. New surgical innovation centers are working to define the field and identify critical aspects of surgical innovation promotion. It is our responsibility as a profession to work to understand innovation in surgery, discover, translate, and commercialize advances to address major clinical problems, and to support the future of our profession consistently and rationally.

    View details for DOI 10.1097/01.sla.0000242706.91771.ce

    View details for Web of Science ID 000242278300012

    View details for PubMedID 17060760

  • The ethics of innovation in pediatric surgery. Seminars in pediatric surgery Riskin, D. J., Longaker, M. T., Krummel, T. M. 2006; 15 (4): 319-323

    Abstract

    Ethical issues in pediatric research have long been debated, and experimentation in pediatric surgery is under intense scrutiny. Extensive legislation and institutional systems that attempt to protect children while supporting necessary research are at times ineffective. Pediatric surgery has less funding and resources for innovation than fields with higher clinical volume. Not unlike pediatrics in general, innovation in pediatric surgery must be beyond criticism. And yet, for the sake of patients, innovation should not only be maintained, but must be encouraged.

    View details for PubMedID 17055963

  • Early-gestation fetal scarless wounds have less lysyl oxidase expression PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2006; 118 (5): 1125-1129

    Abstract

    Lysyl oxidase cross-links collagen and elastin. Because cross-linking likely influences collagen architecture, the authors compared lysyl oxidase expression during scarless and scarring fetal dermal wound repair.Excisional dermal wounds were made on E17 (gestational day 16.5) and E19 (gestational day 18.5) mouse fetuses. Skin and wound RNA was collected at 8, 12, and 24 hours. Quantitative real-time polymerase chain reaction was performed for lysyl oxidase. The effect of transforming growth factor (TGF)-beta1 on lysyl oxidase expression in fetal fibroblasts was tested. Confluent primary fetal and postnatal fibroblast cultures were stimulated with TGF-beta1 for 24 hours, and lysyl oxidase expression was quantitated by performing real-time polymerase chain reaction. Lysyl oxidase expression was also quantitated in unwounded fetal skin to determine its expression profile during development.E17 and E19 fetal skin had approximately 2-fold greater lysyl oxidase expression than postnatal skin (p < 0.01), and fetal fibroblasts had greater baseline lysyl oxidase expression than postnatal fibroblasts. After TGF-beta1 stimulation, fetal and postnatal fibroblasts responded with increases in lysyl oxidase expression. In E17 early-gestation scarless fetal wounds, lysyl oxidase had small increases (<1.5-fold) in expression from 1 to 12 hours. In late-gestation E19 scarring fetal wounds, lysyl oxidase increased 1.8-fold at 8 hours and 2-fold at 12 hours, which was significantly greater than the changes observed in E17 scarless wounds (p < 0.01 for each).Lysyl oxidase has greater expression in E19 late-gestation wounds that heal with scar compared with E17 early-gestation scarless wounds. This suggests a role for lysyl oxidase in scar formation.

    View details for DOI 10.1097/01.prs.0000221056.27536.db

    View details for Web of Science ID 000241327600008

    View details for PubMedID 17016177

  • Differential gene expression between juvenile and adult dura mater: A window into what genes play a role in the regeneration of membranous bone PLASTIC AND RECONSTRUCTIVE SURGERY Wan, D. C., Aalami, O. O., Wang, Z., Nacamuli, R. P., Lorget, F., Derynck, R., Longaker, M. T. 2006; 118 (4): 851-861

    Abstract

    Although reossification of large calvarial defects is possible in children, adults lack this tissue engineering capacity. In this study, the authors compared the differences in gene expression between juvenile and adult dura mater using a mouse cDNA microarray with 42,000 unique elements.Non-suture-associated parietal bone was harvested from 6-day-old and 60-day-old mice. The dura mater was carefully dissected from the calvarial disk and snap-frozen. RNA was extracted from pooled dura mater for microarray analysis. The 25 most differentially expressed genes were listed, as were selected bone-related genes. In addition, quantitative real-time reverse-transcriptase polymerase chain reaction confirmation of selected genes-BMP-2, BMP-4, and BMP-7; and osteopontin (OP), osteocalcin (OC), and FGFR-1-was performed.Juvenile dura mater expressed significantly greater amounts of BMP-2 and OP. Minimal difference in OC expression was observed between juvenile and adult dura mater. Extracellular matrix proteins (Col3a1, 5a1, 6a1, and fibronectin 1), osteoblast differentiation markers (Runx2/Cbfa1, Itm2a, and FGFR-1), and the growth factor Ptn were among other genes with greater expression in juvenile dura mater. Markers of osteoclasts (Acp5, MMP9, Ctsk) and the multiple candidate gene Ntrk2 were also expressed at higher levels in the juvenile dura mater.These findings suggest a more differentiated osteoprogenitor population to exist along with a greater presence of osteoclasts in the juvenile dura mater relative to adults. In addition to establishing a baseline difference in gene expression between juvenile and adult dura mater, new genes potentially critical to the regenerative potential of juvenile calvaria were identified.

    View details for DOI 10.1097/01.prs.0000232366.23897.2b

    View details for Web of Science ID 000240700100004

    View details for PubMedID 16980845

  • Novel techniques in hernia repair. Current surgery Antony, A. K., Herrera, F. A., Easter, D. W., Longaker, M. T., Lorenz, H. P. 2006; 63 (5): 306-309

    View details for PubMedID 16971199

  • Differential transcriptional responses of keloid and normal keratinocytes to serum stimulation JOURNAL OF SURGICAL RESEARCH Xia, W., Phan, T., Lim, I. J., Longaker, M. T., Yang, G. P. 2006; 135 (1): 156-163

    Abstract

    Keloids are benign tumors that occur only in response to injury, for which there is no effective treatment. We demonstrated previously that keloid keratinocytes (KKs) promote fibroblast proliferation more than normal keratinocytes (NKs) and that transforming growth factor (TGF)-beta is a component of that signal. We used the transcriptional response to serum stimulation to examine how TGF-beta expression is stimulated in KKs.Quiescent KKs and NKs were stimulated using serum; harvested using RNA at 0, 1, 6, 12, and 24 h; and analyzed using quantitative real-time polymerase chain reaction. TGF-beta activity in the conditioned medium was measured with an MLEC/PAI-luciferase assay. Inhibition of ERK1/2, p38 kinase, and JNK pathways was performed with PD98059, SB203580, and SP600125, respectively.Increased transcription of TGF-beta2 occurs within 1 h of serum stimulation in KKs but not in NKs. In contrast, TGF-beta3 transcription was suppressed in KKs compared with NKs. No significant differences were observed in the transcriptional response of TGF-beta1. Increased TGF-beta2 mRNA correlated with increased TGF-beta biological activity in the conditioned medium. Inhibition of the ERK, p38 kinase or JNK signal transduction pathways blocked the transcriptional up-regulation of TGF-beta2, TbetaR1, and TbetaR2 in KKs.KKs produce more TGF-beta2 mRNA than NKs in response to serum stimulation, resulting in increased TGF-beta activity in conditioned medium. Combining these results with our previous data lead us to propose a model of keloid formation characterized by an exaggerated response to cellular stress and abnormal epithelial-mesenchymal signaling promoting keloid formation.

    View details for DOI 10.1016/j.jss.2006.01.031

    View details for Web of Science ID 000240203500023

    View details for PubMedID 16564547

  • Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wan, D. C., Shi, Y., Nacamuli, R. P., Quarto, N., Lyons, K. M., Longaker, M. T. 2006; 103 (33): 12335-12340

    Abstract

    Although the multilineage potential of human adipose-derived adult stromal cells (ADAS) has been well described, few published studies have investigated the biological and molecular mechanisms underlying osteogenic differentiation of mouse ADAS. We report here that significant osteogenesis, as determined by gene expression and histological analysis, is induced only when mouse ADAS are cultured in the presence of retinoic acid with or without recombinant human bone morphogenetic protein (BMP)-2 supplementation. Furthermore, a dynamic expression profile for the BMP receptor (BMPR) isoform IB was observed, with dramatic up-regulation during osteogenesis. Western blot analysis revealed that retinoic acid enhanced levels of BMPR-IB protein during the first 7 days of osteogenic differentiation and that RNAi-mediated suppression of BMPR-IB dramatically impaired the ability of ADAS to form bone in vitro. In contrast, absence of BMPR-IA did not significantly diminish ADAS osteogenesis. Our data therefore demonstrate that the osteogenic commitment of multipotent mouse ADAS requires retinoic acid, which enhances expression of the critical BMPR-IB isoform.

    View details for DOI 10.1073/pnas.0604849103

    View details for Web of Science ID 000239867500026

    View details for PubMedID 16894153

  • Functions of vitamin D, retinoic acid, and dexamethasone in mouse adipose-derived mesenchymal cells TISSUE ENGINEERING Malladi, P., Xu, Y., Yang, G. P., Longaker, M. T. 2006; 12 (7): 2031-2040

    Abstract

    Adipose-derived mesenchymal cells (AMCs) offer great promise for tissue engineering of bone. Previously, 1,25-dihydroxyvitamin D3, retinoic acid (RA), and dexamethasone had been shown to promote osteogenesis in bone marrow-derived mesenchymal cells (BMSCs). To study the osteogenic characteristics of mouse AMCs, we applied these 3 hormones alone and in combination to the AMCs and examined markers of osteogenic differentiation. Interestingly, vitamin D and RA demonstrated a consistent, dose-dependent enhancement of osteogenesis and upregulated osteoblast specific markers including osteopontin and osteocalcin. However, in AMCs, dexamethasone clearly inhibited osteogenic differentiation in a dose dependent fashion and greatly increased the adipogenic marker peroxisome proliferator activated receptor gamma (PPAgamma). In summary, we show in vitro that vitamin D and RA are potential candidates to serve as enhancers of osteogenesis of AMCs and may be incorporated into future cell-based strategies for bone tissue engineering.

    View details for Web of Science ID 000239571800029

    View details for PubMedID 16889531

  • Connective tissue growth factor-specific monoclonal antibody therapy inhibits pancreatic tumor growth and metastasis CANCER RESEARCH Dornhoefer, N., Spong, S., Bennewith, K., Salim, A., Klaus, S., Kambham, N., Wong, C., Kaper, F., Sutphin, P., Nacalumi, R., Hoeckel, M., Le, Q., Longaker, M., Yang, G., Koong, A., Giaccia, A. 2006; 66 (11): 5816-5827

    Abstract

    Pancreatic cancer is highly aggressive and refractory to most existing therapies. Past studies have shown that connective tissue growth factor (CTGF) expression is elevated in human pancreatic adenocarcinomas and some pancreatic cancer cell lines. To address whether and how CTGF influences tumor growth, we generated pancreatic tumor cell lines that overexpress different levels of human CTGF. The effect of CTGF overexpression on cell proliferation was measured in vitro in monolayer culture, suspension culture, or soft agar, and in vivo in tumor xenografts. Although there was no effect of CTGF expression on proliferation in two-dimensional cultures, anchorage-independent growth (AIG) was enhanced. The capacity of CTGF to enhance AIG in vitro was linked to enhanced pancreatic tumor growth in vivo when these cells were implanted s.c. in nude mice. Administration of a neutralizing CTGF-specific monoclonal antibody, FG-3019, had no effect on monolayer cell proliferation, but blocked AIG in soft agar. Consistent with this observation, anti-CTGF treatment of mice bearing established CTGF-expressing tumors abrogated CTGF-dependent tumor growth and inhibited lymph node metastases without any toxicity observed in normal tissue. Together, these studies implicate CTGF as a new target in pancreatic cancer and suggest that inhibition of CTGF with a human monoclonal antibody may control primary and metastatic tumor growth.

    View details for DOI 10.1158/0008-5472.CAN-06-0081

    View details for Web of Science ID 000238003100038

    View details for PubMedID 16740721

  • Wnt-4 expression is increased in fibroblasts after TGF-beta 1 stimulation and during fetal and postnatal wound repair PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2006; 117 (7): 2297-2301

    Abstract

    Wnt-4 is a mitogen expressed during postnatal repair and scar formation; however, its expression profile during scarless repair is unknown. Transforming growth factor (TGF)-beta1 has high expression during healing with scar formation. Whether TGF-beta1 directly influences Wnt-4 expression in fetal or postnatal fibroblasts has not been examined.Primary fetal and postnatal mouse fibroblasts were stimulated with TGF-beta1 and Wnt-4 expression quantitated by real-time polymerase chain reaction. Fetal E17 and postnatal mouse excisional wounds were also analyzed for Wnt-4 expression by real-time polymerase chain reaction.In E17 fibroblasts after TGF-beta1 stimulation, Wnt-4 expression increased 4-fold at 1 hour (p < 0.05) and peaked with an 11-fold increase at 2 hours (p < 0.05). By 24 hours, expression decreased to 2-fold baseline levels (p < 0.05). In postnatal fibroblasts, Wnt-4 expression also increased after TGF-beta stimulation, but peak expression was larger and relatively delayed, with a 17-fold increase at 12 hours (p < 0.005). Expression levels at 24 hours were still 4-fold greater than baseline (p < 0.05). In E17 fetal skin, Wnt-4 expression was 3.5-fold greater compared with 3-week-old mice (p < 0.005). Small increases in Wnt-4 expression (less than 2-fold) occurred during both fetal scarless and postnatal scarring mouse wound repair.The authors' data suggest that TGF-beta directly increases Wnt-4 expression in fetal and postnatal fibroblasts and that Wnt-4 is increased in both fetal and postnatal repair.

    View details for DOI 10.1097/01.prs.0000218708.16909.31

    View details for Web of Science ID 000238431500029

    View details for PubMedID 16772932

  • FGF-2 inhibits osteogenesis in mouse adipose tissue-derived stromal cells and sustains their proliferative and osteogenic potential state TISSUE ENGINEERING Quarto, N., Longaker, M. T. 2006; 12 (6): 1405-1418

    Abstract

    In addition to adult bone marrow-derived mesenchymal stem cells, more recently, a second large stromal compartment found in adipose tissue has received attention and is believed to contain multipotent cells. In vitro, adipose tissue-derived stromal cells (ADS) can differentiate down osteogenic, chondrogenic, myogenic, adipose, and even neuronal pathways. In this article, we explore the effect of fibroblast growth factors 2 (FGF-2) on ADS cells. Our results demonstrated that FGF-2 inhibits osteogenesis in ADS cells. The osteogenic inhibitory effects is dose-dependent and reversible, thus suggesting that the lack of osteogenesis observed in ADS cells exposed to FGF-2 is not due to a negative selection triggered by this factor on a subpopulation of osteoblast progenitors. Furthermore, either overexpression of FGF-2, or continuous FGF-2 treatment sustain the proliferative and osteogenic potential state of ADS cells. Therefore, FGF-2 appears to be a positive regulator of osteoprogenitor cells and a negative regulator of osteoblast differentiation in ADS cells. These FGF-2 functional characteristics may assist with cell selection and enrichment for the purpose of bone tissue engineering.

    View details for Web of Science ID 000239570400003

    View details for PubMedID 16846339

  • Fetal and adult fibroblasts have similar TGF-beta-mediated, Smad-dependent signaling pathways PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2006; 117 (7): 2277-2283

    Abstract

    The scarless fetal skin-healing mechanism is mediated in part by the fibroblast and involves differential expression of transforming growth factor (TGF)-beta isoforms 1 and 3. The authors hypothesized that fetal and adult fibroblasts respond differently to TGF-beta isoform-specific stimulation, which may influence whether wounds scar. Connective tissue growth factor (CTGF), Smad3, and Smad7 are TGF-beta target genes. Expression of these targets was quantitated after TGF-beta1 and -beta3 stimulation of fetal and adult fibroblasts.Primary mouse fibroblast cultures at gestational day 16.5 (E17), 18.5 (E19), and 6 weeks (adult) were stimulated with TGF-beta1 or TGF-beta3. Quantitative polymerase chain reaction was performed for CTGF, Smad3, and Smad7 expression.CTGF was reduced four-fold in E17 and E19 compared with adult fibroblasts (p < 0.005). After TGF-beta1 stimulation, CTGF expression increased more than 60-fold in both E17 and E19 (p < 0.01), which was three-fold greater than that in adult fibroblasts (p < 0.005). TGF-beta3 induced more than 70-fold, 50-fold, and 20-fold increases in CTGF expression in E17, E19, and adult fibroblasts, respectively (p < 0.01 for each). Both TGF-beta1 and -beta3 decreased Smad3 expression and increased Smad7 expression in each fibroblast type, suggesting that intact TGF-beta-mediated signaling pathways were present.Fetal (E17 and E19) fibroblasts have lower CTGF expression compared with adult fibroblasts. However, fetal fibroblasts have larger increases in CTGF expression after TGF-beta1 or -beta3 stimulation. Fetal and adult mouse fibroblasts have similar TGF-beta1 and TGF-beta3 transcriptional regulation of Smad3 and Smad7. This suggests that scarless healing is likely not mediated by different Smad-dependent transcriptional responses to TGF-beta isoforms in the fetal E17 fibroblast.

    View details for DOI 10.1097/01.prs.0000224299.16523.76

    View details for Web of Science ID 000238431500026

    View details for PubMedID 16772929

  • An in vivo mouse excisional wound model of scarless healing PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. P. 2006; 117 (7): 2292-2296

    Abstract

    The purpose of this study was to develop a reproducible murine model of fetal scarless wound healing.One-millimeter excisional wounds were made in fetal skin at gestational days 16.5 (E17) and 18.5 (E19) (term = day 21.5, or E22) and marked with India ink. Fetal mortality was less than 30 percent in E17 fetuses and 0 percent in E19 fetuses. Control postnatal 2-mm open wounds were made in 3-week-old mice.At 48 hours, E17 skin wounds had healed completely. E19 skin wounds also healed but were marked by skin irregularity at the wound site. Histologically, E17 wounds had fine reticular collagen architecture by trichrome staining and hair follicle regeneration. In contrast, E19 wounds healed with collagen deposition and scar formation and no hair follicle regeneration.The authors have developed a reliable mouse model of fetal scarless repair to help elucidate the mechanism of scarless wound healing to take advantage of genetically modified animals. The knowledge gained may be used to manipulate scarring in the adult to produce a more fetal-like wound.

    View details for DOI 10.1097/01.prs.0000219340.47232.eb

    View details for Web of Science ID 000238431500028

    View details for PubMedID 16772931

  • Mitogenic and chondrogenic effects of fibroblast growth factor-2 in adipose-derived mesenchymal cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Chiou, M., Xu, Y., Longaker, M. T. 2006; 343 (2): 644-652

    Abstract

    Adipose-derived mesenchymal cells (AMCs) have demonstrated a great capacity for differentiating into bone, cartilage, and fat. Studies using bone marrow-derived mesenchymal cells (BMSCs) have shown that fibroblast growth factor (FGF)-2, a potent mitogenic factor, plays an important role in tissue engineering due to its effects in proliferation and differentiation for mesenchymal cells. The aim of this study was to investigate the function of FGF-2 in AMC chondrogenic differentiation and its possible contributions to cell-based therapeutics in skeletal tissue regeneration. Data demonstrated that FGF-2 significantly promoted the proliferation of AMCs and enhanced chondrogenesis in three-dimensional micromass culture. Moreover, priming AMCs with treatment of FGF-2 at 10 ng/ml demonstrated that cells underwent chondrogenic phenotypic differentiation, possibly by inducing N-Cadherin, FGF-receptor 2, and transcription factor Sox9. Our results indicated that FGF-2 potentiates chondrogenesis in AMCs, similar to its functions in BMSCs, suggesting the versatile potential applications of FGF-2 in skeletal regeneration and cartilage repair.

    View details for DOI 10.1016/j.bbrc.2006.02.171

    View details for Web of Science ID 000236659800042

    View details for PubMedID 16554022

  • Effect of reduced oxygen tension on chondrogenesis and osteogenesis in adipose-derived mesenchymal cells AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Malladi, P., Xu, Y., Chiou, M., Giaccia, A. J., Longaker, M. T. 2006; 290 (4): C1139-C1145

    Abstract

    Recent studies have demonstrated that adipose-derived mesenchymal cells (AMCs) offer great promise for cell-based therapies because of their ability to differentiate toward bone, cartilage, and fat. Given that cartilage is an avascular tissue and that mesenchymal cells experience hypoxia during prechondrogenic condensation in endochondral ossification, the goal of this study was to understand the influence of oxygen tension on AMC differentiation into bone and cartilage. In vitro chondrogenesis was induced using a three-dimensional micromass culture model supplemented with TGF-beta1. Collagen II production and extracellular matrix proteoglycans were assessed with immunohistochemistry and Alcian blue staining, respectively. Strikingly, micromasses differentiated in reduced oxygen tension (2% O(2)) showed markedly decreased chondrogenesis. Osteogenesis was induced using osteogenic medium supplemented with retinoic acid or vitamin D and was assessed with alkaline phosphatase activity and mineralization. AMCs differentiated in both 21 and 2% O(2) environments. However, osteogenesis was severely diminished in a low-oxygen environment. These data demonstrated that hypoxia strongly inhibits in vitro chondrogenesis and osteogenesis in AMCs.

    View details for DOI 10.1152/ajpcell.00415.2005

    View details for Web of Science ID 000236573300023

    View details for PubMedID 16291817

  • Craniofacial bone tissue engineering. Dental clinics of North America Wan, D. C., Nacamuli, R. P., Longaker, M. T. 2006; 50 (2): 175-?

    Abstract

    Repair and reconstruction of the craniofacial skeleton represents a significant biomedical burden, with thousands of procedures per-formed annually secondary to injuries and congenital malformations. Given the multitude of current approaches, the need for more effective strategies to repair these bone deficits is apparent. This article explores two major modalities for craniofacial bone tissue engineering: distraction osteogenesis and cellular based therapies. Current understanding of the guiding principles for each of these modalities is elaborated on along with the knowledge gained from clinical and investigative studies. By laying this foundation, future directions for craniofacial distraction and cell-based bone engineering have emerged with great promise for the advancement of clinical practice.

    View details for PubMedID 16530056

  • Absence of the p53 tumor suppressor gene promotes osteogenesis in mesenchymal stem cells JOURNAL OF PEDIATRIC SURGERY Tataria, M., Quarto, N., Longaker, M. T., Sylvester, K. G. 2006; 41 (4): 624-632

    Abstract

    Osteosarcoma arises predominantly in the metaphyseal growth plate of children during the growth spurt years. These tumors develop during physiological growth from an expanding cell population, suggesting that the transformed cell is a bone-forming progenitor. An absence of the p53 oncogene has been implicated in the origin and progression of osteosarcoma, and because mesenchymal stem cells (MSCs) are the physiological osteogenic progenitor cell population, we hypothesized that a p53-/- mutation would enhance bone differentiation of MSC in a mouse model of in vitro osteogenesis.Clonal MSC populations were derived from p53-/- mice. P53-/- and wild-type cells were placed in osteogenic culture and assessed via Alizarin Red quantification and alkaline phosphatase staining. The osteogenic marker genes Cbfa1, osteopontin, and osteocalcin were assessed by quantitative real time polymerase chain reaction during differentiation.Bone nodule formation and alkaline phosphatase staining was accelerated and enhanced in the p53-/- cells. The early and intermediate osteogenic markers, Cbfa1 and osteopontin, were upregulated in p53-/- MSCs compared with wild-type cells during osteogenesis. The terminal osteogenic marker gene osteocalcin was paradoxically lower in p53-/- MSCs indicating impaired terminal differentiation.The p53-/- mutation enhances and accelerates early osteogenesis in MSCs, but prevents terminal differentiation toward a mature osteocyte phenotype. These findings may have important implications for the regulation of the MSC compartment during the derivation of osteosarcoma in children.

    View details for DOI 10.1016/j.jpedsurg.2005.12.001

    View details for Web of Science ID 000237015800002

    View details for PubMedID 16567167

  • Guided tissue regeneration enhances bone formation in a rat model of failed osteogenesis PLASTIC AND RECONSTRUCTIVE SURGERY Fang, T. D., Nacamuli, R. P., Song, H. J., Fong, K. D., Shi, Y. Y., Longaker, M. T. 2006; 117 (4): 1177-1185

    Abstract

    Guided tissue regeneration is a technique that uses barrier materials to enhance tissue regeneration. Although previously demonstrated to be an effective way of enhancing craniofacial osteogenesis in several animal models, the ability of guided tissue regeneration to augment bone formation in the context of distraction osteogenesis is unknown. In the current study, the authors applied the principle of guided tissue regeneration to their rat mandibular distraction osteogenesis model in an attempt to enhance bone regeneration.Twelve (n = 6 per group) adult Sprague-Dawley rats underwent routine gradual distraction (5 days' latency, 4-mm distraction over 8 days, 4 to 6 weeks of consolidation) and acute distraction (immediate lengthening to 4 mm, 6 to 8 weeks of consolidation). An additional 10 animals underwent acute distraction followed by application of bioabsorbable Gore Resolut XT membranes (acute distraction plus guided tissue regeneration). Membranes were completely wrapped around the distraction gap. Animals were killed 6 and 8 weeks postoperatively and mandibles analyzed radiographically and histologically.Quantitative histomorphometric analyses were performed to compare relative bone formation between all three groups. Gradual distraction mandibles achieved bony union by 6 weeks with 86 percent bone formation, which increased to 98 percent by 8 weeks. Acute distraction mandibles healed with a fibrous nonunion and only 37 percent bone formation by 8 weeks. In contrast, acute distraction plus guided tissue regeneration-treated mandibles formed significantly more bone than acute distraction mandibles by 6 weeks (57 percent) and achieved bony bridging by 8 weeks, with 88 percent new bone formation.The authors' data demonstrate that guided tissue regeneration can significantly enhance bone formation in a fibrous nonunion model of mandibular distraction osteogenesis.

    View details for DOI 10.1097/01.prs.0000204581.59190.53

    View details for Web of Science ID 000236697800016

    View details for PubMedID 16582784

  • P38 MAP kinase mediates transforming growth factor-beta 2 transcription in human keloid fibroblasts AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY Xia, W., Longaker, M. T., Yang, G. P. 2006; 290 (3): R501-R508

    Abstract

    Keloids are abnormal fibrous growths of the dermis that develop only in response to wounding and represent a form of benign skin tumor. Previous studies have shown increased protein levels of TGF-beta in keloid tissue, suggesting a strong association with keloid formation leading us to examine mechanisms for why it is more highly expressed in keloids. Here, we use serum stimulation as an in vitro model to mimic a component of the wound microenvironment and examine differential gene expression in keloid human fibroblasts (KFs) vs. normal human fibroblasts (NFs). Transcription of TGF-beta2 was rapid and peaked between 1 and 6 h after serum stimulation in KFs vs. NFs. We confirmed increased TGF-beta activity in the conditioned medium from KFs, but not NFs. Inhibition of second messenger signaling pathways demonstrated that only the p38 MAPK inhibitor SB-203580 could block upregulation of TGF-beta2 following serum stimulation in KFs. Immunoblotting demonstrated that p38 MAPK was phosphorylated within 15 min and was maintained at a high level in KFs but not in NFs. The transcription factors activating transcription factor-2 and Elk-1 are activated by p38 MAPK, and also showed rapid and prolonged phosphorylation kinetics in KFs but not in NFs. In conclusion, increased TGF-beta2 transcription in response to serum stimulation in KFs appears to be mediated by the p38 MAPK pathway. This suggests the mechanism of keloid pathogenesis may be due in part to an inherent difference in how the fibroblasts respond to wounding.

    View details for DOI 10.1152/ajpregu.00472.2005

    View details for Web of Science ID 000235210300003

    View details for PubMedID 16467496

  • The ISCFS: A body for clinical, educational, and research innovation JOURNAL OF CRANIOFACIAL SURGERY Longaker, M. T., Wan, D. C. 2006; 17 (2): 215-216

    View details for Web of Science ID 000236747300002

    View details for PubMedID 16633164

  • Relationships between tissue dilatation and differentiation in distraction osteogenesis MATRIX BIOLOGY Morgan, E. F., Longaker, M. T., Carter, D. R. 2006; 25 (2): 94-103

    Abstract

    Mechanical factors modulate the morphogenesis and regeneration of mesenchymally derived tissues via processes mediated by the extracellular matrix (ECM). In distraction osteogenesis, large volumes of new bone are created through discrete applications of tensile displacement across an osteotomy gap. Although many studies have characterized the matrix, cellular and molecular biology of distraction osteogenesis, little is known about relationships between these biological phenomena and the local physical cues generated by distraction. Accordingly, the goal of this study was to characterize the local physical environment created within the osteotomy gap during long bone distraction osteogenesis. Using a computational approach, we quantified spatial and temporal profiles of three previously identified mechanical stimuli for tissue differentiation-pressure, tensile strain and fluid flow-as well as another candidate stimulus-tissue dilatation (volumetric strain). Whereas pressure and fluid velocity throughout the regenerate decayed to less than 31% of initial values within 20 min following distraction, tissue dilatation increased with time, reaching steady state values as high as 43% strain. This dilatation created large reductions and large gradients in cell and ECM densities. When combined with previous findings regarding the effects of strain and of cell and ECM densities on cell migration, proliferation and differentiation, these results indicate two mechanisms by which tissue dilatation may be a key stimulus for bone regeneration: (1) stretching of cells and (2) altering cell and ECM densities. These results are used to suggest experiments that can provide a more mechanistic understanding of the role of tissue dilatation in bone regeneration.

    View details for DOI 10.1016/j.matbio.2005.10.006

    View details for Web of Science ID 000236135400005

    View details for PubMedID 16330195

  • Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers PLOS MEDICINE Chi, J. T., Wang, Z., Nuyten, D. S., Rodriguez, E. H., Schaner, M. E., Salim, A., Wang, Y., Kristensen, G. B., Helland, A., Borresen-Dale, A. L., Giaccia, A., Longaker, M. T., Hastie, T., Yang, G. P., Van de Vijver, M. J., Brown, P. O. 2006; 3 (3): 395-409

    Abstract

    Inadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases.We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use.The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.

    View details for DOI 10.1371/journal.pmed.0030047

    View details for Web of Science ID 000236897500020

    View details for PubMedID 16417408

  • Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation JOURNAL OF CELLULAR PHYSIOLOGY Wang, Z., Fong, K. D., Phan, T. T., Lim, I. J., Longaker, M. T., Yang, G. P. 2006; 206 (2): 510-517

    Abstract

    Clinicians have observed that keloids preferentially form in body areas subject to increased skin tension. We hypothesized a difference exists in the transcriptional response of keloid fibroblasts to mechanical strain compared with normal fibroblasts. Normal and keloid fibroblasts were seeded in a device calibrated to deliver a known level of equibiaxial strain. We examined the transcriptional response of TGF-beta isoforms and collagen Ialpha, genes differentially expressed in keloids. Keloid fibroblasts produced more mRNA for TGF-beta1, TGF-beta2, and collagen Ialpha after mechanical strain compared to normals, and this was correlated with protein production. Inhibiting the major mechanical signal transduction pathway with the ERK inhibitor, U0126, blocked upregulation of gene expression. In addition, keloid fibroblasts formed more focal adhesion complexes as measured by immunofluorescence for focal adhesion kinase, integrin beta1, and vinculin. Finally, there is increased activation of focal adhesion kinase when we detected the phosphorylated form of focal adhesion kinase with immunofluorescence and immunoblotting. In summary, keloid fibroblasts have an exaggerated response to mechanical strain compared to normal fibroblasts leading to increased production of pro-fibrotic growth factors. This may be one molecular mechanism for the development of keloids.

    View details for DOI 10.1002/jcp.20486

    View details for Web of Science ID 000234458300028

    View details for PubMedID 16155910

  • Expression of a novel gene, MafB, in Dupuytren's disease JOURNAL OF HAND SURGERY-AMERICAN VOLUME Lee, L. C., Zhang, A. Y., Chong, A. K., Pham, H., Longaker, M. T., Chang, J. 2006; 31A (2): 211-218

    Abstract

    Dupuytren's disease (DD) is characterized by fibroblastic proliferation of the palmar fascia, often leading to flexion contracture in the hand. Although there is a strong genetic component the genome-wide expression of novel genes is not known. The purpose of this study was to use DNA microarray technology to identify upregulated genes in DD.Human tissue samples were harvested from 3 patient sources: DD cord tissue (n = 20), normal-appearing adjacent control fascia (n = 15), and palmar fascia from patients having carpal tunnel release (n = 15). DNA microarray analysis was performed on amplified sample RNA. Novel genes were compared with known gene functions. A candidate gene of interest was studied further by using immunohistochemistry on DD tissue samples and controls.Several novel genes not described previously in the study of DD were upregulated significantly, including MafB, collagen type V, alpha-2 (COL5A2), collagen type VIII, alpha-1 (COL8A1), contactin I (CNTN1), and leucine-rich repeat containing 17 (LRRC17). These upregulated genes were compared with their known gene-expression profiles in other tissues and their purported functions. MafB was found to be of particular interest because of its prominent role in tissue development and cellular differentiation. MafB immunohistochemistry showed positive staining in 50% of the DD specimens but complete absence of MafB in all control tissues (adjacent control fascia, carpal tunnel fascia). Co-localization experiments with MafB and alpha-smooth muscle actin showed staining properties in similar regions but these 2 proteins were not confined solely to the same cells.Microarray analysis of DD tissue has identified significant upregulated gene expression of MafB. MafB protein also is found in Dupuytren's cords but not in control fascia. Co-localization data suggest that the association of MafB with DD is not related exclusively to myofibroblast proliferation. Because of its role in fibroblastic transformation in other models MafB and its relationship to the pathogenesis of DD deserves further study.

    View details for DOI 10.1016/j.jhsa.2005.09.007

    View details for Web of Science ID 000235558900007

    View details for PubMedID 16473681

  • Adipose-derived mesenchymal cells (AMCs): a promising future for skeletal tissue engineering. Biotechnology & genetic engineering reviews Xu, Y., Malladi, P., Wagner, D. R., Tataria, M., Chiou, M., Sylvester, K. G., Longaker, M. T. 2006; 23: 291-308

    View details for PubMedID 22530513

  • Skin wounds in the MRL/MPJ mouse heal with scar WOUND REPAIR AND REGENERATION Colwell, A. S., Krummel, T. M., Kong, W., Longaker, M. T., Lorenz, H. P. 2006; 14 (1): 81-90

    Abstract

    Adult MRL/MpJ mice regenerate cartilage during repair of through-and-through ear punch wounds. However, the ability of this mouse strain to heal isolated cutaneous wounds by regeneration or with scar is unknown. The purpose of this study was to characterize the rate of reepithelialization and collagen architecture in dermal wounds from MRL/MpJ mice compared with C57bl/6 and Balb/c strains. Full-thickness incisional (5 mm) and excisional (2 mm diameter) skin wounds were made on the dorsum of 7-week-old MRL/MpJ, C57bl/6, and Balb/c mice. Ear punch wounds were made simultaneously on each animal. Reepithelialization was complete by 48 hours for incisional skin wounds in each strain. All excisional wounds showed incomplete reepithelialization at 24, 48, and 72 hours. At 14 days, all skin wounds had grossly healed. In contrast to the ear wounds made in C57bl/6 and Balb/c mice, MRL/MpJ ear wounds were completely healed by day 28. Dorsal skin wound sections at 14 and 28 days revealed dense collagen deposition and similar degrees of fibrosis between the three strains of mice. In conclusion, in contrast to wound healing in the ear, MRL/MpJ mouse dorsal cutaneous wounds heal similarly to C57bl/6 and Balb/c mice with dermal collagen deposition and scar formation.

    View details for DOI 10.1111/j.1743-6109.2005.00092.x

    View details for Web of Science ID 000235220000012

    View details for PubMedID 16476076

  • The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging PLASTIC AND RECONSTRUCTIVE SURGERY Shi, Y. Y., Nacamuli, R. P., Salim, A., Longaker, M. T. 2005; 116 (6): 1686-1696

    Abstract

    Adipose-derived mesenchymal cells are multipotent progenitor cells derived from the vascular-stromal compartment of adipose tissue. Although we have recently shown that these cells, from both juvenile and adult animals, are capable of forming bone in vivo, a detailed examination of the differences in the biology of these two populations (and in particular their ability to form bone) has not been performed.Adipose-derived mesenchymal cells were harvested from juvenile (6-day-old) and adult (60-day-old) mice. Differences in cellular attachment, proliferation, and proliferating cell nuclear antigen production were assessed. The ability of cells to undergo adipogenic differentiation was determined by Oil Red O staining. Early osteogenic differentiation was determined with alkaline phosphatase staining, and terminal differentiation with von Kossa staining as well as determination of extracellular matrix calcium content. All experiments were performed in triplicate.Greater attachment, proliferation, and proliferating cell nuclear antigen production were seen in juvenile as compared with adult adipose-derived mesenchymal cells. The juvenile cells underwent significantly greater adipogenic differentiation than did adult cells (p < 0.001). Interestingly, the adult cells were capable of robust early and terminal osteogenic differentiation, with levels of all three osteo-genic assays being similar to those seen in juvenile cells. Differences were not statistically significant.Although biologic differences exist between adipose-derived mesenchymal cells from juveniles and adults, the osteogenic capacity of these cells appears to be minimally affected by donor age. This suggests that these cells may be a particularly useful cellular resource in the design of cell-based therapies for skeletal regeneration in an aging population.

    View details for DOI 10.1097/01.prs.0000185606.03222.a9

    View details for Web of Science ID 000233119900017

    View details for PubMedID 16267433

  • Age-dependent properties and quasi-static strain in the rat sagittal suture JOURNAL OF BIOMECHANICS Henderson, J. H., Chang, L. Y., Song, H. M., Longaker, M. T., Carter, D. R. 2005; 38 (11): 2294-2301

    Abstract

    We measured the morphology of and performed tensile tests on sagittal sutures from rats of postnatal age 2 to 60 days. Using the properties measured ex vivo and a pressure vessel-based analysis, we estimated the quasi-static strain that had existed in the suture in vivo from 2 to 60 days. Sutural thickness, width, and stiffness per length were notable properties found to be age dependent. Sutural thickness increased 4.5-fold (0.11-0.50mm) between 2 and 60 days. Sutural width increased transiently between 2 and 20 days, peaking around 8 days; at 8 days, mean sutural width was 75% larger than mean sutural width at two days (0.35+/-0.07 (SD) vs. 0.20+/-0.06 mm). Sutural stiffness per length increased 4.4-fold (8.77-38.3N/mm/mm) between 2 and 60 days. The quasi-static sutural strain estimated to exist in vivo averaged 270+/-190 muepsilon between 2 and 60 days and was not age dependent. These findings provide data on the age-dependent sutural properties of infant to mature rats and provide the first estimate of quasi-static sutural strain in vivo in the rat. The findings show that during development the rat sagittal suture, as a structure, changes significantly and is exposed to quasi-static tensile strain in vivo due to intracranial pressure.

    View details for DOI 10.1016/j.jbiomech.2004.07.037

    View details for Web of Science ID 000232456400019

    View details for PubMedID 16154417

  • Bone induction in craniofacial defects. Orthodontics & craniofacial research Nacamuli, R. P., Longaker, M. T. 2005; 8 (4): 259-266

    Abstract

    Reconstruction of craniofacial bony deficiencies, whether acquired through trauma or as a result of treatment for disease, is a chronic problem. Although numerous approaches utilizing a wide array of materials ranging from alloplastic materials to autogenous bone grafts have been employed to achieve bony replacement, no ideal clinical approach exists. In this brief review, we will provide an overview of current approaches to treating craniofacial bony defects. We will then discuss advances being made in the design of scaffolding materials and potential candidate cell types with which to design tissue-engineered constructs for craniofacial skeletal repair.

    View details for PubMedID 16238606

  • Expression and possible mechanisms of regulation of BMP3 in rat cranial sutures PLASTIC AND RECONSTRUCTIVE SURGERY Nacamuli, R. P., Fong, K. D., Lenton, K. A., Song, H. M., Fang, T. D., Salim, A., Longaker, M. T. 2005; 116 (5): 1353-1362

    Abstract

    Clinical genetics data and investigative studies have contributed greatly to our understanding of the role of numerous genes in craniosynostosis. Recent studies have introduced antagonists of osteogenesis as potential key regulators of suture fusion and patency. The authors investigated the expression pattern of the bone morphogenetic protein antagonist BMP3 in rat cranial sutures and the factors regulating its expression in vitro.Microarray analysis was performed on rat posterior frontal and sagittal cranial sutures at 5, 10, 15, 20, and 30 days of life (n = 30 per group). Gene expression was confirmed using quantitative real-time reverse transcriptase polymerase chain reaction. Regulation of BMP3 expression was determined using primary rat calvarial osteoblasts stimulated with recombinant human fibroblast growth factor 2 or recombinant human transforming growth factor beta1, or cultured with primary rat nonsuture dura mater. Gene expression was quantified with quantitative real-time reverse transcriptase polymerase chain reaction.BMP3 expression in the posterior frontal suture decreased over the time course analyzed, whereas it increased in the sagittal suture. Notably, BMP3 expression was higher in the patent sagittal suture during the window of posterior frontal suture fusion. Stimulation of osteoblasts with recombinant human fibroblast growth factor 2 led to a rapid and sustained suppression of BMP3 expression (85 percent, p < 0.01) when compared with controls. Co-culture with dural cells decreased BMP3 mRNA by 50 percent compared with controls (p < 0.01).BMP3 is expressed in rat cranial sutures in a temporal pattern suggesting involvement in cranial suture patency and fusion. Furthermore, BMP3 is regulated in calvarial osteoblasts by recombinant human fibroblast growth factor 2 and by paracrine signaling from dura mater. These data add to our knowledge of the role of osteogenic antagonists in cranial suture biology.

    View details for DOI 10.1097/01.prs.0000182223.85978.34

    View details for Web of Science ID 000232421100023

    View details for PubMedID 16217479

  • Hypertrophic scar fibroblasts have increased connective tissue growth factor expression after transforming growth factor-beta stimulation PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Phan, T. T., Kong, W., Longaker, M. T., Lorenz, P. H. 2005; 116 (5): 1387-1390

    Abstract

    Hypertrophic scars and keloids respond to dermal disruption with excessive collagen deposition and increased transforming growth factor (TFG)-beta expression. Connective tissue growth factor (CTGF) is a downstream mediator of TGF-beta activity that is associated with scar and fibrosis. The authors hypothesize that there is increased expression of CTGF by hypertrophic scar and keloid fibroblasts in response to TGF-beta stimulation.Primary fibroblasts were isolated in culture from human hypertrophic scar (n = 2), keloid (n = 2), and normal skin (n = 2). After 18 hours of serum starvation, the cells were stimulated with 10 ng/ml of TGF-beta1, TGF-beta2, and TGF-beta3 for 24 hours. Quantitative real-time polymerase chain reaction was performed on extracted RNA samples to assay for CTGF mRNA expression.Baseline CTGF expression was increased 20-fold in unstimulated hypertrophic scar fibroblasts and 15-fold in keloid fibroblasts compared with normal fibroblasts. CTGF expression increased greater than 150-fold when stimulated with TGF-beta1 (p < 0.002) and greater than 100-fold when stimulated by TGF-beta2 or TGF-beta3 compared with normal fibroblasts (p < 0.02 and p < 0.002, respectively). CTGF expression was greatest after TGF-beta1 stimulation in hypertrophic scar fibroblasts compared with TGF-beta2 (p < 0.04) and TGF-beta3 (p < 0.02). Keloid fibroblast CTGF expression also increased greater than 100-fold after stimulation with TGF-beta1 (p = 0.16) and greater than 75-fold after addition of TGF-beta2 and TGF-beta3 (p = 0.06 and p = 0.22, respectively).Hypertrophic scar fibroblasts have both intrinsic up-regulation of CTGF transcription and an exaggerated capacity for CTGF transcription in response to TGF-beta stimulation. These data suggest that blockage of CTGF activity may reduce pathologic scar formation.

    View details for DOI 10.1097/01.prs.0000182343.99694.28

    View details for Web of Science ID 000232421100027

    View details for PubMedID 16217483

  • Microarray analysis of mechanical shear effects on flexor tendon cells PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Trindade, M. C., Wang, Z., Nacamuli, R. P., Pham, H., Fang, T. D., Song, H. J., Smith, L., Longaker, M. T., Chang, J. 2005; 116 (5): 1393-1404

    Abstract

    Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. The authors hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing.To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis.Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, the authors also found up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with the authors' microarray data.The authors demonstrate an overall antifibrotic expression pattern in response to shear stress in tendon cells that may provide insight into the mechanisms by which early mobilization decreases adhesion formation without impaired tendon healing.

    View details for DOI 10.1097/01.prs.0000182345.86453.4f

    View details for Web of Science ID 000232421100029

    View details for PubMedID 16217485

  • Gene profiling of cells expressing different FGF-2 forms GENE Quarto, N., Fong, K. D., Longaker, M. T. 2005; 356: 49-68

    Abstract

    Fibroblast Growth Factor-2 (FGF-2) induces cell proliferation, cell migration, embryonic development, cell differentiation, angiogenesis and malignant transformation. The four forms of FGF-2 (Low Molecular Weight) and (High Molecular Weights) are alternative translation products, and have a different subcellular localization: the high molecular weight (HMWFGF-2) forms are nuclear while the low molecular weight form, (LMWFGF-2) is mainly cytoplasmic. Our previous work demonstrated NIH 3T3 cells expressing different FGF-2 forms, displayed a different phenotype, suggesting that nuclear and cytoplasmic forms of FGF-2 may have different functions. Here we report a cDNA microarray-based study in NIH 3T3 fibroblasts expressing different FGF-2 forms. Several candidate genes that affect cell-cycle, tumor suppression, adhesion and transcription were identified as possible mediators of the HMWFGF-2 phenotype and signaling pattern. These results demonstrated that HMWFGF-2 and LMWFGF-2 target the expression of different genes. Particularly, our data suggest that HMWFGF-2 forms may function as inducers of growth inhibition and tumor suppression activities.

    View details for DOI 10.1016/j.gene.2005.05.014

    View details for Web of Science ID 000231750700006

    View details for PubMedID 16023796

  • Adipose-derived mesenchymal cells as a potential cell source for skeletal regeneration CURRENT OPINION IN MOLECULAR THERAPEUTICS Xu, Y., Malladi, P., Wagner, D. R., Longaker, M. T. 2005; 7 (4): 300-305

    Abstract

    Recent studies suggest that adipose tissue contains pluripotent cells that are similar to those derived from other tissues, such as bone marrow. Mesenchymal cells isolated from adipose tissue are capable of differentiating along osteogenic, chondrogenic, myogenic, adipogenic and possibly neuronal lineages. Current knowledge of adipose-derived mesenchymal cells is reviewed, with a particular focus on efforts to direct these cells towards bone formation. Cell-based therapies using adipose tissue are anticipated to be of great clinical interest for skeletal tissue repair and regeneration.

    View details for Web of Science ID 000231167800003

    View details for PubMedID 16121695

  • Angiogenesis is required for successful bone induction during distraction osteogenesis JOURNAL OF BONE AND MINERAL RESEARCH Fang, T. D., Salim, A., Xia, W., Nacamuli, R. P., Guccione, S., Song, H. M., Carano, R. A., Filvaroff, E. H., Bednarski, M. D., Giaccia, A. J., Longaker, M. T. 2005; 20 (7): 1114-1124

    Abstract

    The role of angiogenesis during mechanically induced bone formation is incompletely understood. The relationship between the mechanical environment, angiogenesis, and bone formation was determined in a rat distraction osteogenesis model. Disruption of either the mechanical environment or endothelial cell proliferation blocked angiogenesis and bone formation. This study further defines the role of the mechanical environment and angiogenesis during distraction osteogenesis.Whereas successful fracture repair requires a coordinated and complex transcriptional program that integrates mechanotransductive signaling, angiogenesis, and osteogenesis, the interdependence of these processes is not fully understood. In this study, we use a system of bony regeneration known as mandibular distraction osteogenesis (DO) in which a controlled mechanical stimulus promotes bone induction after an osteotomy and gradual separation of the osteotomy edges to examine the relationship between the mechanical environment, angiogenesis, and osteogenesis.Adult Sprague-Dawley rats were treated with gradual distraction, gradual distraction plus the angiogenic inhibitor TNP-470, or acute distraction (a model of failed bony regeneration). Animals were killed at the end of distraction (day 13) or at the end of consolidation (day 41) and examined with muCT, histology, and immunohistochemistry for angiogenesis and bone formation (n = 4 per time-point per group). An additional group of animals (n = 6 per time-point per group) was processed for microarray analysis at days 5, 9, 13, 21, and 41.Either TNP-470 administration or disruption of the mechanical environment prevented normal osteogenesis and resulted in a fibrous nonunion. Subsequent analysis of the regenerate showed an absence of angiogenesis by gross histology and immunohistochemical localization of platelet endothelial cell adhesion molecule in the groups that failed to heal. Microarray analysis revealed distinct patterns of expression of genes associated with osteogenesis, angiogenesis, and hypoxia in each of the three groups. Our findings confirm the interdependence of the mechanical environment, angiogenesis, and osteogenesis during DO, and suggest that induction of proangiogenic genes and the proper mechanical environment are both necessary to support new vasculature for bone induction in DO.

    View details for DOI 10.1359/JBMR.050301

    View details for Web of Science ID 000230134100005

    View details for PubMedID 15940364

  • Age-dependent residual tensile strains are present in the dura mater of rats JOURNAL OF THE ROYAL SOCIETY INTERFACE Henderson, J. H., Nacamuli, R. P., Zhao, B., Longaker, M. T., Carter, D. R. 2005; 2 (3): 159-167

    Abstract

    The objectives of this study were to determine whether residual tensile strains exist in the dura mater of mammals in vivo, and whether the strains are age-dependent. We made incisions in the parietal dura mater of immature and mature rats, and measured the retraction of the dura mater from each incision. We then used a finite-element model to calculate the strain present in the parietal dura mater of each rat. We found that age-dependent residual tensile strains are present in the dura mater of rats. The mean average residual strain of the immature rats was significantly larger than that of the mature rats (4.96+/-1.54% (s.d.) versus 0.39+/-0.13%, p<0.0001), with the mean strain calculated in the mature rats of the order of the minimum measurement that could be made using our experimental approach. In addition, in the immature rats mean residual strain in the longitudinal direction was significantly larger than mean residual strain in the transverse direction (6.11+/-3.62% versus 3.82+/-2.64%, p=0.0218). Our findings show that age-dependent residual tensile strains exist in the dura mater of rats. We speculate that these strains may reflect the rate and direction of cranial growth and may also influence cranial healing.

    View details for DOI 10.1098/rsif.2005.0035

    View details for Web of Science ID 000234341800004

    View details for PubMedID 16849176

  • Differential transcriptional expression profiles of juvenile and adult calvarial bone PLASTIC AND RECONSTRUCTIVE SURGERY Aalami, O. O., Nacamuli, R. P., Salim, A., Fong, K. D., Lenton, K. A., Song, H. M., Fang, T. D., Longaker, M. T. 2005; 115 (7): 1986-1994

    Abstract

    It has widely been observed that young children are capable of reossifying large calvarial defects, while adults lack this endogenous tissue-engineering capacity. The ability of juvenile animals to regenerate calvarial defects has been investigated in multiple animal models, including mice. In this study, the authors used cDNA microarrays to investigate the expression of osteogenesis-associated genes upstream and downstream of Runx2 in juvenile and adult mouse calvaria.Nonsuture-associated parietal bone discs were harvested from 6-day-old (n = 50) and 60-day-old (n = 35) male CD-1 mice. After separation of the underlying dura mater and overlying pericranium, the calvarial discs were snap-frozen and RNA was extracted from pooled samples of calvaria for microarray analysis. Genes analyzed included cytokines, receptors, and cell-surface and matrix proteins both upstream and downstream of Runx2.Genes associated with the Runx2 pathway had notably higher levels in the juvenile versus adult calvaria. All genes except for osteocalcin were expressed at least twofold higher in the juvenile calvaria. This pattern was validated with quantitative real-time polymerase chain reaction. In addition, mRNA for potent osteoinductive growth factors was present at higher levels in the juvenile compared with the adult calvaria.These findings reflect a genomic environment of active osteoblast differentiation and ossification in the juvenile calvaria compared with the adult "quiescent" calvarial tissue. These data suggest that a decreased osteogenic potential of adult calvarial osteoblasts may, in part, explain the inability of adult animals to heal calvarial defects.

    View details for DOI 10.1097/01.PRS.0000163323.66318.73

    View details for Web of Science ID 000229497700025

    View details for PubMedID 15923847

  • Mechanobiology of mandibular distraction osteogenesis: finite element analyses with a rat model JOURNAL OF ORTHOPAEDIC RESEARCH Loboa, E. G., Fang, T. D., Parker, D. W., Warren, S. M., Fong, K. D., Longaker, M. T., Carter, D. R. 2005; 23 (3): 663-670

    Abstract

    Three-dimensional finite element (FE) analyses were performed to characterize the local mechanical environment created within the tissue regenerate during mandibular distraction osteogenesis (DO) in a rat model. Finite element models were created from three-dimensional computed tomography image data of rat hemi-mandibles at four different time points during an optimal distraction osteogenesis protocol (i.e., most successful protocol for bone formation): end latency (post-operative day (POD) 5), distraction day 2 (POD 7), distraction day 5 (POD 10), and distraction day 8 (POD 13). A 0.25 mm distraction was simulated and the resulting hydrostatic stresses and maximum principal tensile strains were determined within the tissue regenerate. When compared to previous histological findings, finite element analyses showed that tensile strains up to 13% corresponded to regions of new bone formation and regions of periosteal hydrostatic pressure with magnitudes less than 17 kPa corresponded to locations of cartilage formation. Tensile strains within the center of the gap were much higher, leading us to conclude that tissue damage would occur there if the tissue was not compliant enough to withstand such high strains, and that this damage would trigger formation of new mesenchymal tissue. These data were consistent with histological evidence showing mesenchymal tissue present in the center of the gap throughout distraction. Finite element analyses performed at different time points during distraction were instrumental in determining the changes in hydrostatic stress and tensile strain fields throughout distraction, providing a mechanical environment rationale for the different levels of bone formation in end latency, and distraction day 2, 5, and 8 specimens.

    View details for DOI 10.1016/j.orthres.2004.09.010

    View details for Web of Science ID 000229375000022

    View details for PubMedID 15885489

  • Sox9 neural crest determinant gene controls patterning and closure of the posterior frontal cranial suture DEVELOPMENTAL BIOLOGY Sahar, D. E., Longaker, M. T., Quarto, N. 2005; 280 (2): 344-361

    Abstract

    Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification.

    View details for DOI 10.1016/j.ydbio.2005.01.022

    View details for Web of Science ID 000228377600007

    View details for PubMedID 15882577

  • FGF-2 acts through an ERK1/2 intracellular pathway to affect osteoblast differentiation PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Mathy, J. A., Warren, S. M., Nacamuli, R. P., Song, H. M., Lenton, K., Fong, K. D., Fang, D. T., Longaker, M. T. 2005; 115 (3): 838-852

    Abstract

    An abundance of genetic and experimental data have suggested that fibroblast growth factor (FGF) signaling plays a central role in physiological and pathological cranial suture fusion. Although alterations in the differentiation and proliferation of sutural osteoblasts may be a key mediator of this process, the mechanisms by which FGF signaling regulates osteoblast differentiation remain incompletely understood. In the current study, the authors show that recombinant human FGF-2 alters osteoblastic expression of bone morphogenetic protein-2 and Msx-2 in vitro to favor cellular differentiation and osteoinduction. The ERK1/2 intracellular signaling cascade was shown to be necessary for recombinant human FGF-2-mediated bone morphogenetic protein-2 transcriptional changes. Furthermore, the cellular production of an intermediate transcriptional modifier was found to be necessary for the recombinant human FGF-2-mediated gene expression changes in bone morphogenetic protein-2 and Msx-2. Together, these findings offer new insight into the mechanisms by which FGF-2 modulates osteoblast biology.

    View details for DOI 10.1097/01.PRS.0000153035.73507.7B

    View details for Web of Science ID 000227432100023

    View details for PubMedID 15731686

  • Bone morphogenetic protein 2 and retinoic acid accelerate in vivo bone formation, osteoclast recruitment, and bone turnover TISSUE ENGINEERING Cowan, C. M., Aalami, O. O., Shi, Y. Y., Chou, Y. F., Mari, C., Thomas, R., Quarto, N., Nacamuli, R. P., Contag, C. H., Wu, B., Longaker, M. T. 2005; 11 (3-4): 645-658

    Abstract

    Reconstruction of craniofacial defects presents a substantial biomedical burden, and requires complex surgery. Interestingly, children after age 2 years and adults are unable to heal large skull defects. This nonhealing paradigm provides an excellent model system for craniofacial skeletal tissueengineering strategies. Previous studies have documented the in vivo osteogenic potential of adipose-derived stromal (ADS) cells and bone marrow-derived stromal (BMS) cells. This study investigates the ability to accelerate in vivo osteogenesis on ex vivo recombinant human bone morphogenetic protein 2 (BMP-2) and retinoic acid stimulation. Mouse osteoblasts, ADS cells, and BMS cells were seeded onto apatite-coated PLGA scaffolds, stimulated with rhBMP-2 and retinoic acid ex vivo for 4 weeks, and subsequently implanted into critically sized (4 mm) calvarial defects. Samples were harvested after 2, 4, 8, and 12 weeks. Areas of complete bony bridging were noted as early as 2 weeks in vivo; however, osteoclasts were attracted to the scaffold as identified by calcitonin receptor staining and tartrate-resistant acid phosphatase activity staining. Although the optimal method of in vitro osteogenic priming for mesenchymal cells remains unknown, these results provide evidence that BMP-2 and retinoic acid stimulation of multipotent cells ex vivo can subsequently induce significant quantities of bone formation within a short time period in vivo.

    View details for Web of Science ID 000228999400031

    View details for PubMedID 15869441

  • New developments in pediatric plastic surgery research CLINICS IN PLASTIC SURGERY Nacamuli, R. P., Wan, D. C., Lenton, K. A., Longaker, M. T. 2005; 32 (1): 123-?

    Abstract

    Pediatric plastic surgery research is a rapidly expanding field. Unique in many ways, researchers in this field stand at the union of multiple scientific specialties, including biomedical engineering, tissue engineering, polymer science, molecular biology, developmental biology, and genetics. The goal of this scientific effort is to translate research advances into improved treatments for children with congenital and acquired defects. Although the last decade has seen a dramatic acceleration in research related to pediatric plastic surgery, the next 10 years will no doubt lead to novel treatment strategies with improved clinical outcomes.

    View details for DOI 10.1016/j.cps.2004.10.003

    View details for Web of Science ID 000226935600013

    View details for PubMedID 15636770

  • Cranial suture biology CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 66 Lenton, K. A., Nacamuli, R. P., Wan, D. C., Helms, J. A., Longaker, M. T. 2005; 66: 287-?

    View details for Web of Science ID 000228212000009

    View details for PubMedID 15797457

  • Increased angiogenesis and expression of vascular endothelial growth factor during scarless repair PLASTIC AND RECONSTRUCTIVE SURGERY Colwell, A. S., Beanes, S. R., Soo, C., Dang, C., Ting, K., Longaker, M. T., Atkinson, J. B., Lorenz, H. P. 2005; 115 (1): 204-212

    Abstract

    Vascular endothelial growth factor (VEGF) is a dimeric heparin-binding glycoprotein that is a potent endothelial cell-specific mitogen with increased expression during adult cutaneous wound healing. VEGF activity is mediated by two receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), which are expressed primarily in vascular endothelial cells. Initiation of profibrotic cytokine expression likely coordinates the transition from scarless healing to scar formation in fetal wounds. Angiogenesis is an important component of the scarring repair process, but the function of VEGF and degree of angiogenesis during scarless repair has not been investigated. We hypothesize that VEGF and its receptors are differentially expressed in scarless compared with scarring fetal wounds because VEGF is implicated in angiogenesis during skin development and adult wound healing. Excisional wounds were created on fetal rats at gestational ages 16.5 days (E16) and 18.5 days (E18) (term = 21.5 days). Wounds were harvested at 24 and 72 hours (n = 12 wounds per time point). Nonwounded fetal skin (E17, E19, and E21) was used as control. Reduced-cycle, specific-primer, reverse-transcriptase polymerase chain reaction was performed to determine the expression of VEGF and its receptors, VEGFR-1 and VEGFR-2. Wounds at 72 hours and fetal skin controls were examined under high-power microscopy for blood vessel counts. Unpaired two-tailed t test was used (p < 0.05 was considered significant). VEGF expression increased 2.4-fold (p < 0.001) during normal skin development from E17 to E19. In scarless wounds (E16), VEGF expression increased 2.8-fold (p < 0.02) at 72 hours. No increased expression occurred in the scarring wounds (E18). VEGFR-1 and VEGFR-2 expression increased over 2-fold during normal skin development from E17 to E21. However, each was down-regulated 30 to 50 percent in scarless (E16) and scarring (E18) wounds. There is a 2-fold increase in mean vessel counts per high-power field in scarless (E16) wounds at 72 hours compared with age-matched control skin (p < 0.02) and a 1.7-fold increase in mean vessel count in scarring fetal wounds (E18) compared with age-matched control skin (p < 0.05). There is no difference in the total number of vessels found in scarless versus scarring wounds or between 19.5-day versus 21.5-day fetal skin. VEGF and its receptors, VEGFR-1 and VEGFR-2, increase expression during skin development and dermal differentiation. VEGF expression quickly elevates during scarless compared with scarring repair, which likely contributes to the more rapid scarless fetal repair rate. Similar numbers of new ves-sels are formed during scarless and scarring fetal repair.

    View details for DOI 10.1097/01.PRS.0000138252.51581.22

    View details for Web of Science ID 000226058400028

    View details for PubMedID 15622252

  • The zebrafish (Danio rerio): a model system for cranial suture Patterning CELLS TISSUES ORGANS Quarto, N., Longaker, M. T. 2005; 181 (2): 109-118

    Abstract

    The zebrafish (Danio rerio) is an alluring model system currently used to study early embryonic development, organogenesis and gene functional analysis. However, few studies have been devoted to post-embryonic development. We have explored the possibility of using this organism to analyze how cranial suture patterning occurs. This study reports on the establishment of the zebrafish skull vault anatomy, calvarial osteogenesis, and cranial suture morphology. Our results demonstrate that the anatomy of the zebrafish cranial vault and cranial sutures is very similar to that of mammalian organisms. Indeed, the zebrafish represents a versatile and valuable model system for the study of the biogenesis of cranial sutures.

    View details for DOI 10.1159/000091100

    View details for Web of Science ID 000235916100005

    View details for PubMedID 16534205

  • Mammalian fetal organ regeneration REGENERATIVE MEDICINE I: THEORIES, MODELS AND METHODS Colwell, A. S., Longaker, M. T., Lorenz, H. P. 2005; 93: 83-100

    Abstract

    The developing fetus has the remarkable ability to heal dermal skin wounds by regenerating normal epidermis and dermis with restoration of the extracellular matrix architecture, strength, and function. The biology responsible for scarless wound healing in skin is a paradigm for ideal tissue repair. This regenerative capacity is lost in late gestation when fetal wounds heal with fibrosis and scar. Early in gestation, fetal skin is developing at a rapid pace in a unique environment. Investigation of normal skin embryogenesis and comparison between early scarless and late scarring fetal wounds has revealed distinct differences in inflammatory response, cellular mediators, wound contraction, cytokines, growth factors, and extracellular matrix modulators. The knowledge gained from comparative observational studies has served as a base for experimental interventions in animal models to induce or ameliorate scar. Although much progress has been made over the past decade, the mechanism of fetal wound healing remains largely unknown and attempts to mimic the scarless wound phenotype have not been completely successful. Identification of more key genes involved in skin regeneration may have implications in adult skin wounds and repair in other organ systems.

    View details for DOI 10.1007/b99972

    View details for Web of Science ID 000228468300004

    View details for PubMedID 15791945

  • High-dose retinoic acid modulates rat calvarial osteoblast biology JOURNAL OF CELLULAR PHYSIOLOGY Song, H. M., Nacamuli, R. P., Xia, W., Bari, A. S., Shi, Y. Y., Fang, T. D., Longaker, M. T. 2005; 202 (1): 255-262

    Abstract

    Retinoic acid has been shown to adversely affect craniofacial development. Cleft palate and craniosynostosis are two examples of craniofacial defects associated with prenatal exposure to this agent. Although the effects of retinoic acid on cephalic neural crest-derived tissues have previously been studied, the specific effects of retinoic acid on the cellular biology of osteoblasts remain unclear. The purpose of this study was to analyze in detail the effects of pharmacologic doses of retinoic acid on the differentiation and proliferation of osteoblasts derived from an intramembranous source. Primary rat calvarial osteoblasts were established in culture and treated with 1 or 10 microM all-trans-retinoic acid. Retinoic acid treatment markedly increased expression of osteopontin up to 48 h after stimulation. Consistent with this early stage of differentiation, both mRNA and protein analysis of FGF receptor isoforms demonstrated a switch in predominance from fibroblast growth factor receptor 2 (fgfr2) to fgfr1. Analysis of PCNA protein confirmed inhibition of proliferation by retinoic acid. To determine whether these alterations in osteoblast biology would lead to increased differentiation, we examined short term [alkaline phosphatase (AP) activity] and long term (von Kossa staining) surrogates of bone formation in vitro. These assays confirmed that retinoic acid increased osteogenesis, with a 4-fold increase in bone nodule formation in cells treated with 10 microM retinoic acid after 28 days. Overall, our results demonstrated that pharmacologic doses of all-trans-retinoic acid decreased osteoblast proliferation and increased differentiation, suggesting that retinoic acid may effect craniofacial development by pathologically enhancing osteogenesis.

    View details for DOI 10.1002/jcp.20115

    View details for Web of Science ID 000225514300027

    View details for PubMedID 15389522

  • Cyclophilin C-associated protein is a mediator for fibronectin fragment-induced matrix metalloproteinase-13 expression JOURNAL OF BIOLOGICAL CHEMISTRY Kong, W. Y., Longaker, M. T., Lorenz, H. P. 2004; 279 (53): 55334-55340

    Abstract

    The function of cyclophilin C-associated protein (CyC-AP) on expression of extracellular matrix and matrix metalloproteinases (MMPs) was studied in CyC-AP-null mice. Fibronectin showed increased expression of the 53- and 29-kDa fragments in skin and wounds from CyC-AP-null mice. Type I collagen had an initial degraded pattern in the skin of CyC-AP-null mice, which did not occur in wild-type mice. MMP-3, MMP-13, MMP-14, and tumor necrosis factor-alpha (TNFalpha) had a higher expression in CyC-AP-null skin. During wound healing, MMP-13 and TNFalpha were stimulated to an even higher level, suggesting they are regulated by multiple factors. To understand the regulatory mechanisms of the up-regulated MMPs, the direct effects of TNFalpha, IL-1beta, 45-kDa fibronectin fragment (FN-45), and the 70-kDa fibronectin fragments (FN-70) on the expression of MMPs were studied. MMP-13 expression increased significantly in both CyC-AP-null and wild-type dermal fibroblasts after treatment with IL-1beta or with TNFalpha. However, MMP-13 expression did not increase in CyC-AP-null fibroblasts but did increase only in wild-type fibroblasts after FN-45 and FN-70 treatment. MMP-3 activation was induced by FN-45 and did not show a difference between CyC-AP-null and wild-type fibroblasts, suggesting different regulatory pathways for FN-45 on MMP-13 and MMP-3 expression. Our data are the first to demonstrate that deletion of CyC-AP can abolish fibronectin fragment-induced MMP-13 expression through an unknown mechanism. CyC-AP is an important factor for the regulation of MMP-13 expression.

    View details for DOI 10.1074/jbc.M410804200

    View details for Web of Science ID 000225960800045

    View details for PubMedID 15507443

  • Quantitative transcriptional analysis of fusing and nonfusing cranial suture complexes in mice PLASTIC AND RECONSTRUCTIVE SURGERY Nacamuli, R. P., Song, H. J., Fang, T. D., Fong, K. D., Mathy, J. A., Shi, Y. Y., Salim, A., Longaker, M. T. 2004; 114 (7): 1818-1825

    Abstract

    Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior frontal and sagittal suture complexes (including the associated dura mater, suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior frontal suture as compared with the sagittal suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior frontal suture in mice.

    View details for DOI 10.1097/01.PRS.0000143578.41666.2C

    View details for Web of Science ID 000225578900018

    View details for PubMedID 15577353

  • Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts JOURNAL OF BIOLOGICAL CHEMISTRY Salim, A., Nacamuli, R. P., Morgan, E. F., Giaccia, A. J., Longaker, M. T. 2004; 279 (38): 40007-40016

    Abstract

    Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

    View details for DOI 10.1074/jbc.M403715200

    View details for Web of Science ID 000223791500098

    View details for PubMedID 15263007

  • Applications of a mouse model of calvarial healing: Differences in regenerative abilities of juveniles and adults PLASTIC AND RECONSTRUCTIVE SURGERY Aalami, O. O., Nacamuli, R. P., Lenton, K. A., Cowan, C. M., Fang, T. D., Fong, K. D., Shi, Y. Y., Song, H. M., Sahar, D. E., Longaker, M. T. 2004; 114 (3): 713-720

    Abstract

    Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors' analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing: <5 percent in all groups; p < 0.05). All three defect sizes were found to be critical in the adult, whereas significant healing was seen regardless of the size of the defect in juvenile mice. The establishment of this model will facilitate further, detailed evaluation of the molecular biology underlying the different regenerative abilities of juvenile versus adult mice and enhance research into membranous bone induction by making available powerful tools such as knockout and transgenic animals.

    View details for DOI 10.1097/01.PRS.0000131016.12754.30

    View details for Web of Science ID 000223436200015

    View details for PubMedID 15318051

  • Complex epithelial-mesenchymal interactions modulate transforming growth factor-beta expression in keloid-derived cells WOUND REPAIR AND REGENERATION Xia, W., Phan, T. T., Lim, I. J., Longaker, M. T., Yang, G. P. 2004; 12 (5): 546-556

    Abstract

    Keloids are proliferative dermal growths representing a pathologic wound healing response. We have previously demonstrated that coculture of fibroblasts derived from either keloid or normal skin have an elevated proliferation rate when cocultured with keloid-derived keratinocytes vs. normal keratinocytes. In these studies, we examined the contribution of transforming growth factor-beta (TGF-beta) to this phenomenon using a two-chamber coculture system. Fibroblast proliferation in coculture was slower with the addition of a pan-TGF-beta neutralizing antibody. Keloid keratinocytes in coculture expressed more TGF-beta1, -beta3, and TGF-beta receptor 1 than normal keratinocytes. Keloid fibroblasts cocultured with keloid keratinocytes expressed more mRNA for TGF-beta1, -beta2, TGF-beta receptor 1, and Smad2. Keloid fibroblasts also produced more type I collagen, connective tissue growth factor, and insulin-like growth factor-II/mannose-6-phosphate receptor when cocultured with keloid keratinocytes vs. normal keratinocytes. Levels of total and activated TGF-beta activity increased when fibroblasts were cocultured with keratinocytes, correlating with the changes in transcriptional activity of TGF-beta. In conclusion, we find a complex paracrine interaction regulates TGF-beta mRNA expression and activation between keratinocytes and fibroblasts. These data suggest that keloid pathogenesis may result from both an increased TGF-beta production and activation by the keloid keratinocyte, and elevated TGF-beta expression, utilization, and signaling in keloid fibroblasts.

    View details for Web of Science ID 000224025400007

    View details for PubMedID 15453837

  • Donor-derived, liver-specific protein expression after bone marrow transplantation TRANSPLANTATION Jenkins, D. D., Streetz, K., Tataria, M., Sahar, D., Kurobe, M., Longaker, M. T., Kay, M. A., Sylvester, K. G. 2004; 78 (4): 530-536

    Abstract

    Bone marrow transplantation (BMT) may represent a novel mechanism to deliver a functional gene to a deficient liver. Bone marrow-derived hepatocytes are rare and without a defined contribution to liver function. Consequently, the clinical significance of BMT to treat liver disease is unclear. We sought to quantify bone marrow-derived hepatocyte protein expression after BMT and determine whether the process is inducible with liver injury.Mice transgenic for human alpha-1 antitrypsin (hAAT) under a hepatocyte-specific promoter were used as bone marrow donors. Adenoviral transduction of modified urokinase plasminogen activator (Ad-muPA) was used to induce liver injury. Eight weeks after lethal irradiation and BMT, recipients were stratified into two groups: BMT alone (n = 5) and BMT + Ad-muPA (n= 10). Both groups of animals were bled before (t = 0) and at 2, 4, 8, and 16 weeks after Ad-muPA administration, and the serum samples were assessed for hAAT by enzyme-linked immunosorbent assay.Transgenic donor mice expressed 5 to 10 mg/mL of hAAT. Recipients of BMT alone expressed less than 80 ng/mL of hAAT over all time periods. Animals receiving BMT + Ad-muPA showed sustained and stable hAAT expression of approximately 200 ng/mL. Differences were statistically significant at each time point.Serum protein levels from liver-specific transgene expression are detectable and persist after BMT. Expression is low, but inducible with liver injury. We are currently developing strategies to augment donor-derived, liver-specific protein expression after BMT.

    View details for DOI 10.1097/01.TP.0000130180.42573.BI

    View details for Web of Science ID 000223486500008

    View details for PubMedID 15446311

  • Stem cell differentiation NATURE BIOTECHNOLOGY Salim, A., Giaccia, A. J., Longaker, M. T. 2004; 22 (7): 804-805

    View details for DOI 10.1038/nbt0704-803d

    View details for Web of Science ID 000222533000013

    View details for PubMedID 15229534

  • Creation and characterization of a mouse model of mandibular distraction osteogenesis BONE Fang, T. D., Nacamuli, R. P., Song, H. M., Fong, K. D., Warren, S. M., Salim, A., Carano, R. A., Filvaroff, E. H., Longaker, M. T. 2004; 34 (6): 1004-1012

    Abstract

    While the histological and ultrastructural changes associated with distraction osteogenesis have been extensively characterized using various animal models, the molecular mechanisms governing this technique remain poorly understood. In the current study, for the first time, we describe a mouse mandibular distraction osteogenesis model. Development of this model will allow assessment of factors involved in normal vs. abnormal healing (especially in non-unions) of craniofacial skeletal elements. Complete osteotomies were created on the right hemimandibles of 51 adult male CD-1 mice and customized distraction devices attached. Thirty-three animals underwent gradual distraction (5 days latency, distraction at 0.2 mm BID x 8 days, 28 days consolidation), while the remaining 18 mice underwent acute lengthening (immediate distraction to 3.2 mm) at the time of surgery. Mandibles were harvested at time points corresponding to the latent (POD 5), distraction (POD 9, 13), and consolidation (POD 28, 41) periods and processed for histological or quantitative real-time RT-PCR analysis. Specimens from each group were processed for microCT analysis. Histological and radiological data demonstrated that all mandibles undergoing gradual distraction achieved complete bony union by the end of consolidation, while those undergoing acute lengthening formed a fibrous non-union. Quantitative real-time RT-PCR demonstrated upregulation of mRNA for VEGF, FGF-2, collagen I, and osteopontin during gradual distraction but not during acute lengthening. These data validate our novel mouse mandibular distraction model and demonstrate its utility in elucidating the molecular mechanisms regulating bone formation during distraction osteogenesis as compared to those that are expressed during the formation of fibrous non-unions.

    View details for DOI 10.1016/j.bone.2004.02.011

    View details for Web of Science ID 000222219600009

    View details for PubMedID 15193546

  • Apoptosis in a rodent model of cranial suture fusion: In situ imaging and gene expression analysis PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Song, H. M., Nacamuli, R. P., Franc, B. L., Mari, C., Fang, T. D., Warren, S. M., Contag, C. H., Blankenberg, F. G., Longaker, M. T. 2004; 113 (7): 2037-2047

    Abstract

    Craniosynostosis, the premature fusion of cranial sutures, is one of the most common craniofacial anomalies, with a reported incidence of up to one in 2500 live births. Despite its prevalence, the cause of craniosynostosis remains unknown. Previously, apoptosis has been postulated to be a contributing factor in the pathogenesis of craniosynostosis, although the role of programmed cell death in cranial sutures is poorly understood. To address this problem, the authors used an established rodent model of posterior-frontal suture fusion and sagittal suture patency to globally examine apoptosis in cranial sutures. Apoptosis was evaluated by systemically coinjecting Sprague-Dawley rats with both fluorescent and technetium-99m-labeled annexin V at time points before, during, and after the period of predicted posterior-frontal suture fusion to determine the magnitude and time course of overall apoptotic activity in both fusing and patent sutures. Using these novel in situ imaging techniques, the authors observed a significant increase in the overall levels of apoptosis in both the posterior-frontal and sagittal suture complexes during the period of predicted posterior-frontal suture fusion. To further explore this increase in apoptotic activity, they used microarray technology to study apoptosis-related genes within the suture complex. Interestingly, there was activation of distinct apoptotic pathways in the posterior-frontal and sagittal sutures during the period of predicted posterior-frontal suture fusion. Whereas increased transcription of genes associated with the mitochondria-mediated apoptotic pathway occurred in the posterior-frontal suture during fusion, activation of genes associated with the death receptor-mediated apoptotic pathway predominated in the patent sagittal suture during the same time period. These data suggest that although overall apoptotic activity in rat patent and fusing sutures is similar, the pathways mediating apoptosis within each suture are distinct.

    View details for DOI 10.1097/01.prs.000012118201199.c1

    View details for Web of Science ID 000221917700021

    View details for PubMedID 15253194

  • Adipose-derived adult stromal cells heal critical-size mouse calvarial defects NATURE BIOTECHNOLOGY Cowan, C. M., Shi, Y. Y., Aalami, O. O., Chou, Y. F., Mari, C., Thomas, R., Quarto, N., Contag, C. H., Wu, B., Longaker, M. T. 2004; 22 (5): 560-567

    Abstract

    In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84-99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

    View details for DOI 10.1038/nbt958

    View details for Web of Science ID 000221159700024

    View details for PubMedID 15077117

  • Mechanisms of murine cranial suture patency mediated by a dominant negative transforming growth factor-beta receptor adenovirus PLASTIC AND RECONSTRUCTIVE SURGERY Song, H. J., Fong, D., Nacamuli, R. P., Warren, S. M., Fang, T. D., Mathy, J. A., Cowan, C. M., Aalami, O. O., Longaker, M. T. 2004; 113 (6): 1685-1697

    Abstract

    Using a physiologic model of mouse cranial suture fusion, the authors' laboratory has previously demonstrated that transforming growth factor (TGF)-betas appear to be more abundantly expressed in the suture complex of the fusing posterior frontal compared with the patent sagittal suture. Furthermore, the authors have shown that by blocking TGF-beta signaling with a replication-deficient adenovirus encoding a defective, dominant negative type II TGF-beta receptor (AdDN-TbetaRII), posterior frontal suture fusion was inhibited. In this study, the authors attempt to further elucidate the role of TGF-beta in cranial suture fusion by investigating possible mechanisms of AdDN-TbetaRII-mediated cranial suture patency using both an established organ culture model and a novel in vitro co-culture system that recapitulates the in vivo anatomic dura mater/cranial suture relationship. In this article, the authors demonstrate that blocking TGF-beta signaling with the AdDN-TbetaRII construct led to inhibition of cellular proliferation in the suture mesenchyme and subjacent dura mater during the early period of predicted posterior frontal suture fusion. Interestingly, co-culture experiments revealed that transfecting osteoblasts with AdDN-TbetaRII led to alterations in the gene expression levels of two important bone-related molecules (Msx2 and osteopontin). Inhibiting TGF-beta signaling prevented time-dependent suppression of Msx2 and prevented induction of osteopontin, thereby retarding osteoblast differentiation. Furthermore, the authors demonstrated that the AdDN-TbetaRII construct was capable of blocking TGF-beta -mediated up-regulation of collagen IalphaI, an extracellular matrix molecule important for bone formation. Collectively, these data strongly suggest that AdDN-TbetaRII maintains posterior frontal patency, in part by altering early events in de novo bone formation, including cellular proliferation and early extracellular matrix production.

    View details for DOI 10.1097/001.PRS.000117363.43699.5B

    View details for Web of Science ID 000221141000019

    View details for PubMedID 15114130

  • In vitro murine posterior frontal suture fate is age-dependent: Implications for cranial suture biology PLASTIC AND RECONSTRUCTIVE SURGERY Song, H. J., Sahar, D. E., Fong, K. D., Nacamuli, R. P., Fang, T. D., Mathy, J. A., Aalami, O. O., Warren, S. M., Longaker, M. T. 2004; 113 (4): 1192-1204

    Abstract

    In CD-1 mice, the posterior frontal suture (analogous to the human metopic suture) fuses while all other cranial sutures remain patent. In an in vitro organ culture model, the authors previously demonstrated that posterior frontal sutures explanted immediately before the onset of suture fusion (at 25 days old) mimic in vivo physiologic fusion. In the first portion of this study, the authors defined how early in development the posterior frontal suture fuses in their tension-free, serum-free organ culture system by serially analyzing posterior frontal suture fusion from calvariae explanted at different stages of postnatal development. Their results revealed a divergence of suture fate leading to abnormal patency or physiologic fusion between the first and second weeks of life, respectively, despite viability and continued growth of the calvarial explants in vitro. From these data, the authors postulated that the gene expression patterns present in the suture complex at the time of explant may determine whether the posterior frontal suture fuses or remains patent in organ culture. Therefore, to elucidate potentially important differences in gene expression within this "window of opportunity," they performed a cDNA microarray analysis on 5-day-old and 15-day-old posterior frontal and sagittal whole suture complexes corresponding to the age ranges for unsuccessful (1 to 7 days old) and successful (14 to 21 days old) in vitro posterior frontal suture fusion. Overall, their microarray results reveal interesting differential expression patterns of candidate genes in different categories, including angiogenic cytokines and mechanosensitive genes potentially important in cranial suture biology.

    View details for DOI 10.1097/01.PRS.0000110203.90911.63

    View details for Web of Science ID 000220618600011

    View details for PubMedID 15083020

  • Inhibition of TGF-beta-induced collagen production in rabbit flexor tendons. journal of hand surgery Zhang, A. Y., Pham, H., Ho, F., Teng, K., Longaker, M. T., Chang, J. 2004; 29 (2): 230-235

    Abstract

    Postoperative adhesions frequently compromise the success of flexor tendon repair. Manipulation of growth factors responsible for scar formation may be a method of decreasing adhesion formation. Transforming growth factor beta (TGF-beta) is a key cytokine in the pathogenesis of tissue fibrosis. The purpose of this study was to examine the effectiveness of TGF-beta neutralizing antibody in blocking TGF-beta-induced collagen I production in rabbit flexor tendons in vitro.Sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were obtained from rabbit flexor tendons. Each cell culture was supplemented with 1 ng/mL of TGF-beta along with increasing doses of TGF-beta neutralizing antibody (0.1-2.0 microg/mL). Collagen I production was measured by enzyme-linked immunoabsorbent assay and TGF-beta bioactivity was measured by the luciferase assay. Results were compared with TGF-beta alone and unsupplemented controls.The addition of neutralizing antibody significantly reduced TGF-beta-induced collagen I production in a dose-dependent manner in all 3 cell cultures. TGF-beta bioactivity was also reduced by its neutralizing antibody.This study shows that TGF-beta inhibition through its neutralizing antibody was effective in cultured flexor tendon cells. The results encourage further experiments that use such agents to modulate flexor tendon wound healing in in vivo models in the hope of eventually blocking the effect of TGF-beta on flexor tendons clinically.

    View details for PubMedID 15043894

  • [The molecular biology of distraction osteogenesis]. Revue de stomatologie et de chirurgie maxillo-faciale Boulétreau, P., Longaker, M. T. 2004; 105 (1): 23-25

    Abstract

    Distraction osteogenesis has become a mainstay in bone engineering and the recent application of this technique to the membranous craniofacial skeleton has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, if the biomechanical, histological and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, our laboratory has described a rat mandibular distraction model that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. In this Article, we present the hypotheses and current research that have furthered our knowledge of the molecular mechanisms that govern distraction osteogenesis. Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (Transforming Growth Factor-B, Bone Morphogenetic Proteins, Insulin-like Growth Factor-1, Fibroblast Growth Factor-2) during distraction osteogenesis have been best characterized and will be discussed in this text. We believe that novel systems like the rat model will facilitate our understanding of the biomolecular mechanisms that mediate membranous distraction osteogenesis and will ultimately guide the development of targeted-strategies designed to accelerate bone healing.

    View details for PubMedID 15041867

  • Mechanobiology of mandibular distraction osteogenesis: experimental analyses with a rat model BONE Loboa, E. G., Fang, T. D., Warren, S. M., Lindsey, D. P., Fong, K. D., Longaker, M. T., Carter, D. R. 2004; 34 (2): 336-343

    Abstract

    We analyzed mechanobiological influences on successful distraction osteogenesis (DO). Mandibular distraction surgeries were performed on 15 adult male Sprague-Dawley rats. Animals underwent gradual distraction (GD), progressive lengthening by small increments (5-day latency followed by 0.25 mm distractions twice daily for 8 days followed by 28-day maturation period). Distracted hemimandibles were harvested on postoperative days (POD) 5, 7, 10, 13, and 41. Load-displacement curves were then recorded for ex vivo distractions of 0.25 mm and stresses determined. Histologically, new bone formation appeared in GD specimens on distraction day 2 (POD 7), filling 50-60% of the gap by distraction day 8 (POD 13), with nearly complete bony bridging at end maturation (POD 41). Average tensile strains imposed by each incremental distraction ranged from approximately 10% to 12.5% during distraction days 2-8 and were associated with bone apposition rates of about 260 microm/day. Because this GD protocol was previously determined to be optimal for DO, we conclude that strains within this range provide an excellent environment for de novo bone apposition. Distraction caused tissue damage in distraction day 2, 5, and 8 specimens as evidenced by distinct drops in the load/displacement curves. Taken together, our interpretation of these data is that daily distractions cause daily tissue damage which triggers new mesenchymal tissue formation.

    View details for DOI 10.1016/j.bone.2003.10.012

    View details for Web of Science ID 000220014000011

    View details for PubMedID 14962812

  • Flexor tendon wound healing in vitro: Lactate up-regulation of TGF-beta expression and functional activity PLASTIC AND RECONSTRUCTIVE SURGERY Yalamanchi, N., Klein, M. B., Pham, H. M., Longaker, M. T., Chang, J. 2004; 113 (2): 625-632

    Abstract

    Flexor tendon wound healing in zone II is complicated by adhesions to the surrounding fibro-osseous sheath. These adhesions can significantly alter tendon gliding and ultimately hand function. Lactate and transforming growth factor-beta (TGF-beta) are two important mediators of wound healing that have been demonstrated to independently increase collagen production by cells of the tendon sheath, epitenon, and endotenon. This study examined the effects of lactate on TGF-beta peptide and receptor production by flexor tendon cells. Tendon sheath fibroblasts, epitenon tenocytes, and endotenon tenocytes were isolated from rabbit flexor tendons and cultured separately. Cell cultures were supplemented with 50 mM lactate, and the expression of three TGF-beta peptide isoforms (beta1, beta2, and beta3) and three receptor isoforms (R1, R2, and R3) was quantified with enzyme-linked immunosorbent assays. TGF-beta functional activity was also assessed with the addition of tendon cell conditioned media to mink lung epithelial cells transfected with a luciferase reporter gene expression construct responsive to TGF-beta. Supplementation of the cell culture medium with lactate significantly (p < 0.05) increased the expression of all TGF-beta peptide and receptor isoforms in all three cell lines. Tendon sheath fibroblasts exhibited the greatest increases in beta1 and beta2 peptide isoform expression (30 and 23 percent, respectively), whereas endotenon tenocytes demonstrated the greatest increase in beta3 peptide expression (32 percent). Epitenon tenocytes exhibited the greatest increases in receptor isoform R1 and R2 expression (17 and 19 percent, respectively). All three tendon cell types demonstrated significant (p < 0.05) increases in TGF-beta functional activity when exposed to lactate. Epitenon tenocytes demonstrated the greatest increase in activity (>4 times control values), whereas tendon sheath fibroblasts demonstrated the highest overall levels of total TGF-beta functional activity. Lactate significantly increased TGF-beta peptide (beta1, beta2, and beta3) expression, receptor (R1, R2, and R3) expression, and functional activity, suggesting a common pathway regulating tendon cell collagen production. Modulation of lactate and TGF-beta levels may provide a means of modulating the effects of TGF-beta on adhesion formation in flexor tendon wound healing.

    View details for DOI 10.1097/01.PRS.0000101529.47062.34

    View details for Web of Science ID 000220063300023

    View details for PubMedID 14758225

  • Sutural bone deposition rate and strain magnitude during cranial development BONE Henderson, J. H., Longaker, M. T., Carter, D. R. 2004; 34 (2): 271-280

    Abstract

    It is widely believed that rapid growth of the human brain generates tensile strain in cranial sutures, and that this strain influences the rate of bone deposition at the sutural margins during development. We developed general theoretical techniques for estimating sutural bone deposition rate and strain magnitude during mammalian cranial development. A geometry-based analysis was developed to estimate sutural bone deposition rate. A quasi-static stress analysis was developed to estimate sutural strain magnitude. We applied these techniques to the special case of normal cranial development in humans. The results of the bone deposition rate analysis indicate that average human sutural bone deposition rate is on the order of 100 microm/day at 1 month of age and decreases in an approximately exponential fashion during the first 4 years of life. The results of the strain analysis indicate that sutural strain magnitude is highly dependent on the assumed stiffness of the sutures, with estimated strain at 1 month of age ranging from approximately 20 to 400 microstrain. Regardless of the assumed stiffness of the sutures, the results indicate that sutural strain magnitude is small and decreases in an approximately exponential fashion during the first 4 years of life. The finding that both sutural bone deposition rate and strain magnitude decrease with increasing age is consistent with quasi-static tensile strain in sutures influencing sutural osteoblast activity in a dose-dependent manner. However, the small magnitude of the predicted strains suggests that tissue level strains in sutures may be too small to directly influence osteoblast biology. In light of these results, we suggest other biomechanical mechanisms, such as a tension-induced angiogenic environment in the sutures or mechanotransduction in the underlying dura mater, through which tension across sutures may regulate the rate of bone deposition in sutures.

    View details for DOI 10.1016/j.bone.2003.10.007

    View details for Web of Science ID 000220014000004

    View details for PubMedID 14962805

  • Tissue-engineered bone using mesenchymal stem cells and a biodegradable scaffold JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Nacamuli, R. K., Song, H. J., Longaker, M. T. 2004; 15 (1): 34-37

    View details for Web of Science ID 000225848500011

    View details for PubMedID 14704559

  • Stem cells - Review and update ARCHIVES OF SURGERY Sylvester, K. G., Longaker, M. T. 2004; 139 (1): 93-99

    Abstract

    Regenerative medicine and emerging biotechnologies stand to revolutionize the practice of medicine. Advancements in stem cell biology, including embryonic and postnatal somatic stem cells, have made the prospect of tissue regeneration a potential clinical reality. Short of reproductive cloning, these same technologies, properly used, could allow for the creation of replacement tissue for the deficient host. To provide a concise review for surgeons on the current science and biology of stem cells, we surveyed the scientific literature, MEDLINE, and relevant political headlines that illuminate the stem cell discussion; the issues are summarized in this review. Building on this conceptual framework, the related issues of clinical promise and the political debate enveloping this emerging technology are examined. A basic understanding of stem cell biology is paramount to stay informed of this emerging technology and the national debate.

    View details for Web of Science ID 000187998500022

    View details for PubMedID 14718284

  • From scarless fetal wounds to keloids: Molecular studies in wound healing WOUND REPAIR AND REGENERATION Yang, G. P., Lim, I. J., Phan, T. T., Lorenz, H. P., Longaker, M. T. 2003; 11 (6): 411-418

    Abstract

    Surgical researchers were among the first to describe the different phases of wound healing and the events in tissue repair and regeneration that were taking place during each phase. The understanding of these events has been significantly enhanced in recent years by modern techniques in molecular and cellular biology. In this article, we discuss new findings in scarless fetal repair, angiogenesis in wound healing, and keloid pathogenesis. This serves to highlight the advances that have been made and also how much remains to be understood.

    View details for Web of Science ID 000186779200006

    View details for PubMedID 14617279

  • Mechanical strain affects dura mater biological processes: Implications for immature calvarial healing PLASTIC AND RECONSTRUCTIVE SURGERY Fong, K. D., Warren, S. M., Loboa, E. G., Henderson, J. H., Fang, T. D., Cowan, C. M., Carter, D. R., Longaker, M. T. 2003; 112 (5): 1312-1327

    Abstract

    The human brain grows rapidly during the first 2 years of life. This growth generates tensile strain in the overlying dura mater and neurocranium. Interestingly, it is largely during this 2-year growth period that infants are able to reossify calvarial defects. This clinical observation is important because it suggests that calvarial healing is most robust during the period of active intracranial volume expansion. With a rat model, it was previously demonstrated that immature dura mater proliferates more rapidly and produces more osteogenic cytokines and markers of osteoblast differentiation than does mature dura mater. It was therefore hypothesized that mechanical strain generated by the growing brain induces immature dura mater proliferation and increases osteogenic cytokine expression necessary for growth and healing of the overlying calvaria. Human and rat (n = 40) intracranial volume expansion was calculated as a function of age. These calculations demonstrated that 83 percent of human intracranial volume expansion is complete by 2 years of age and 90 percent of Sprague-Dawley rat intracranial volume expansion is achieved by 2 months of age. Next, the maximal daily circumferential tensile strains that could be generated in immature rat dura mater were calculated, and the corresponding daily biaxial tensile strains in the dura mater during this 2-month period were determined. With the use of a three-parameter monomolecular growth curve, it was calculated that rat dura mater experiences daily equibiaxial strains of at most 9.7 percent and 0.1 percent at birth (day 0) and 60 days of age, respectively. Because it was noted that immature dural cells may experience tensile strains as high as approximately 10 percent, neonatal rat dural cells were subjected to 10 percent equibiaxial strain in vitro, and dural cell proliferation and gene expression profiles were analyzed. When exposed to mechanical strain, immature dural cells rapidly proliferated (5.8-fold increase in proliferating cell nuclear antigen expression at 24 hours). Moreover, mechanical strain induced marked up-regulation of dural cell osteogenic cytokine production; transforming growth factor-beta1 messenger RNA levels increased 3.4-fold at 3 hours and fibroblast growth factor-2 protein levels increased 4.5-fold at 24 hours and 5.6-fold at 48 hours. Finally, mechanical strain increased dural cell expression of markers of osteoblast differentiation (2.8-fold increase in osteopontin levels at 3 hours). These findings suggest that mechanical strain can induce changes in dura mater biological processes and gene expression that may play important roles in coordinating the growth and healing of the neonatal calvaria.

    View details for DOI 10.1097/01.PRS.0000079860.14734.D6

    View details for Web of Science ID 000220062700014

    View details for PubMedID 14504515

  • Markers of osteoblast differentiation in fusing and nonfusing cranial sutures PLASTIC AND RECONSTRUCTIVE SURGERY Nacamuli, R. P., Fong, K. D., Warren, S. M., Fang, T. D., Song, H. M., Helms, J. A., Longaker, M. T. 2003; 112 (5): 1328-1335

    Abstract

    Accumulating clinical genetic data support the hypothesis that alterations in osteoblast differentiation are closely associated with craniosynostoses. Gain-of-function mutations in FGFR1, FGFR2, FGFR3, and Msx2 and loss-of-function mutations in Twist are examples of such alterations. Several studies have examined how these mutations alter the expression patterns for transcription factors such as Runx2 and noncollagenous extracellular matrix molecules such as osteopontin and osteocalcin. One limitation of such studies is that they examine samples derived from craniosynostotic patients with sutures that have already fused, thus missing the dynamic osteogenic process of suture fusion. In this study, in situ hybridization was used to localize Runx2, osteopontin, and osteocalcin expression in the sagittal and posterior frontal sutures in mice (n = 20), before (day 13), during (days 23, 33, and 43), and after (day 53) the period of physiological posterior frontal suture fusion. The data demonstrated similar patterns of expression in fusing (posterior frontal) and nonfusing (sagittal) sutures. The expression of all three genes was primarily concentrated in the osteogenic fronts of both sutures and decreased with time. Notably, none of the three genes was expressed in the mesenchyme of either fusing or nonfusing sutures. The data suggest that the molecular signals leading to bone formation along the osteogenic fronts in fusing and nonfusing sutures are similar, raising the possibility that other factors, such as antagonists of osteogenesis, might have a role in maintaining suture patency.

    View details for DOI 10.1097/01.PRS.0000079826.24086.CD

    View details for Web of Science ID 000220062700015

    View details for PubMedID 14504516

  • Tissue engineering and regenerative medicine CLINICS IN PLASTIC SURGERY Jenkins, D. D., Yang, G. P., Lorenz, H. P., Longaker, M. T., Sylvester, K. G. 2003; 30 (4): 581-?

    Abstract

    Regenerative medicine is evolving toward a powerful new paradigm of functional restoration. With the ethical use of gene therapy or through the manipulation of autologous tissues, improved tissue replacements may soon be available. The promise of engineered whole organs, although fraught with technical hurdles, remains on the horizon. As these advances occur, physicians and surgeons of the twenty-first century will possess ever more powerful tools to restore form and function.

    View details for DOI 10.1016/S0094-1298(03)00076-2

    View details for Web of Science ID 000186313400011

    View details for PubMedID 14621306

  • Age-related changes in the biomolecular mechanisms of clvarial osteoblast biology affect fibroblast growth factor-2 signaling and osteogenesis JOURNAL OF BIOLOGICAL CHEMISTRY Cowan, C. M., Quarto, N., Warren, S. M., Salim, A., Longaker, M. T. 2003; 278 (34): 32005-32013

    Abstract

    The ability of immature animals to orchestrate successful calvarial ossification has been well described. This capacity is markedly attenuated in mature animals and humans greater than 2 years of age. Few studies have investigated biological differences between juvenile and adult osteoblasts that mediate successful osteogenesis. To identify possible mechanisms for this clinical observation, we investigated cellular and molecular differences between primary osteoblasts derived from juvenile (2-day-old) and adult (60-day-old) rat calvaria. Data demonstrated that juvenile osteoblasts contain a subpopulation of less differentiated cells as observed by spindle-like morphology and decreased osteocalcin production. Juvenile, compared with adult, osteoblasts showed increased proliferation and adhesion. Furthermore, following rhFGF-2 stimulation juvenile osteoblasts increased expression of collagen I alpha 1 (5-fold), osteopontin (13-fold), and osteocalcin (16-fold), compared with relatively unchanged adult osteoblasts. Additionally, juvenile osteoblasts organized and produced more matrix proteins and formed 41-fold more bone nodules. Alternatively, adult osteoblasts produced more FGF-2 and preferentially translated the high molecular weight (22 kDa) form. Although adult osteoblasts transcribed more FGF-R1 and juvenile osteoblasts transcribed more FGF-R2 at baseline levels, juvenile osteoblasts translated more FGF-R1 and -R2 and showed increased phosphorylation. Collectively, these findings begin to explain why juvenile, but not adult, osteoblasts successfully heal calvarial defects.

    View details for DOI 10.1074/jbc.M304698200

    View details for Web of Science ID 000184782100070

    View details for PubMedID 12788918

  • FGF-2 stimulation affects calvarial osteoblast biology: Quantitative analysis of nine genes important for cranial suture biology by real-time reverse transcription polymerase chain reaction PLASTIC AND RECONSTRUCTIVE SURGERY Mathy, J. A., Lenton, K., Nacamuli, R. P., Fong, K. D., Song, H. M., Fang, T. D., Yang, G. P., Longaker, M. T. 2003; 112 (2): 528-539

    Abstract

    Appropriately timed closure of the cranial sutures is a critical factor in normal postnatal morphogenesis of the cranial vault. Suture patency is necessary to permit rapid neonatal expansion of the cerebral hemispheres, and later ossification is important for bony protection of the cerebrum. Premature suture ossification (craniosynostosis) leads to myriad adverse functional and developmental consequences. Several murine studies have implicated dura-derived fibroblast growth factor-2 (FGF-2) paracrine signaling as a critical factor promoting physiologic posterior frontal suture fusion. In this study, the authors used real-time reverse transcription polymerase chain reaction (RT-PCR) to study an in vitro system that models the in vivo stimulation of suture calvarial osteoblasts by dura-derived FGF-2. The authors advocate real-time RT-PCR as a powerful and rapid technique that offers advantages in the highly sensitive, specific, and reproducible analyses of nine genes known to be important in cranial suture biology. The genes studied were growth factors [FGF-2, transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3], growth factor receptors (FGF-R1, FGF-R2, TGF-beta RI, and TGF-beta RII), and a marker of osteoblast differentiation (Co1-I alpha I). These analyses provide a "snapshot" of several important genes involved in suture fusion that is more inclusive and quantitative than that which has been previously reported.

    View details for DOI 10.1097/01.PRS.0000070729.05978.BB

    View details for Web of Science ID 000184532700020

    View details for PubMedID 12900611

  • The influence of temperature on the degradation rate of LactoSorb Copolymer. Pietrzak WS, Kumar M, Eppley BL. J Craniofac Surg 2003; 14:176-183. journal of craniofacial surgery Fong, K. D., Nacamuli, R. P., Longaker, M. T. 2003; 14 (4): 594-595

    View details for PubMedID 12947922

  • Porous polymethylmethacrylate as bone substitute in the craniofacial area. Bruens ML, Pieterman H, de Wijn JR, et al. J Craniofac Surg 2003; 14:63-68. journal of craniofacial surgery Lenton, K. A., Nacamuli, R. P., Longaker, M. T. 2003; 14 (4): 596-598

    View details for PubMedID 12947924

  • In utero surgery for cleft lip/palate: Minimizing the "ripple effect" of scarring JOURNAL OF CRANIOFACIAL SURGERY Lorenz, H. P., Longaker, M. T. 2003; 14 (4): 504-511

    Abstract

    Surgical intervention is currently performed on highly selected fetuses with anatomical deformities that have a high mortality or severe morbidity when treated postnatally. In the future, in utero surgical intervention for non-life-threatening disease may become possible as fetal surgery becomes safer for the mother and fetus. Fetal cleft repair is an attractive intervention for plastic surgeons because it affords the potential to provide a scarless repair and correct the primary deformity. Furthermore, scarless fetal lip and palate repairs may prevent the ripple effect of postnatal scarring with its resultant secondary dentoalveolar and midface growth deformities. These potential benefits can dramatically reduce the number of postnatal reconstructive procedures in children with facial clefts. The rationale for a prenatal treatment approach to the patient with cleft lip/palate and the experimental evidence to support in utero intervention are discussed in this article.

    View details for Web of Science ID 000184409100019

    View details for PubMedID 12867864

  • Molecular cloning and expression of keratinocyte proline-rich protein, a novel squamous epithelial marker isolated during skin development JOURNAL OF BIOLOGICAL CHEMISTRY Kong, W. Y., Longaker, M. T., Lorenz, H. P. 2003; 278 (25): 22781-22786

    Abstract

    We describe a novel rat cDNA named keratinocyte proline-rich protein (KPRP) isolated by RNA differential display during skin development. We determine that KPRP is expressed in stratified squamous epithelium, and its approximately 2.8-kb cDNA encodes a 699-amino acid protein with high proline content (19%). KPRP is an insoluble protein, similar to most epidermal terminal differentiation-associated proteins. Immunoblot of the protein lysate from keratinocytes, using strong reducing conditions, demonstrates two KPRP bands of approximately 76 and 55 kDa size. KPRP is expressed in stratified squamous epithelia of skin, tongue, and esophagus. The initiation of KPRP expression in fetal rat skin at E17, E18, E19, E20, and E21 was analyzed by reverse transcription-PCR. Fetal skin at E19 and later expresses KPRP. In situ hybridization of skin from E18, E19, and 4-day-old neonatal rats demonstrates that interfollicular and follicular keratinocytes express KPRP. Anti-KPRP antibody demonstrates KPRP protein localizes to all layers of stratified epithelia in skin, tongue, and esophagus. In cultured dermal keratinocytes, KPRP is diffusely distributed throughout the cytoplasm with denser staining adjacent to the nuclear and plasma membranes. Additionally, immunoreactive intracellular granules are observed during keratinocyte detachment from their plastic substrate. Rat KPRP has 89% homology to a mouse genomic DNA sequence and 56% homology to a human hypothetical protein. We conclude that KPRP may be a new epidermal terminal differentiation-related protein expressed in stratified squamous epithelia. KPRP is expressed by fetal dermal keratinocytes during late gestation and is a new marker of maturing epidermis during fetal skin development.

    View details for DOI 10.1074/jbc.M210488200

    View details for Web of Science ID 000183503900081

    View details for PubMedID 12668678

  • New strategies for craniofacial repair and replacement: A brief review JOURNAL OF CRANIOFACIAL SURGERY Fong, K. D., Nacamuli, R. P., Song, H. J., Warren, S. M., Lorenz, H. P., Longaker, M. T. 2003; 14 (3): 333-339

    Abstract

    Craniofacial anomalies can severely affect the appearance, function, and psychosocial well being of patients; thus, tissue engineers are developing new techniques to functionally and aesthetically rebuild craniofacial structures. In the past decade, there have been tremendous advances in the field of tissue engineering that will substantially alter how surgeons approach craniofacial reconstruction. In this brief review, we highlight some of the preclinical recombinant protein, gene transfer, and cell-based strategies currently being developed to augment endogenous tissue repair or create structures for replacement. In addition, we discuss the importance of studying endogenous models of tissue induction and present some of the current in vitro and in vivo approaches to growing complex tissues/organs for craniofacial reconstruction.

    View details for Web of Science ID 000183221300011

    View details for PubMedID 12826804

  • Regional dura mater differentially regulates osteoblast gene expression JOURNAL OF CRANIOFACIAL SURGERY Warren, S. M., Greenwald, J. A., Nacamuli, R. P., Fong, K. D., Song, H. J., Fang, T. D., Mathy, J. A., Longaker, M. T. 2003; 14 (3): 363-370

    Abstract

    Recent studies have suggested that regionally differentiated dura mater regulates murine cranial suture fate by providing growth factors to the osteoblasts in the overlying suture complex. To determine if regionally differentiated dura mater is capable of effecting changes in osteoblast gene expression, an in vitro coculture system was established in which osteoblast-enriched cell cultures derived from neonatal rat calvaria were grown in serum-free media in the presence of dural cells derived from posterior frontal (PF) or sagittal (SAG) dural tissues, recapitulating the in situ relation between the underlying dura mater and the osteoblasts in the overlying cranial suture. In this study, the changes in osteoblast gene expression induced by signaling from regional dura mater were examined by analyzing total cellular RNA isolated from osteoblasts cocultured with PF or SAG dural cells. The expression of extracellular matrix molecules (alkaline phosphatase, bone sialoprotein, osteopontin, and osteocalcin) and the transcription factor Msx2 was assessed. Consistent with previous data, the findings demonstrate that osteoblasts cocultured with dural cells undergo changes in gene expression indicative of a more differentiated osteoblast. Additionally, the data suggest that regionally differentiated dura mater isolated from the PF suture enhances the expression of osteogenic genes to a greater extent than SAG suture-derived dural cells. These data support an osteoinductive role for suture-derived dural cells in vitro that may have implications for suture biology in vivo.

    View details for Web of Science ID 000183221300015

    View details for PubMedID 12826808

  • Scientific foundations - Equibiaxial tensile strain affects calvarial osteoblast biology JOURNAL OF CRANIOFACIAL SURGERY Fong, K. D., Nacamuli, R. P., Loboa, E. G., Henderson, J. H., Fang, T. D., Song, H. M., Cowan, C. M., Warren, S. M., Carter, D. R., Longaker, M. T. 2003; 14 (3): 348-355

    Abstract

    Mechanical tensile strain is believed to play an important role in regulating calvarial morphogenesis. To better understand the effects of mechanical strain on pathologic calvarial growth, we applied 10% constant equibiaxial tensile strain to neonatal rat calvarial osteoblast cultures and examined cellular proliferation, cytokine production, and extracellular matrix molecule expression. Mechanical strain markedly increased osteoblast proliferation as demonstrated by increased proliferating cell nuclear antigen (PCNA) protein. In addition, both transforming growth factor-beta1 (TGF-beta1) mRNA expression and fibroblast growth factor-2 (FGF-2) protein production were increased with exposure to strain. Moreover, mechanical strain induced expression of the extracellular matrix molecule collagen IalphaI. To further explore the relationship between mechanotransduction, osteogenesis, and angiogenesis, we examined the effect of mechanical strain on calvarial osteoblast expression of vascular endothelial growth factor (VEGF). Interestingly, we found that mechanical strain induced a rapid (within 3 hrs) increase in osteoblast VEGF expression. These data suggest that constant equibiaxial tensile strain-induced mechanotransduction can influence osteoblasts to assume an "osteogenic" and "angiogenic" phenotype, and these findings may have important implications for understanding the mechanisms of pathologic strain-induced calvarial growth.

    View details for Web of Science ID 000183221300013

    View details for PubMedID 12826806

  • The BMP antagonist noggin regulates cranial suture fusion NATURE Warren, S. M., Brunet, L. J., Harland, R. M., Economides, A. N., Longaker, M. T. 2003; 422 (6932): 625-629

    Abstract

    During skull development, the cranial connective tissue framework undergoes intramembranous ossification to form skull bones (calvaria). As the calvarial bones advance to envelop the brain, fibrous sutures form between the calvarial plates. Expansion of the brain is coupled with calvarial growth through a series of tissue interactions within the cranial suture complex. Craniosynostosis, or premature cranial suture fusion, results in an abnormal skull shape, blindness and mental retardation. Recent studies have demonstrated that gain-of-function mutations in fibroblast growth factor receptors (fgfr) are associated with syndromic forms of craniosynostosis. Noggin, an antagonist of bone morphogenetic proteins (BMPs), is required for embryonic neural tube, somites and skeleton patterning. Here we show that noggin is expressed postnatally in the suture mesenchyme of patent, but not fusing, cranial sutures, and that noggin expression is suppressed by FGF2 and syndromic fgfr signalling. Since noggin misexpression prevents cranial suture fusion in vitro and in vivo, we suggest that syndromic fgfr-mediated craniosynostoses may be the result of inappropriate downregulation of noggin expression.

    View details for DOI 10.1038/nature01545

    View details for Web of Science ID 000182111400045

    View details for PubMedID 12687003

  • Tools and techniques for craniofacial tissue engineering TISSUE ENGINEERING Warren, S. M., Fong, K. D., Chen, C. M., Loboa, E. G., Cowan, C. M., Lorenz, H. P., Longaker, M. T. 2003; 9 (2): 187-200

    Abstract

    Craniofacial surgery is an important conduit for tissue-engineering applications. As interdisciplinary collaborations improve, we can expect to see remarkable progress in de novo tissue synthesis, replacement, and repair. Ultimately, we may one day find that gene-modified cell-based tissue-engineering strategies will succeed today's reconstructive strategies. In this review, we highlight the major gene- and cell-based preclinical tools and techniques that are currently being developed to solve common craniofacial problems.

    View details for Web of Science ID 000182336200001

    View details for PubMedID 12740082

  • In utero models of craniofacial surgery WORLD JOURNAL OF SURGERY Harling, T. R., Stelnicki, E. J., Hedrick, M. H., Longaker, M. T. 2003; 27 (1): 108-116

    Abstract

    Fetal surgery is finding ever-increasing applications. At present, fetal surgical techniques are used only to treat diseases associated with high mortality risk or severe morbidity if left untreated prior to birth. Yet as a discipline, in utero therapy is attractive to plastic surgeons because it affords the potential to (1) provide a scarless repair, (2) correct the primary deformity, (3) prevent secondary deformities, and (4) give the parents a "normal"-appearing child at birth. Each of these potential benefits is particularly important in patients with craniofacial anomalies. This article will both review the animal models that have been used to study the potential for intrauterine plastic surgery and provide a synopsis of the benefits that fetal therapy may provide in the treatment of selected craniofacial anomalies.

    View details for DOI 10.1007/s00268-002-6745-2

    View details for Web of Science ID 000180270800017

    View details for PubMedID 12557046

  • Methods for investigating fetal tissue repair. Methods in molecular medicine Peled, Z. M., Warren, S. M., Bouletreau, P. J., Longaker, M. T. 2003; 78: 149-159

    View details for PubMedID 12825269

  • Matrix metalloproteinases and the ontogeny of scarless repair: The other side of the wound healing balance PLASTIC AND RECONSTRUCTIVE SURGERY Peled, Z. M., Phelps, E. D., Updike, D. L., Chang, J., Krummel, T. M., Howard, E. W., Longaker, M. T. 2002; 110 (3): 801-811

    Abstract

    Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process. In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3. Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.

    View details for DOI 10.1097/01.PRS.0000019915.20203.EC

    View details for Web of Science ID 000177332700013

    View details for PubMedID 12172142

  • Adenovirus-mediated transmission of a dominant negative transforming growth factor-beta receptor inhibits in vitro mouse cranial suture fusion PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Spector, J. A., Greenwald, J. A., Ueno, H., Longaker, M. T. 2002; 110 (2): 506-514

    Abstract

    Recent studies have implicated the transforming growth factor (TGF)-beta family in the regulation of pathological sporadic cranial suture fusion. In addition, these studies have shown that TGF-beta is highly expressed by the dura mater underlying fusing murine cranial sutures. The purpose of the present experiments was to analyze the effects of disrupting TGF-beta signaling during programmed mouse cranial suture fusion. Using recombinant DNA technology, a replication-deficient adenovirus encoding a defective TGF-beta receptor (Ad.DN-TbetaRII) capable of blocking TGF-beta biological activity was constructed. Mouse posterior frontal sutures were harvested before the initiation of suture fusion (postnatal day 25), and the dura mater underlying the suture was infected with vehicle, Ad.DN-TbetaRII, or control virus (Ad.LacZ; n = 10 each). Sutures were cultured for 14 or 30 days in an organ culture system and analyzed macroscopically and histologically.X-gal staining of Ad.LacZ-infected sutures 14 days after culture revealed strong staining of cells localized to the dura mater. Macroscopic analysis revealed complete sutural fusion in vehicle and Ad.LacZ-infected sutures. In contrast, Ad.DN-TBRII-infected sutures demonstrated nearly complete patency. Histological analysis confirmed our macroscopic observations with sutural fusion in 81.3 +/- 10 percent and 74.5 +/- 9 percent of vehicle and Ad.LacZ-infected sutures, respectively, versus 38.1 +/- 12 percent (p < 0.001) in Ad.DN-TbetaRII-infected sutures. In addition, transfection with the Ad.DN-TbetaRII virus resulted in a significant attenuation of anterior-to-posterior suture fusion, with the majority of fused sections localized to anterior sections. These data strongly implicate TGF-beta biological activity in the dura mater underlying the posterior frontal suture in the regulation of programmed sutural fusion. In addition, this study demonstrates the utility of adenovirus-mediated gene transfer in preventing programmed sutural fusion.

    View details for Web of Science ID 000177043900022

    View details for PubMedID 12142669

  • Flexor tendon healing in vitro: effects of TGF-beta on tendon cell collagen production. journal of hand surgery Klein, M. B., Yalamanchi, N., Pham, H., Longaker, M. T., Chang, J. 2002; 27 (4): 615-620

    Abstract

    Flexor tendon healing is complicated by adhesions to the surrounding sheath. Transforming growth factor beta (TGF-beta) is a cytokine with numerous activities related to wound healing. We examined the effects of TGF-beta-1, -2 and -3 on tendon cell proliferation and collagen production. Three separate cell lines--sheath fibroblasts, epitenon and endotenon tenocytes--were isolated from rabbit flexor tendons and cultured separately. Cell culture media was supplemented with 1 or 5 ng/mL of TGF-beta-1, -2, or -3. Cell number and collagen I and III production were measured and compared with unsupplemented control cultures. The addition of TGF-beta to cell culture media resulted in a decrease in cell number in all 3 lines that did not reach statistical significance. There was a significant increase (p <.05) in collagen I and III production with the addition of all 3 TGF-beta isoforms. Modulation of TGF-beta production may provide a mechanism to modulate adhesion formation clinically.

    View details for PubMedID 12132085

  • Factors in the fracture microenvironment induce primary osteoblast angiogenic cytokine production PLASTIC AND RECONSTRUCTIVE SURGERY Bouletreau, P. J., Warren, S. M., Spector, J. A., Steinbrech, D. S., Mehrara, B. J., Longaker, M. T. 2002; 110 (1): 139-148

    Abstract

    Neoangiogenesis is essential for successful wound repair. Platelets are among the earliest cells recruited to a site of skeletal injury and are thought to provide numerous factors critical to successful repair. The release of platelet-derived growth factor (PDGF) after skeletal injury increases osteoblast proliferation, chemotaxis, and collagen synthesis; however, its angiogenic effect on osteoblast biology remains unknown. The purpose of this study was to investigate the effect of recombinant human (rh)PDGF-BB on the synthesis of vascular endothelial growth factor (VEGF) by primary neonatal rat calvarial osteoblasts. Furthermore, the authors investigated whether PDGF works in concert with hypoxia, another component of the fracture microenvironment, to additively or synergistically induce VEGF production. Osteoblast cultures were stimulated with varying concentrations of rhPDGF-BB (1, 10, 50, and 100 ng/ml) in normoxic and hypoxic (<1% oxygen) conditions for 0, 3, 6, 12, and 24 hours, and VEGF gene expression was analyzed by Northern blot analysis. To determine whether rhPDGF-BB-induced VEGF messenger RNA (mRNA) expression was transcriptionally mediated or required de novo protein synthesis, transcription, and translation, studies were performed using actinomycin D and cycloheximide, respectively. Treatment with 50 ng/ml rhPDGF-BB resulted in a 2.4-fold increase in VEGF mRNA expression after 3 hours. Interestingly, rhPDGF-BB and hypoxia seemed to have an additive effect, resulting in a 3.7-fold increase in VEGF mRNA expression after 6 hours in primary neonatal rat calvarial osteoblasts. Furthermore, by using actinomycin D and cycloheximide, the authors demonstrated that the rhPDGF-BB-induced VEGF mRNA expression was transcriptionally mediate and not dependent on de novo protein synthesis. These data demonstrate that rhPDGF-BB transcriptionally increases osteoblasts VEGF mRNA expression in vitro. Furthermore, the semiquantitative results suggest that rhPDGF-BB and hypoxia act additively to increase VEGF mRNA expression. It is postulated that similar mechanisms may occur in vivo, at a site of skeletal injury, to induce neoangiogenesis and promote fracture repair.

    View details for Web of Science ID 000176399000025

    View details for PubMedID 12087245

  • Re: Sequence analysis of fibroblast growth factor receptor 2 (FGFR2) in Japanese patients with craniosynostosis. Sakai et al. J Craniofac. Surg. 2001, 12: 580-585. journal of craniofacial surgery Warren, S. M., Longaker, M. T. 2002; 13 (4): 597-599

    View details for PubMedID 12140430

  • Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: Implications for fracture healing PLASTIC AND RECONSTRUCTIVE SURGERY Bouletreau, P. J., Warren, S. M., Spector, J. A., Peled, Z. M., Gerrets, R. P., Greenwald, J. A., Longaker, M. T. 2002; 109 (7): 2384-2397

    Abstract

    The endothelium is a metabolically active secretory tissue, capable of responding to a wide array of environmental stimuli. Hypoxia and vascular endothelial growth factor (VEGF) are two components of the putative fracture microenvironment. This study investigated the role of hypoxia and VEGF on endothelial cell activation as it relates to the bone repair process. It was hypothesized that endothelial cells may have an important osteogenic role in fracture healing through the production of bone morphogenetic protein-2 (BMP-2), an osteogenic cytokine at the fracture site. Therefore, BMP-2 mRNA and protein expression in endothelial cells under hypoxia and/or VEGF treatment was studied. The authors observed a 2-fold to 3-fold up-regulation of BMP-2 mRNA expression in bovine capillary endothelial cells and human microvascular endothelial cells stimulated with hypoxia or rhVEGF. Furthermore, the combined effects of hypoxia and rhVEGF appeared to be additive on BMP-2 mRNA expression in bovine capillary endothelial cells. Actinomycin D and cycloheximide studies suggested that the increased mRNA expression was transcriptionally regulated. BMP-2 protein expression was up-regulated after 24 and 48 hours of treatment with either hypoxia or rhVEGF in bovine capillary endothelial cells. Surprisingly, the data suggest that endothelial cells may play not only an angiogenic role but also an osteogenic role by a direct stimulation of the osteoblasts, through the enhanced expression of a potent osteogenic factor, BMP-2, at the fracture site.

    View details for Web of Science ID 000175761500033

    View details for PubMedID 12045566

  • Transport distraction osteogenesis: A new method to heal adult calvarial defects PLASTIC AND RECONSTRUCTIVE SURGERY Bouletreau, P. J., Warren, S. M., Paccione, M. F., Spector, J. A., McCarthy, J. G., Longaker, M. T. 2002; 109 (3): 1074-1084

    Abstract

    Popularized by Gavril Ilizarov in the 1960s, monofocal distraction osteogenesis has become a well-established method of endogenous bone engineering. This revolutionary surgical technique has significantly augmented the available reconstructive orthopedic and craniomaxillofacial procedures. Bifocal distraction osteogenesis, or bone transportation, is a modification of monofocal distraction that involves moving a free segment of living bone to fill an intercalary bone defect. Bifocal distraction has been applied successfully to reconstruct complex mandibular and long bone defects. Because traumatic or postsurgical calvarial defects do not spontaneously heal in humans older than 18 to 24 months of age, we hypothesized that bifocal distraction osteogenesis could be applied to the skull to close critical size calvarial defects. Critical size (15 x 15 mm) calvarial defects were created in eight New Zealand White rabbits. Next, a 15-mm x 10-mm calvarial box osteotomy was created just anterior to the skull defect. This osteotomy created a free bone segment that could be transported. A custom-made transport distraction device was fixed into place and the skin incision was closed. After a 4-day latency period, the distraction device was activated (0.5 mm once daily for 30 days) in seven animals; the distraction device in one animal was not activated and served as a control. All animals underwent 30 days of consolidation and were then killed. Radiographs and computed tomographic scans were performed at the following time points: end of latency period (postoperative day 4), mid-distraction (postoperative day 19), and end of consolidation period (postoperative day 64). Gross and histologic analysis was performed to evaluate the quality of the bony regenerate. The control animal healed with a fibrous union. Complete closure of the skull defects was observed in five of seven rabbits at the end of the consolidation period. One animal was removed from the study because of an early loosening of the distraction device, and one was removed because of device failure. Of the remaining five animals that completed the distraction protocol, radiographs and computerized tomographic scans showed successful ossification in all five rabbits at the end of the consolidation period. This study suggests that transport distraction osteogenesis is a promising technique that may be applied to a variety of commonly encountered craniofacial problems such as nonhealing calvarial defects.

    View details for Web of Science ID 000174211900046

    View details for PubMedID 11884839

  • The molecular biology of distraction osteogenesis JOURNAL OF CRANIO-MAXILLOFACIAL SURGERY Bouletreau, P. J., Warren, S. M., Longaker, M. T. 2002; 30 (1): 1-11

    Abstract

    Distraction osteogenesis has become a mainstay in bone tissue engineering and has significantly improved our armamentarium for reconstructive craniomaxillofacial procedures. However, although the biomechanical, histological, and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in the interfragmental gap of gradually distracted bone segments remain largely unclear. Recently, a rat model of mandibular distraction was described that provides an excellent environment for deciphering the molecular mechanisms that mediate distraction osteogenesis. This article presents the hypotheses and current research that have furthered knowledge of the molecular mechanisms that govern distraction osteogenesis. Recent studies have implicated a growing number of cytokines that are intimately involved in the regulation of bone synthesis and turnover. The gene regulation of numerous cytokines (transforming growth factor-beta1, -beta2, -beta3, bone morphogenetic proteins, insulin-like growth factor-1, fibroblast growth factor-2) and extracellular matrix proteins (osteonectin, osteopontin) during distraction osteogenesis have been best characterized and are discussed in this article. It is believed that understanding the biomolecular mechanisms that mediate membranous distraction osteogenesis may guide the development of targeted strategies designed to improve distraction osteogenesis and accelerate bone healing.

    View details for DOI 10.1054/jems.2001.0263

    View details for Web of Science ID 000177084200001

    View details for PubMedID 12064876

  • Co-culture of osteoblasts, with immature dural cells causes an increased rate and degree of osteoblast differentiation PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Greenwald, J. A., Warren, S. M., Bouletreau, P. J., Crisera, F. E., Mehrara, B. J., Longaker, M. T. 2002; 109 (2): 631-642

    Abstract

    For decades surgeons have exploited the ability of infants to reossify large calvarial defects. To demonstrate the role of dura mater-osteoblast communication during the process of calvarial reossification, the authors used a novel in vitro system that recapitulates the in vivo anatomic relationship of these cell populations. Primary cultures of osteoblast cells from 2-day-old Sprague-Dawley rat pups were grown on six-well plates, and cultures of immature, non-suture-associated dura mater cells from 6-day-old Sprague-Dawley rat pups were grown on Transwell inserts. When the osteoblast and dura mater cell cultures reached confluence, they were combined. This Transwell co-culture system permitted the two cell populations to grow together in the same well, but it prevented direct cell-to-cell contact. Therefore, the authors were able to determine, for the first time, whether paracrine signaling from immature, non-suture-associated dura mater could influence the biologic activity of osteoblasts. Osteoblasts co-cultured with dural cells proliferated significantly faster after 2 days (2.1 x 10(5) +/- 2.4 x 10(4) versus 1.4 x 10(5) +/- 2.2 x 10(4), p < or = 0.05) and 4 days (3.1 x 10(5) +/- 5 x 10(4) versus 2.2 x 10(5) +/- 4.0 x 10(4), p < or = 0.01) than did osteoblasts cultured alone. After 20 days, co-cultured osteoblasts expressed greater amounts of mRNA for several markers of osteoblast differentiation, including collagen I alpha I (4-fold), alkaline phosphatase (2.5-fold), osteopontin (3-fold), and osteocalcin (4-fold), than did osteoblasts cultured alone. After 30 days, co-cultured osteoblasts produced bone nodules that were significantly greater both in number (324 +/- 29 nodules versus 252 +/- 29 nodules per well, p , < or = 0.04) and total area of nodules (65 +/- 11 mm(2) versus 24 +/- 1.6 mm(2), p < or = 0.003) than osteoblasts cultured alone. To begin to understand how dural cells effect changes in osteoblast gene expression, the authors compared the expression of candidate genes, transforming growth factor beta 1 and fibroblast growth factor 2, in dural cells and osteoblasts before and after 5 days of culture. Interestingly, the dura mater produced marked amounts of these osteogenic cytokines compared with osteoblasts.The described co-culture system demonstrated that co-cultured osteoblasts proliferated more rapidly and experienced an increased rate and degree of cellular maturation than did osteoblasts cultured alone. The authors hypothesize that this effect was due to paracrine signaling (e.g., transforming growth factor beta 1 and fibroblast growth factor 2) from the dura mater, and they are investigating those mechanisms in ongoing experiments. Collectively these data verify that immature, non-suture-associated dura mater can influence the biologic activity of osteoblasts. Moreover, the production of cytokines derived from the dura mater (e.g., transforming growth factor beta 1 and fibroblast growth factor 2), and they may begin to explain why immature animals and infants with intact dura mater can reossify large calvarial defects.

    View details for Web of Science ID 000173678000033

    View details for PubMedID 11818846

  • Dura mater biology: Autocrine and paracrine effects of fibroblast growth factor 2 PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Greenwald, J. A., Warren, S. M., Bouletreau, P. J., Detch, R. C., Fagenholz, P. J., Crisera, F. E., Longaker, M. T. 2002; 109 (2): 645-654

    Abstract

    The dura mater, the outermost layer of the meninges, is thought to be essential for calvarial morphogenesis, postnatal suture fusion, and osseous repair of calvarial defects. Despite numerous studies illustrating the fundamental role of the dura mater, there is little information about the autocrine and paracrine mechanisms regulating dural cell biology during calvarial ossification. Previous work conducted in the authors' laboratory demonstrated that non-suture-associated dural cells from 6-day-old rat pups expressed high levels of fibroblast growth factor 2 (FGF-2), whereas dural cells from 60-day-old adult rats expressed very little FGF-2. Because young mammals can successfully heal large calvarial defects, the authors sought to investigate the autocrine and/or paracrine effects of FGF-2 on the proliferation, gene expression, and alkaline phosphatase production of dural cells. Cultures of non-suture-associated dural cells were established from 6-day-old Sprague-Dawley rat pups and then stimulated with recombinant human FGF-2 (rhFGF-2; 10 ng/ml). Dural cells stimulated with rhFGF-2 proliferated significantly faster than untreated dural cells at 24 hours (2.1 x 10(5) +/- 3.2 x 10(4) versus 1.1 x 10(5) +/- 1.8 x 10(4), p < or = 0.001) and 48 hours (2.3 x 10(5) +/- 4.2 x 10(4) versus 1.2 x 10(5) +/- 1.3 x 10(4), p < or = 0.001). Moreover, dural cells stimulated with rhFGF-2 expressed 7-fold more proliferating cell nuclear antigen than did control cultures. Treatment with rhFGF-2 increased dural cell expression of genes important for skeletal repair: FGF-2 (7-fold), transforming growth factor beta 1 (3-fold), transforming growth factor beta 3 (4-fold), and type I collagen (4-fold). Furthermore, rhFGF-2 increased dural cell expression of osteopontin (2-fold), a "late" marker of osteoblastic differentiation. Interestingly, dural cell alkaline phosphatase activity, an "earlier" marker of osteoblast differentiation, was significantly decreased by treatment with rhFGF-2 compared with control cultures at 24 hours (0.005 +/- 0.001 versus 0.01 +/- 0.003, p < or = 0.01) and 48 hours (0.004 +/- 0.0009 versus 0.01 +/- 0.0009). Together these data provide insight into the autocrine and paracrine effects of FGF-2 on the biology of the dura mater.

    View details for Web of Science ID 000173678000035

    View details for PubMedID 11818848

  • Overhealing, underhealing, and skin regeneration: a new perspective on wound healing. Asian journal of surgery Peled, Z. M., Galiano, R., Chin, G. S., Chang, J., Krummel, T. M., Longaker, M. T. 2002; 25 (1): 102-110

    View details for PubMedID 17585454

  • The pathogenesis of craniosynostosis in the fetus YONSEI MEDICAL JOURNAL Warren, S. M., Longaker, M. T. 2001; 42 (6): 646-659

    Abstract

    Craniosynostosis occurs in approximately 1:2000 live births. It may affect the coronal, sagittal, metopic and lambdoid sutures in isolation or in combination. Although non-syndromic synostoses are more common, over 150 genetic syndromes have been identified. Recent advances in genetic mapping have linked chromosomal mutations with craniosynostotic syndromes. Despite the identification of these genetic mutations, the fundamental biomolecular mechanisms mediating cranial suture biology remain unknown. Today, many laboratories are investigating murine cranial suture biology as a model for human cranial suture development and fusion. Normal murine cranial suture biology is very complex, but evidence suggests that the dura mater provides the biomolecular blueprints (e.g. the soluble growth factors), which guide the fate of the pleuripotent osteogenic fronts. While our knowledge of these dura-derived signals has increased dramatically in the last decade, we have barely begun to understand the fundamental mechanisms that mediate cranial suture fusion or patency. Interestingly, recent advances in both premature human and programmed murine suture fusion have revealed unexpected results, and have generated more questions than answers.

    View details for Web of Science ID 000173016100009

    View details for PubMedID 11754148

  • Gene expression of transforming growth factor beta isoforms in interposition nerve grafting JOURNAL OF HAND SURGERY-AMERICAN VOLUME Karanas, Y. L., Bogdan, M. A., Lineaweaver, W. C., Hentz, V. R., Longaker, M. T., Chang, J. 2001; 26A (6): 1082-1087

    Abstract

    Scar production and neuroma formation at nerve graft coaptation sites may limit axonal regeneration and impair functional outcome. Transforming growth factor beta (TGF-beta) is a family of growth factors that is involved in scar formation, wound healing, and nerve regeneration. Fifteen adult Sprague-Dawley rats underwent autogenous nerve grafting. The nerve grafts were analyzed by in situ hybridization to determine the temporal and spatial expression of TGF-beta1 and TGF-beta3 messenger RNA (mRNA). The grafted nerves showed increased expression of TGF-beta1 and TGF-beta3 mRNA in the nerve and the surrounding connective tissue during the first postoperative week. These data suggest that modulation of TGF-beta levels in the first postoperative week may be effective in helping to control scar formation and improve nerve regeneration.

    View details for Web of Science ID 000172412500014

  • Fetal wound healing: Progress report and future directions SURGERY Longaker, N. T., Peled, Z. M., Chang, J., Krummel, T. M. 2001; 130 (5): 785-787

    View details for Web of Science ID 000172026000002

    View details for PubMedID 11685186

  • The ontogeny of scarless healing II: EGF and PDGF-B gene expression in fetal rat skin and fibroblasts as a function of gestational age ANNALS OF PLASTIC SURGERY Peled, Z. M., Rhee, S. J., HSU, M., Chang, J., Krummel, T. M., Longaker, M. T. 2001; 47 (4): 417-424

    Abstract

    Twenty years ago, surgeons noted the ability of early-gestation fetal skin to heal in a scarless manner. Since that time, numerous investigators have attempted to elucidate the mechanisms behind this phenomenon. As a result of this effort, it is now well established that many animals undergo a transition late in development from scarless cutaneous healing to a scar-forming, adultlike phenotype. The authors have been interested in the role played by cytokines known to be involved in the adult wound-healing process and how they relate to scarless repair. They therefore asked the following question: Are genes for epidermal growth factor (EGF) and platelet-derived growth factor-B (PDGF-B) expressed differentially as a function of gestational age in fetal rat skin and dermal fibroblasts? To answer this question, skin from fetal Sprague-Dawley rats (N = 56) at time points that represented both the scarless and scar-forming periods of rat gestation was harvested. In addition, fibroblasts derived from fetal rat skin were cultured in vitro at similar times. These cells were expanded in culture and, when confluent, total ribonucleic acid from both fibroblasts and whole skin was extracted and subjected to Northern blot analysis with probes for EGF and PDGF-B. Results demonstrated that neither EGF nor PDGF-B gene expression changed markedly as a function of gestational age in fetal fibroblasts alone. In whole skin, however, both EGF and PDGF-B demonstrated a marked decrease in gene expression with increasing gestational age. Furthermore, the most striking decrease in gene expression for both cytokines came between 16 and 18 days of gestation-the transition point between scarless and scar-forming repair in the fetal rat. These data suggest that EGF and PDGF may play a role in the mechanism of scarless cutaneous repair. Moreover, it appears that fetal fibroblasts are not the cell type responsible for this differential gene expression. These results raise questions about the unique cytokine milieu likely to be present during the time of scarless healing and the cells that ultimately guide the mechanisms leading to skin regeneration.

    View details for Web of Science ID 000171407400010

    View details for PubMedID 11601578

  • New directions in plastic surgery research CLINICS IN PLASTIC SURGERY Warren, S. M., Longaker, M. T. 2001; 28 (4): 719-?

    Abstract

    Plastic surgery research affords tremendous opportunities in a variety of affluent mode systems. Only recently have researchers applied molecular biologic techniques to common plastic surgery problems. For example, investigating the fundamental biomolecular mechanisms of normal palate and cranial suture morphogenesis will improve the understanding of the etiopathogenesis of CLP and craniosynostosis and facilitate the development of biologically-based interventions. Furthermore, as interdisciplinary collaborations improve, surgeons can expect to see remarkable progress in de novo tissue synthesis, replacement, and repair. Ultimately, they may one day find that gene-modified endogenous tissue engineering will succeed today's biocompatible scaffolds and allogeneic or zenogeneic replacement strategies. In general, plastic surgeons can look forward to the development of highly effective biomolecular treatments for clinical problems such as complex wound repair, prolific scarring, bone deficits (or surpluses), and organ system replacement or repair. Researchers believe that biologically-based strategies like these will be combined with technical advances that harness minimally invasive approaches. Together, clinicians expect these new tactics will reduce morbidity and improve the results of clinical problems treated by plastic surgeons.

    View details for Web of Science ID 000171901500008

    View details for PubMedID 11727856

  • A mouse model of mandibular osteotomy healing JOURNAL OF CRANIOFACIAL SURGERY Paccione, M. F., Warren, S. M., Spector, J. A., Greenwald, J. A., Bouletreau, P. J., Longaker, M. T. 2001; 12 (5): 444-450

    Abstract

    The purpose of this study was to establish a novel mouse model of membranous osteotomy healing. By applying this model to transgenic mice or using in situ hybridization techniques, we can subsequently investigate candidate genes that are believed to be important in membranous osteotomy healing. In the current study, 20 adult male CD-1 mice underwent a full-thickness osteotomy between the second and third molars of the right hemimandible using a 3-mm diamond disc and copious irrigation. Compo-Post pins were secured into the mandible, 2 mm anterior and posterior to the osteotomy. After the soft tissues were reapproximated and the skin was closed, an acrylic external fixator was attached to the exposed posts for stabilization. The animals were killed on postoperative day number 7, 10, 14, and 28 (n=5 animals per time point). The right hemimandibles were decalcified and embedded in paraffin for histologic evaluation or immunohistochemistry localizing osteocalcin. At 7 days after the osteotomy, early intramembranous bone formation could be seen extending from either edge of the osteotomized bone. By 10 days, an increasing number of small blood vessels could be seen within and around the osteotomy. At 14 days, the bone edges were in close approximation, and by 28 days the callus had been replaced by actively remodeling woven bone in all specimens examined. Immunohistochemistry demonstrated that osteocalcin expression correlated temporally with the transition from a soft to a hard callus. Furthermore, osteocalcin was spatially confined to osteoblasts actively laying down new osteoid or remodeling bone. This study describes a novel mouse model of membranous osteotomy healing that can be used as a paradigm for future osteotomy healing studies investigating candidate genes critical for osteogenesis and successful bone repair.

    View details for Web of Science ID 000170944000008

    View details for PubMedID 11572249

  • Hypoxia regulates osteoblast gene expression JOURNAL OF SURGICAL RESEARCH Warren, S. M., Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Greenwald, J. A., Spector, J. A., Bouletreau, P. J., Longaker, M. T. 2001; 99 (1): 147-155

    Abstract

    Vascular disruption secondary to fracture creates a hypoxic gradient of injury wherein the oxygen tension at the center of the wound is very low. In vivo this hypoxic microenvironment stimulates the expression of a variety of cytokines from inflammatory cells, fibroblasts, endothelial cells, and osteoblasts. In order to begin to dissect this complex system, we have examined the effects of hypoxia on isolated osteoblast gene expression in vitro. Understanding gene expression in this system may facilitate the development of targeted therapeutic modalities designed to accelerate fracture repair and reduce complications. Using an established model of in vitro hypoxia, we have analyzed the expression of genes involved in bone matrix production and turnover. Subconfluent neonatal rat calvarial osteoblasts were exposed to hypoxia (pO(2) = 35-40 mm Hg) and total cellular RNA was collected at 0, 3, 6, 24, and 48 h. Northern analysis was used to analyze the expression patterns of (1) transforming growth factors (TGFs)-beta1, -beta2, and -beta3 and their type I receptor; (2) collagens I and III; and (3) tissue inhibitor of metalloproteinase-1. We have demonstrated a marked elevation of TGF-beta1 gene expression within 3 h of hypoxia. Although neither TGF-beta2 nor TGF-beta3 expression was affected by hypoxia, the TGF-beta type I receptor was substantially upregulated within 6 h. In addition, extracellular matrix scaffolding molecules (collagens I and III) were markedly, but differentially, upregulated. Finally, we have demonstrated that the expression of an inhibitor of extracellular matrix turnover, the tissue inhibitor of metalloproteinase-1, was strikingly decreased in response to hypoxia. These results imply that hypoxia can affect osseous healing by altering the expression of cytokines, bone-specific extracellular matrix molecules, and their regulators.

    View details for DOI 10.1006/jsre.2001.6128

    View details for Web of Science ID 000169838100021

    View details for PubMedID 11421617

  • Role of TGF-beta signaling in the regulation of programmed cranial suture fusion JOURNAL OF CRANIOFACIAL SURGERY Longaker, M. T. 2001; 12 (4): 389-390

    View details for Web of Science ID 000169712600016

    View details for PubMedID 11482626

  • Distraction osteogenesis of the craniofacial skeleton PLASTIC AND RECONSTRUCTIVE SURGERY McCarthy, J. G., Stelnicki, E. J., Mehrara, B. J., Longaker, M. T. 2001; 107 (7): 1812-1827

    Abstract

    Distraction osteogenesis is becoming the treatment of choice for the surgical correction of hypoplasias of the craniofacial skeleton. Its principle is based on the studies of Ilizarov, who showed that osteogenesis can be induced if bone is expanded (distracted) along its long axis at the rate of 1 mm per day. This process induces new bone formation along the vector of pull without requiring the use of a bone graft. The technique also provides the added benefit of expanding the overlying soft tissues, which are frequently deficient in these patients. This article reviews the authors' 11-year clinical and research experience with mandibular distraction osteogenesis. It highlights the indications and contraindications of the technique and emphasizes the critical role that basic science research has played in its evolution.

    View details for Web of Science ID 000169013300029

    View details for PubMedID 11391207

  • Ontogeny of expression of transforming growth factor-beta 1 (TGF-beta 1), TGF-beta 3, and TGF-beta receptors I and II in fetal rat fibroblasts and skin PLASTIC AND RECONSTRUCTIVE SURGERY HSU, M., Peled, Z. M., Chin, G. S., Liu, W., Longaker, M. T. 2001; 107 (7): 1787-1794

    Abstract

    Fetal cutaneous wounds that occur in early gestation heal without scar formation. Although much work has been done to characterize the role of transforming growth factor-beta (TGF-beta) isoforms in the adult wound repair process, their function in fetal scarless wound repair is not well understood. The authors hypothesized that the pattern of expression for TGF-beta isoforms and their receptors may influence the phenotypic transition from scarless to scar-forming repair observed during fetal gestation. Using time-dated fetal Sprague-Dawley rat fibroblasts and unwounded skin at gestational ages 14, 16, 18, and 21 days postcoitum of the scarless (< or =16 days) and scar-forming (>16 days) periods of gestation (term = 21.5 days), the authors analyzed the endogenous messenger RNA (mRNA) levels of TGF-beta 1 and TGF-beta 3 and their signaling receptors TGF-beta-RI and TGF-beta-RII. Northern blot analyses in both fibroblasts and unwounded skin revealed that levels of TGF-beta 1 were not differentially expressed, whereas more TGF-beta 3 mRNA transcript was found in early than in late gestation. Fibroblast expression of TGF-beta-RI showed no substantial differences, whereas expression of TGF-beta-RII increased during gestation. In contrast, expression of both TGF-beta-RI and TGF-beta-RII in unwounded skin showed decreasing levels as a function of gestational age. The differential levels of TGF-beta 1 and TGF-beta 3 suggest that the ratio of these cytokines may provide a predominantly antiscarring or profibrotic signal upon wounding during the scar-free or scar-forming periods of gestation, respectively. Furthermore, lower amounts of the ligand-binding TGF-beta-RII seen in early gestation fibroblasts suggest a decreased ability to perceive ligand during the period of scarless repair.

    View details for Web of Science ID 000169013300023

    View details for PubMedID 11391201

  • Discoidin domain receptors and their ligand, collagen, are temporally regulated in fetal rat fibroblasts in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Chin, G. S., Lee, S., HSU, M., Liu, W., Kim, W. J., Levinson, H., Longaker, M. T. 2001; 107 (3): 769-776

    Abstract

    The biochemical regulation of collagen deposition during adult cutaneous wound repair is poorly understood. Likewise, how collagen is perceived and modulated in fetal scarless healing remains unknown. Recently, discoidin domain receptors-1 and 2 (DDR1 and DDR2) with tyrosine kinase activity have been identified as novel receptors for collagen. In light of these findings, it was speculated that the production of collagen receptors DDR1 and DDR2 by fetal fibroblasts may be temporally regulated to correlate with the ontogeny of embryonic scar formation. More specifically, because DDRs directly bind collagen and transmit the signals intracellularly, it was hypothesized that they may play an important role in fetal scarless healing by ultimately regulating and modulating collagen production and organization. As part of a fundamental assessment to elucidate the role of DDRs in scarless fetal wound repair, the endogenous expression of DDR1, DDR2, collagen I, and total collagen, as a function of fetal Sprague-Dawley rat skin fibroblasts of different gestational ages, representing scar-free (E16.5) periods was determined. Using explanted dermal fibroblasts of gestational days E13.5, E16.5, E18.5, and E21.5 (term gestation = 21.5 days) fetuses (n = 92), [3H]proline incorporation assay and Northern and Western blotting analysis were performed to compare the expressions of these molecules with scar-free and scar-forming stages of embryonic development. These results revealed a pattern of increasing collagen production with increasing gestational ages, whereas DDR1 expression decreased with increasing gestational age. This observation suggests that elevated levels of DDR1 may play an important role in scarless tissue regeneration by early gestation fetal fibroblasts. In contrast, DDR2 was expressed by fetal rat fibroblasts at a similar level throughout gestation. These data demonstrate for the first time the temporal expression of collagen and DDR tyrosine kinases in fetal rat fibroblasts as a function of gestational ages. Overall, these data suggest that differential temporal expression of the above-mentioned molecules during fetal skin development may play an important role in the ontogeny of scar formation. Future studies will involve the characterization of the biomolecular functions of these receptor kinases during fetal wound repair.

    View details for Web of Science ID 000167122900018

    View details for PubMedID 11304604

  • Rat mandibular distraction osteogenesis: Latency, rate, and rhythm determine the adaptive response JOURNAL OF CRANIOFACIAL SURGERY Paccione, M. F., Mehrara, B. J., Warren, S. M., Greenwald, J. A., Spector, J. A., Luchs, J. S., Longaker, M. T. 2001; 12 (2): 175-182

    Abstract

    Distraction osteogenesis is a well-established technique of endogenous tissue engineering. The biomechanical factors thought to affect the quality of the distraction regenerate include the latency, rate, rhythm, and consolidation period. In an effort to understand the impact of these parameters on regenerate bone formation, this study was designed to decipher the most adaptive response in a rat model of mandibular distraction osteogenesis. Ninety-six adult Sprague-Dawley rats were divided into 16 subgroups (n = 6 per subgroup) based on variations in the distraction parameters (i.e., latency, rate, and rhythm). After a 28-day consolidation period, the mandibles were harvested, decalcified, and sectioned. A standardized histologic ranking system was used to evaluate the effect of each protocol on the adaptive response of the regenerate bone. In this study, we have demonstrated that the latency period dramatically affects the success of distraction osteogenesis. Furthermore, distraction rates up to 0.50 mm per day stimulated excellent regenerate bone formation, whereas greater distraction rates produced a fibrous union. Finally, higher frequency distraction (i.e., increased rhythm) appeared to accelerate regenerate bone formation. We believe that defining the critical parameters of this model will improve future analysis of gene expression during rat mandibular distraction osteogenesis and may facilitate the development of biologically based strategies designed to enhance regenerate bone formation.

    View details for Web of Science ID 000170020400015

    View details for PubMedID 11314629

  • Osteoblast gene expression is differentially regulated by TGF-beta isoforms JOURNAL OF CRANIOFACIAL SURGERY Fagenholz, P. J., Warren, S. M., Greenwald, J. A., Bouletreau, P. J., Spector, J. A., Crisera, F. E., Longaker, M. T. 2001; 12 (2): 183-190

    Abstract

    The transforming growth factor beta (TGF-beta) superfamily encompasses a number of important growth factors including several TGF-beta isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-beta 1, -beta 2, and -beta 3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair. Numerous studies suggest that each isoform has unique in vivo functions; however, the effects of these TGF-beta isoforms on osteoblast gene expression and maturation have never been directly compared. In the current study, we treated undifferentiated neonatal rat calvaria osteoblast-enriched cell cultures with 2.5 ng/ml of each TGF-beta isoform and analyzed gene expression at 0, 3, 6, and 24 hours. We demonstrated unique isoform-specific regulation of endogenous TGF-beta 1 and type I collagen mRNA transcription. To assess the effects of extended TGF-beta treatment on osteoblast maturation, we differentiated osteoblast cultures in the presence of 2.5 ng/ml of each TGF-beta isoform. Analysis of collagen I, alkaline phosphatase, and osteocalcin demonstrated that each TGF-beta isoform uniquely suppressed the transcription of these osteoblast differentiation markers. Interestingly, TGF-beta isoform treatment increased osteopontin expression in primary osteoblasts after 4 and 10 days of differentiation. To our knowledge, these data provide the first direct comparison of the effects of the TGF-beta isoforms on osteoblast gene expression in vitro. Furthermore, these data suggest that TGF-beta isoforms may exert their unique in vivo effects by differentially regulating osteoblast cytokine secretion, extracellular matrix production, and the rate of cellular maturation.

    View details for Web of Science ID 000170020400016

    View details for PubMedID 11314630

  • New developments in cranial suture research PLASTIC AND RECONSTRUCTIVE SURGERY Warren, S. M., Greenwald, J. A., Spector, J. A., Bouletreau, P., Mehrara, B. J., Longaker, M. T. 2001; 107 (2): 523-540

    View details for Web of Science ID 000166825100034

    View details for PubMedID 11214072

  • Rat mandibular distraction osteogenesis: Part III. Gradual distraction versus acute lengthening PLASTIC AND RECONSTRUCTIVE SURGERY Warren, S. M., Mehrara, B. J., Steinbrech, D. S., Paccione, M. F., Greenwald, J. A., Spector, J. A., Longaker, M. T. 2001; 107 (2): 441-453

    Abstract

    Distraction osteogenesis is a well-established method of endogenous tissue engineering. This technique has significantly augmented our armamentarium of reconstructive craniofacial procedures. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been extensively described, the molecular mechanisms governing successful membranous distraction remain unknown. Using an established rat model, the molecular differences between successful (i.e., osseous union with gradual distraction) and ineffective (i.e., fibrous union with acute lengthening) membranous bone lengthening was analyzed. Herein, the first insight into the molecular mechanisms of successful membranous bone distraction is provided. In addition, these data provide the foundation for future targeted therapeutic manipulations designed to improve osseous regeneration. Vertical mandibular osteotomies were created in 52 adult male Sprague-Dawley rats, and the animals were fitted with customized distraction devices. Twenty-six animals underwent immediate acute lengthening (3 mm; a length previously shown to result in fibrous union) and 26 animals were gradually distracted (after a 3-day latency period, animals were distracted 0.25 mm twice daily for 6 days; total = 3 mm). Four mandibular regenerates were harvested from each group for RNA analysis on 5, 7, 9, 23, and 37 days postoperatively (n = 40). Two mandibular regenerates were also harvested from each group and prepared for immunohistochemistry on postoperative days 5, 7, and 37 (n = 12). In addition to the 52 experimental animals, 4 control rats underwent sham operations (skin incision only) and mandibular RNA was immediately collected. Control and experimental specimens were analyzed for collagen I, osteocalcin, tissue inhibitor of metalloproteinase-1, and vascular endothelial growth factor mRNA and protein expression. In this study, marked elevation of critical extracellular matrix molecules (osteocalcin and collagen I) during the consolidation phase of gradual distraction compared with acute lengthening is demonstrated. In addition, the expression of an inhibitor of extracellular matrix turnover, tissue inhibitor of metalloproteinase-1, remained strikingly elevated in gradually distracted animals. Finally, this study demonstrated that neither gradual distraction nor acute lengthening appreciably alters vascular endothelial growth factor expression. These results suggest that gradual distraction osteogenesis promotes successful osseous bone repair by regulating the expression of bone-specific extracellular matrix molecules. In contrast, decreased production or increased turnover of bone scaffolding proteins (i.e., collagen) or regulators of mineralization (i.e., osteocalcin) may lead to fibrous union during acute lengthening.

    View details for Web of Science ID 000166825100021

    View details for PubMedID 11214060

  • In vivo modulation of FGF biological activity alters cranial suture fate AMERICAN JOURNAL OF PATHOLOGY Greenwald, J. A., Mehrara, B. J., Spector, J. A., Warren, S. M., Fagenholz, P. J., Smith, L. P., Bouletreau, P. J., Crisera, F. E., Ueno, H., Longaker, M. T. 2001; 158 (2): 441-?

    Abstract

    Gain-of-function mutations in fibroblast growth factor receptors have been identified in numerous syndromes associated with premature cranial suture fusion. Murine models in which the posterior frontal suture undergoes programmed fusion after birth while all other sutures remain patent provide an ideal model to study the biomolecular mechanisms that govern cranial suture fusion. Using adenoviral vectors and targeted in utero injections in rats, we demonstrate that physiological posterior frontal suture fusion is inhibited using a dominant-negative fibroblast growth factor receptor-1 construct, whereas the normally patent coronal suture fuses when infected with a construct that increases basic fibroblast growth factor biological activity. Our data may facilitate the development of novel, less invasive treatment options for children with craniosynostosis.

    View details for Web of Science ID 000166925900014

    View details for PubMedID 11159182

  • Expression of bone morphogenetic proteins during membranous bone healing PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Luchs, J. S., Mehrara, B. J., Greenwald, J. A., Smith, L. P., Longaker, M. T. 2001; 107 (1): 124-134

    Abstract

    For the reconstructive plastic surgeon, knowledge of the molecular biology underlying membranous fracture healing is becoming increasingly vital. Understanding the complex patterns of gene expression manifested during the course of membranous fracture repair will be crucial to designing therapies that augment poor fracture healing or that expedite normal osseous repair by strategic manipulation of the normal course of gene expression. In the current study, we present a rat model of membranous bone repair. This model has great utility because of its technical simplicity, reproducibility, and relatively low cost. Furthermore, it is a powerful tool for analysis of the molecular regulation of membranous bone repair by immunolocalization and/or in situ hybridization techniques. In this study, an osteotomy was made within the caudal half of the hemimandible, thus producing a stable bone defect without the need for external or internal fixation. The healing process was then catalogued histologically in 28 Sprague-Dawley rats that were serially killed at 1, 2, 3, 4, 5, 6, and 8 weeks after operation. Furthermore, using this novel model, we analyzed, within the context of membranous bone healing, the temporal and spatial expression patterns of several members of the bone morphogenetic protein (BMP) family, known to be critical regulators of cells of osteoblast lineage. Our data suggest that BMP-2/-4 and BMP-7, also known as osteogenic protein-1 (OP-1), are expressed by osteoblasts, osteoclasts, and other more primitive mesenchymal cells within the fracture callus during the early stages of membranous fracture healing. These proteins continue to be expressed during the process of bone remodeling, albeit less prominently. The return of BMP-2/-4 and OP-1 immunostaining to baseline intensity coincides with the histological appearance of mature lamellar bone. Taken together, these data underscore the potentially important regulatory role played by the bone morphogenetic proteins in the process of membranous bone repair.

    View details for Web of Science ID 000166100000018

    View details for PubMedID 11176610

  • Understanding the molecular mechanisms governing distraction osteogenesis CRANIOFACIAL SURGERY 9 Mehrara, B., Warren, S., Bouletreau, P., Paccione, M., Longaker, M. 2001: 97-100
  • Immavure dura mater paracrine signaling increases osteoblast proliferation and differentiation CRANIOFACIAL SURGERY 9 Fagenholz, P., Spector, J., Greenwald, J., Warren, S., Detch, R., Mehrara, B., Longaker, M. 2001: 81-84
  • The autocrine and paracrine effects of FGF-2 on dura mater: Implications for calvarial growth and re-ossification CRANIOFACIAL SURGERY 9 Smith, L., Spector, J., Greenwald, J., Warren, S., Bouletreau, P., Detch, R., Fagenholz, P., Crisera, F., Longaker, M. 2001: 121-124
  • Regional dura mater determines fate of overlying cranial suture CRANIOFACIAL SURGERY 9 Greenwald, J., Mehrara, B., Spector, J., Warren, S., Crisera, F., Fagenholz, P., Bouletreau, P., Longaker, M. 2001: 89-92
  • Latency, rate, and rhythm determine the adaptive response in a rat model of mandibular distraction osteogenesis CRANIOFACIAL SURGERY 9 Paccione, M., Warren, S., Mehrara, B., Bouletreau, P., Longaker, M. 2001: 173-175
  • Regional differentiation of cranial suture-associated dura mater in vivo and in vitro: Implications for suture fusion and patency JOURNAL OF BONE AND MINERAL RESEARCH Greenwald, J. A., Mehrara, B. J., Spector, J. A., Warren, S. M., Crisera, F. E., Fagenholz, P. J., Bouletreau, P. J., Longaker, M. T. 2000; 15 (12): 2413-2430

    Abstract

    Despite its prevalence, the etiopathogenesis of craniosynostosis is poorly understood. To better understand the biomolecular events that occur when normal craniofacial growth development goes awry, we must first investigate the mechanisms of normal suture fusion. Murine models in which the posterior frontal (PF) suture undergoes programmed sutural fusion shortly after birth provide an ideal model to study these mechanisms. In previous studies, our group and others have shown that sutural fate (i.e., fusion vs. patency) is regulated by the dura mater (DM) directly underlying a cranial suture. These studies have led to the hypothesis that calvarial DM is regionally differentiated and that this differentiation guides the development of the overlying suture. To test this hypothesis, we evaluated the messenger RNA (mRNA) expression of osteogenic cytokines (transforming growth factor beta1 [TGF-beta1] and TGF-beta3) and bone-associated extracellular matrix (ECM) molecules (collagen I, collagen III, osteocalcin, and alkaline phosphatase) in freshly isolated, rat dural tissues associated with the PF (programmed to fuse) or sagittal (SAG; remains patent) sutures before histological evidence of sutural fusion (postnatal day 6 [N6]). In addition, osteocalcin protein expression and cellular proliferation were localized using immunohistochemical staining and 5-bromo-2'deoxyuridine (BrdU) incorporation, respectively. We showed that the expression of osteogenic cytokines and bone-associated ECM molecules is potently up-regulated in the DM associated with the PF suture. In addition, we showed that cellular proliferation in the DM associated with the fusing PF suture is significantly less than that found in the patent SAG suture just before the initiation of sutural fusion N6. Interestingly, no differences in cellular proliferation rates were noted in younger animals (embryonic day 18 [E18] and N2). To further analyze regional differentiation of cranial suture-associated dural cells, we established dural cell cultures from fusing and patent rat cranial sutures in N6 rats and evaluated the expression of osteogenic cytokines (TGF-beta1 and fibroblast growth factor 2 [FGF-2]) and collagen I. In addition, we analyzed cellular production of proliferating cell nuclear antigen (PCNA). These studies confirmed our in vivo findings and showed that dural cell cultures derived from the fusing PF suture expressed significantly greater amounts of TGF-beta1, FGF-2, and collagen I. In addition, similar to our in vivo findings, we showed that PF suture-derived dural cells produced significantly less PCNA than SAG suture-derived dural cells. Finally, coculture of dural cells with fetal rat calvarial osteoblastic cells (FRCs) revealed a statistically significant increase in proliferation (*p < 0.001) in FRCs cocultured with SAG suture-derived dural cells as compared with FRCs cocultured alone or with PF suture-derived dural cells. Taken together, these data strongly support the hypothesis that the calvarial DM is regionally differentiated resulting in the up-regulation of osteogenic cytokines and bone ECM molecules in the dural tissues underlying fusing but not patent cranial sutures. Alterations in cytokine expression may govern osteoblastic differentiation and ECM molecule deposition, thus regulating sutural fate. Elucidation of the biomolecular events that occur before normal cranial suture fusion in the rat may increase our understanding of the events that lead to premature cranial suture fusion.

    View details for Web of Science ID 000165463300014

    View details for PubMedID 11127206

  • Gene expression of transforming growth factor-beta 3 and tissue inhibitor of metalloproteinase type 1 during membranous bone healing in rats JOURNAL OF CRANIOFACIAL SURGERY Bouletreau, P. J., Steinbrech, D., Spector, J. A., Warren, S. M., Greenwald, J. A., Mehrara, B. J., Detch, R. C., Longaker, M. T. 2000; 11 (6): 521-526

    Abstract

    A number of growth factors have been implicated in fracture repair. Transforming growth factor-beta 3 (TGF-beta 3) is believed to be involved in osteoblast proliferation, chemotaxis, and collagen synthesis. The collagens act as the scaffolding for new bone matrix formation, whereas tissue inhibitors of metalloproteinases (TIMPs) may help regulate matrix remodeling in bone repair. Despite their hypothesized integral role in fracture repair, the temporal expression of these molecules in membranous bone fracture healing remains unknown. The objective of this study was to assess the temporal pattern of TGF-beta 3 and TIMP type 1 (TIMP-1) expression in rat mandibular fracture healing. Twenty-eight adult male Sprague-Dawley rats underwent a mandibular osteotomy, and the healing regenerate was harvested on postoperative days 3, 5, 7, 9, 23, and 37. Total cellular ribonucleic acid was isolated, and Northern analysis was performed. TGF-beta 3 expression was downregulated dramatically 3 days after the osteotomy and remained less than 20% of control levels throughout repair. In marked contrast, TIMP-1 gene expression, low during early repair, increased more than twofold over control at later time points. Understanding the temporal pattern of gene expression during membranous fracture healing has important clinical implications because elucidating these mechanisms may lead to appropriate biomolecular approaches to augment membranous bone fracture healing.

    View details for Web of Science ID 000167444900003

    View details for PubMedID 11314491

  • The effects of ionizing radiation on osteoblast-like cells in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Dudziak, M. E., Saadeh, P. B., Mehrara, B. J., Steinbrech, D. S., Greenwald, J. A., Gittes, G. K., Longaker, M. T. 2000; 106 (5): 1049-1061

    Abstract

    The well-described detrimental effects of ionizing radiation on the regeneration of bone within a fracture site include decreased osteocyte number, suppressed osteoblast activity, and diminished vascularity. However, the biologic mechanisms underlying osteoradionecrosis and the impaired fracture healing of irradiated bone remain undefined. Ionizing radiation may decrease successful osseous repair by altering cytokine expression profiles resulting from or leading to a change in the osteoblastic differentiation state. These changes may, in turn, cause alterations in osteoblast proliferation and extracellular matrix formation. The purpose of this study was to investigate the effects of ionizing radiation on the proliferation, maturation, and cytokine production of MC3T3-E1 osteoblast-like cells in vitro. Specifically, the authors examined the effects of varying doses of ionizing radiation (0, 40, 400, and 800 cGy) on the expression of transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and alkaline phosphatase. In addition, the authors studied the effects of ionizing radiation on MC3T3-E1 cellular proliferation and the ability of conditioned media obtained from control and irradiated cells to regulate the proliferation of bovine aortic endothelial cells. Finally, the authors evaluated the effects of adenovirus-mediated TGF-beta1 gene therapy in an effort to "rescue" irradiated osteoblasts. The exposure of osteoblast-like cells to ionizing radiation resulted in dose-dependent decreases in cellular proliferation and promoted cellular differentiation (i.e., increased alkaline phosphatase production). Additionally, ionizing radiation caused dose-dependent decreases in total TGF-beta1 and VEGF protein production. Decreases in total TGF-beta1 production were due to a decrease in TGF-beta1 production per cell. In contrast, decreased total VEGF production was secondary to decreases in cellular proliferation, because the cellular production of VEGF by irradiated osteoblasts was moderately increased when VEGF production was corrected for cell number. Additionally, in contrast to control cells (i.e., nonirradiated), conditioned media obtained from irradiated osteoblasts failed to stimulate the proliferation of bovine aortic endothelial cells. Finally, transfection of control and irradiated cells with a replication-deficient TGF-beta1 adenovirus before irradiation resulted in an increase in cellular production of TGF-beta1 protein and VEGF. Interestingly, this intervention did not alter the effects of irradiation on cellular proliferation, which implies that alterations in TGF-beta1 expression do not underlie the deficiencies noted in cellular proliferation. The authors hypothesize that ionizing radiation-induced alterations in the cytokine profiles and differentiation states of osteoblasts may provide insights into the cellular mechanisms underlying osteoradionecrosis and impaired fracture healing.

    View details for Web of Science ID 000089824900015

    View details for PubMedID 11039376

  • A molecular analysis of the isolated rat posterior frontal and sagittal sutures: Differences in gene expression PLASTIC AND RECONSTRUCTIVE SURGERY Spector, J. A., Mehrara, B. J., Greenwald, J. A., Saadeh, P. B., Steinbrech, D. S., Smith, L. P., Longaker, M. T. 2000; 106 (4): 852-861

    Abstract

    Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture.

    View details for Web of Science ID 000089084200017

    View details for PubMedID 11007400

  • Immature versus mature dura mater: II. Differential expression of genes important to calvarial reossification PLASTIC AND RECONSTRUCTIVE SURGERY Greenwald, J. A., Mehrara, B. J., Spector, J. A., Fagenholz, P. J., Saadeh, P. B., Steinbrech, D. S., Gittes, G. K., Longaker, M. T. 2000; 106 (3): 630-638

    Abstract

    The ability of immature animals and newborns to orchestrate successful calvarial reossification is well described. This capacity is markedly attenuated in mature animals and in humans greater than 2 years of age. Previous studies have implicated the dura mater as critical to successful calvarial reossification. The authors have previously reported that immature, but not mature, dural tissues are capable of elaborating a high expression of osteogenic growth factors and extracellular matrix molecules. These findings led to the hypothesis that a differential expression of osteogenic growth factors and extracellular matrix molecules by immature and mature dural tissues may be responsible for the clinically observed phenotypes (i.e., immature animals reossify calvarial defects; mature animals do not). This study continues to explore the hypothesis through an analysis of transforming growth factor (TGF)-beta3, collagen type III, and alkaline phosphatase mRNA expression. Northern blot analysis of total RNA isolated from freshly harvested immature (n = 60) and mature (n = 10) dural tissues demonstrated a greater than three-fold, 18-fold, and nine-fold increase in TGF-beta3, collagen type III, and alkaline phosphatase mRNA expression, respectively, in immature dural tissues as compared with mature dural tissues. Additionally, dural cell cultures derived from immature (n = 60) and mature dura mater (n = 10) were stained for alkaline phosphatase activity to identify the presence of osteoblast-like cells. Alkaline phosphatase staining of immature dural cells revealed a significant increase in the number of alkaline phosphatase-positive cells as compared with mature dural tissues (p < 0.001). In addition to providing osteogenic humoral factors (i.e., growth factors and extracellular matrix molecules), this finding suggests that immature, but not mature, dura mater may provide cellular elements (i.e., osteoblasts) that augment successful calvarial reossification. These studies support the hypothesis that elaboration of osteogenic growth factors (i.e., TGF-beta33) and extracellular matrix molecules (i.e., collagen type III and alkaline phosphatase) by immature, but not mature, dural tissues may be critical for successful calvarial reossification. In addition, these studies suggest for the first time that immature dural tissues may provide cellular elements (i.e., osteoblasts) to augment this process.

    View details for Web of Science ID 000088925700016

    View details for PubMedID 10987470

  • Mechanisms of fibroblast growth factor-2 modulation of vascular endothelial growth factor expression by osteoblastic cells ENDOCRINOLOGY Saadeh, P. B., Mehrara, B. J., Steinbrech, D. S., Spector, J. A., Greenwald, J. A., Chim, G. S., Ueno, H., Gittes, G. K., Longaker, M. T. 2000; 141 (6): 2075-2083

    Abstract

    Normal bone growth and repair is dependent on angiogenesis. Fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), and transforming growth factor-beta (TGFbeta) have all been implicated in the related processes of angiogenesis, growth, development, and repair. The purpose of this study was to investigate the relationships between FGF-2 and both VEGF and TGFbeta in nonimmortalized and clonal osteoblastic cells. Northern blot analysis revealed 6-fold peak increases in VEGF mRNA at 6 h in fetal rat calvarial cells and MC3T3-E1 osteoblastic cells after stimulation with FGF-2. Actinomycin D inhibited these increases in VEGF mRNA, whereas cycloheximide did not. The stability ofVEGF mRNA was not increased after FGF-2 treatment. Furthermore, FGF-2 induced dose-dependent increases in VEGF protein levels (P < 0.01). Although in MC3T3-E1 cells, TGFbeta1 stimulates a 6-fold peak increase in VEGF mRNA after 3 h of stimulation, we found that both TGFbeta2 and TGFbeta3 yielded 2- to 3-fold peak increases in VEGF mRNA levels noted after 6 h of stimulation. Similarly, both TGFbeta2 and TGFbeta3 dose dependently increased VEGF protein production. To determine whether FGF-2-induced increases in VEGF mRNA may have occurred independently of TGFbeta, we disrupted TGFbeta signal transduction (using adenovirus encoding a truncated form of TGFbeta receptor II), which attenuated TGFbeta1 induction of VEGF mRNA, but did not impede FGF-2 induction ofVEGF mRNA. In summary, FGF-2-induced VEGF expression by osteoblastic cells is a dose-dependent event that may be independent of concomitant FGF-2-induced modulation of TGFbeta activity.

    View details for Web of Science ID 000088386100025

    View details for PubMedID 10830293

  • The impact of biomolecular medicine and tissue engineering on plastic surgery in the 21st century PLASTIC AND RECONSTRUCTIVE SURGERY Lorenz, H. P., Hedrick, M. H., Chang, J., Mehrara, B. J., Longaker, M. T. 2000; 105 (7): 2467-2481

    View details for Web of Science ID 000087277300027

    View details for PubMedID 10845305

  • Gene expression of TGF-beta, TGF-beta receptor, and extracellular matrix proteins during membranous bone healing in rats PLASTIC AND RECONSTRUCTIVE SURGERY Steinbrech, D. S., Mehrara, B. J., Rowe, N. M., Dudziak, M. E., Luchs, J. S., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 2000; 105 (6): 2028-2038

    Abstract

    Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.

    View details for Web of Science ID 000086725000018

    View details for PubMedID 10839400

  • "Pumping the regenerate": An evaluation of oscillating distraction osteogenesis in the rodent mandible ANNALS OF PLASTIC SURGERY Greenwald, J. A., Luchs, J. S., Mehrara, B. J., Spector, J. A., Mackool, R. J., McCarthy, J. G., Longaker, M. T. 2000; 44 (5): 516-521

    Abstract

    Mandibular distraction osteogenesis (DO) has become an important technique to lengthen the hypoplastic mandible and to reconstruct osseous defects after ablative surgery. The hallmark of successful DO is the creation of new bone within the distraction gap. Several anecdotal reports have described alternating compressing and lengthening protocols (i.e., "pumping the regenerate") to augment regenerate bone formation. The purpose of this experiment was to analyze formally the effects of an alternating compression/distraction protocol with a traditional distraction protocol. Ten adult male rats underwent unilateral mandibular osteotomy with placement of a custom distractor. After a latency period of 5 days, distraction was initiated at a rate of 0.25 mm twice daily. Animals in the control group (N = 5) were distracted to a length of 5.0 mm for 10 days at a rate of 0.25 mm twice daily. In contrast, animals in the experimental group (N = 5) were distracted to a length of 2.5 mm (at a rate of 0.25 mm twice daily) for 5 days, then compressed 1.0 mm for a 2-day period, and redistracted to a length of 5.0 mm. Regenerate cross-sectional area was evaluated by computed tomography performed after 5 weeks of consolidation. Gross examination and histological analysis were performed by a panel of experienced reviewers. Radiological as well as histological analysis of regenerate cross-sectional area demonstrated no significant differences between experimental (i.e., "pumped") and control groups. Both groups demonstrated excellent regenerate bone formation with no evidence of fibrous union. This study represents the first attempt to investigate the anecdotal technique of pumping the mandibular regenerate. The authors have demonstrated that pumping the regenerate leads to no substantial differences in radiological or histological appearance of regenerate bone formation.

    View details for Web of Science ID 000087001700018

    View details for PubMedID 10805303

  • Expression of adenovirally delivered gene products in healing osseous tissues ANNALS OF PLASTIC SURGERY Spector, J. A., Mehrara, B. J., Luchs, J. S., Greenwald, J. A., Fagenholz, P. J., Saadeh, P. B., Steinbrech, D. S., Longaker, M. T. 2000; 44 (5): 522-528

    Abstract

    Gene therapy has moved from the promise of laboratory investigation to the reality of clinical practice in just the last decade. Various methods for delivery of genes to host cells have been developed and utilized both in vitro and in vivo. From the perspective of the plastic surgeon, gene therapy holds the promise to augment healing in clinical situations that remain difficult to treat, such as chronic wounds, osteoradionecrosis, or possibly to expedite current clinical practices, such as distraction osteogenesis. The authors chose to investigate the potential for gene therapy in osseous tissues using a replication-deficient adenovirus vector to deliver the marker transgene beta-galactosidase. An adenovirus vector is ideal for use in situations in which transgene expression is desired for only a relatively short period of time, such as wound and fracture healing. Utilizing a rat mandibular osteotomy model, they demonstrated that, using an adenoviral vector, foreign genes can be delivered in a simple fashion and can be expressed in a reliable manner within and around the osteotomy site for at least 10 days. Furthermore, there was no evidence of transfection of distant tissues associated with local application of the adenovirus vector. With this information, clinicians may now attempt to deliver osteogenic and angiogenic genes in a site-specific fashion to improve and expedite osseous healing.

    View details for Web of Science ID 000087001700019

    View details for PubMedID 10805304

  • Hypoxia increases insulinlike growth factor gene expression in rat osteoblasts ANNALS OF PLASTIC SURGERY Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Greenwald, J. A., Spector, J. A., Gittes, G. K., Longaker, M. T. 2000; 44 (5): 529-534

    Abstract

    Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.

    View details for Web of Science ID 000087001700021

    View details for PubMedID 10805305

  • Biomolecular mechanisms of calvarial bone induction: Immature versus mature dura mater PLASTIC AND RECONSTRUCTIVE SURGERY Greenwald, J. A., Mehrara, B. J., Spector, J. A., Chin, G. S., Steinbrech, D. S., Saadeh, P. B., Luchs, J. S., Paccione, M. F., Gittes, G. K., Longaker, M. T. 2000; 105 (4): 1382-1392

    Abstract

    The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greater than 2 years of age and in mature animals. The dura mater has been implicated as a regulator of calvarial reossification. To date, however, few studies have attempted to identify biomolecular differences in the dura mater that enable immature, but not mature, dura to induce osteogenesis. The purpose of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, basic fibroblast growth factor, collagen type IalphaI, osteocalcin, and alkaline phosphatase are critical growth factors and extracellular matrix proteins essential for successful osteogenesis. In this study, we have characterized the proliferation rates of immature (6-day-old rats, n = 40) and mature (adult rats, n = 10) dura cell cultures. In addition, we analyzed the expression of transforming growth factor beta-1, basic fibroblast growth factor-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in vitro findings were corroborated with Northern blot analysis of mRNA expression in total cellular RNA isolated from snap-frozen age-matched dural tissues (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of cultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than mature dural cells (p < 0.01). Additionally, immature dural cells produce significantly greater amounts of transforming growth factor beta-1, basic fibroblast growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Northern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transforming growth factor beta-1, osteocalcin, and collagen IalphaI gene expression, respectively, in immature as compared with mature dura mater. Finally, in keeping with their in vivo phenotype, immature dural cells formed large calcified bone nodules in vitro, whereas mature dural cells failed to form bone nodules even with extended culture. These studies suggest that differential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature and mature dura mater. Finally, we believe that the biomolecular bone- and matrix-inducing phenotype of immature dura mater regulates the ability of young children and immature animals to heal calvarial defects.

    View details for Web of Science ID 000085995500018

    View details for PubMedID 10744229

  • VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Greenwald, J. A., Spector, J. A., Gittes, G. K., Longaker, M. T. 2000; 278 (4): C853-C860

    Abstract

    Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone.

    View details for Web of Science ID 000086325900025

    View details for PubMedID 10751333

  • A rat model of gingivoperiosteoplasty JOURNAL OF CRANIOFACIAL SURGERY Mehrara, B. J., Saadeh, P. B., Steinbrech, D. S., Dudziak, M., Grayson, B. H., Cutting, C. B., McCarthy, J. G., Gittes, G. K., Longaker, M. T. 2000; 11 (1): 54-58

    Abstract

    The ability to avoid a subsequent bone graft makes the use of gingivoperiosteoplasty (GPP) at the time of cleft lip repair an attractive technique. The use of GPP, in combination with presurgical orthodontics, has been shown to result in successful bony union in the majority of patients. However, secondary bone grafting is still necessary in 30% to 40% of patients due to persistent alveolar bony defects. The elucidation of methods to improve the success rates of these procedures has been hampered by the lack of reproducible animal models. The purpose of this study was, therefore, to develop a rodent model of GPP that would facilitate the investigation of methods to improve osteogenesis in alveolar defects. We report a surgically produced rat model (9 x 5 x 3-mm alveolar defect) that is reproducible, inexpensive (relative to large-animal models), and simple technically. In addition, healing in this model occurs in a predictable manner during a 12-week period, thus enabling analysis of methods designed to accelerate or facilitate osseous regeneration.

    View details for Web of Science ID 000084778600010

    View details for PubMedID 11314101

  • Transforming growth factor-beta 1 modulates the expression of vascular endothelial growth factor by osteoblasts AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Saadeh, P. B., Mehrara, B. J., Steinbrech, D. S., Dudziak, M. E., Greenwald, J. A., Luchs, J. S., Spector, J. A., Ueno, H., Gittes, G. K., Longaker, M. T. 1999; 277 (4): C628-C637

    Abstract

    Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-beta1 (TGF-beta1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-beta1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-beta1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-beta1 treatment. Actinomycin D inhibited the TGF-beta1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-beta1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-beta1 induction of VEGF mRNA. Additionally, TGF-beta1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells (P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-beta1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-beta1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing.

    View details for Web of Science ID 000083919100005

    View details for PubMedID 10516092

  • Hypoxia regulates VEGF expression and cellular proliferation by osteoblasts in vitro PLASTIC AND RECONSTRUCTIVE SURGERY Steinbrech, D. S., Mehrara, B. J., Saadeh, P. B., Chin, G., Dudziak, M. E., Gerrets, R. P., Gittes, G. K., Longaker, M. T. 1999; 104 (3): 738-747

    Abstract

    Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.

    View details for Web of Science ID 000082004500019

    View details for PubMedID 10456527

  • New developments in craniofacial surgery research CLEFT PALATE-CRANIOFACIAL JOURNAL Mehrara, B. J., Longaker, M. T. 1999; 36 (5): 377-387

    Abstract

    The recent explosion in our understanding of developmental biology and genetics has enhanced our understanding of craniofacial biology. While it is not possible to summarize all new developments in craniofacial research, this article will review three areas: fetal models and surgery for craniofacial disorders, the biology of distraction osteogenesis, and the molecular mechanisms of cranial suture fusion. Numerous models of craniofacial disorders have been described, including small, short gestation and large, long gestation. The benefits and shortcomings of each are discussed. In addition, we discuss recent studies investigating the molecular mechanisms of mandibular distraction osteogenesis. Finally, we present a review of recent advances in the understanding of mechanisms of craniosynostosis, with particular emphasis on the biology of programmed cranial suture fusion in rodents.

    View details for Web of Science ID 000082621300001

    View details for PubMedID 10499398

  • Regional differentiation of rat cranial suture-derived dural cells is dependent on association with fusing and patent cranial sutures PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Greenwald, J., Chin, G. S., Dudziak, M., Sagrioglu, J., Steinbrech, D. S., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 1999; 104 (4): 1003-1013

    Abstract

    A significant body of literature supports a role for the dura mater underlying cranial sutures in the regulation of sutural fate. These studies have implicated regional differentiation of the dura mater based on association with fusing and patent rat cranial sutures. The purpose of these experiments was to isolate and characterize dural cells associated with fusing (posterior frontal) and patent (sagittal) rat cranial sutures. Six-day-old rats were killed, and the dura mater underlying the posterior frontal and sagittal sutures was harvested. Dural cells were briefly trypsinized and allowed to reach confluence. Two litters (10 animals per litter) were used for each set of experiments. Cells were harvested after the first and fifth passages for analysis of vimentin and desmoplakin expression (characteristic of human meningeal cells), cellular proliferation, density at confluence (a measure of cellular contact inhibition), and alkaline phosphatase production. In addition, bone nodule formation and collagen I production were analyzed in first passage cells. The results indicate that suture-derived dural cells can be established and that these cells coexpress vimentin and desmoplakin. In addition, it is demonstrated that first-passage sagittal suture-derived dural cells proliferate significantly faster and have decreased cellular contact inhibition than posterior frontal suture-derived cells (p < 0.01). Finally, it is shown that suture-derived dural cells have osteoblast-like properties, including alkaline phosphatase production, collagen I expression, and bone nodule formation in vitro. The possible mechanisms by which regional differentiation of suture-derived dural cells occur are discussed.

    View details for Web of Science ID 000082302500016

    View details for PubMedID 10654740

  • Basic fibroblast growth factor and transforming growth factor beta-1 expression in the developing dura mater correlates with calvarial bone formation PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Most, D., Chang, J., Bresnick, S., Turk, A., Schendel, S. A., Gittes, G. K., Longaker, M. T. 1999; 104 (2): 435-444

    Abstract

    Numerous studies have found dura mater-calvarial mesenchyme interactions during calvarial bone induction; however, the exact molecular mechanisms governing these inductive events remain unknown. Recent studies have implicated basic fibroblast growth factor (FGF-2) and transforming growth factor-beta1 (TGF-beta1) in regulating bone formation. The purpose of this study was, therefore, to investigate the expression of FGF-2 and TGF-beta1 during calvarial bone formation in rats. Eight rats were killed on embryonic days 14, 18, and 20 and neonatal day 1 (n = 32). Four animals at each time point were analyzed by in situ hybridization, and the remainder were analyzed by immunohistochemistry. The results indicated that the dura mater underlying the developing calvarial bone strongly expressed FGF-2 and TGF-beta1 mRNA at all time points examined. In contrast, minimal growth factor expression was noted in the overlying calvarial mesenchyme until embryonic day 18, but it increased significantly with increasing age. Importantly, FGF-2 and TGF-beta1 mRNA expression in the dura mater underlying the developing calvarium preceded and was significantly greater than expression in the calvarium mesenchyme (p < 0.05). Interestingly, minimal expression of FGF-2 and TGF-beta1 mRNA was noted for all time points in the dura mater underlying the posterior frontal suture and within the posterior frontal suture connective tissue (p < 0.01 when compared with the dura mater underlying the developing calvarium). Immunohistochemical findings closely paralleled mRNA expression, with intense staining for FGF-2 and TGF-beta1 in the dura mater underlying the developing calvarial mesenchyme. Increasing FGF-2 and TGF-beta1 staining was noted within calvarial osteoblasts with increasing age, particularly in cells located near the endocranial surface (i.e., in contact with the developing dura mater). These findings, together with the known biologic functions of FGF-2 and TGF-beta1, implicate these growth factors in the regulation of calvarial bone growth by the developing dura mater. The possible mechanisms of this interaction are discussed.

    View details for Web of Science ID 000081608500017

    View details for PubMedID 10654687

  • Fetal wound repair: where do we go from here? Seminars in pediatric surgery Stelnicki, E. J., Chin, G. S., Gittes, G. K., Longaker, M. T. 1999; 8 (3): 124-130

    Abstract

    In contrast to adult wound healing, early-gestation fetal skin wound healing occurs rapidly, in a regenerative fashion, and without scar formation. The accelerated rate of healing, relative lack of an acute inflammatory response, and an absence of neovascularization distinguishes fetal from adult wound healing. However, this remarkable ability of the fetus to heal without scarring still remains poorly understood. The uncertainties include the role of cytokines, extracellular matrix components, homeobox genes, and certain cell types in the scarless wound repair process. Nevertheless, some strides have been made within the last two decades. This report, discusses the current knowledge of the mechanisms and characteristics of scarless fetal wound healing. Furthermore, to shy away from being just another all inclusive review, the authors point out deficiencies in the knowledge base on this important topic. Last, the future direction of research is discussed that may elucidate the mechanisms regulating the scarless repair phenomena.

    View details for PubMedID 10461325

  • Adenovirus-mediated gene therapy of osteoblasts in vitro and in vivo JOURNAL OF BONE AND MINERAL RESEARCH Mehrara, B. J., Saadeh, P. B., Steinbrech, D. S., Dudziak, M., Spector, J. A., Greenwald, J. A., Gittes, G. K., Longaker, M. T. 1999; 14 (8): 1290-1301

    Abstract

    Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing. Transforming growth factor beta1 (TGF-beta1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair. The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules. Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery. In this study, we investigate the efficacy of replication-deficient adenoviruses containing the human TGF-beta1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo. We demonstrate that adenovirus-mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF-beta1 virus causing a marked up-regulation in TGF-beta1 mRNA expression even 7 days after transfection. Increased TGF-beta1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46-fold increase in TGF-beta1 synthesis as compared with control cells (vehicle- or B-galactosidase-transfected). Moreover, virally produced TGF-beta1 was functionally active and regulated the expression of collagen IalphaI (5-fold increase) and the vascular endothelial growth factor (2.5-fold increase). Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus-mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection. Finally, we show that delivery of the hTGF-beta1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone.

    View details for Web of Science ID 000081682000005

    View details for PubMedID 10457261

  • Fibroblast response to hypoxia: The relationship between angiogenesis and matrix regulation JOURNAL OF SURGICAL RESEARCH Steinbrech, D. S., Longaker, M. T., Mehrara, B. J., Saadeh, P. B., Chin, G. S., Gerrets, R. P., Chau, D. C., Rowe, N. M., Gittes, G. K. 1999; 84 (2): 127-133

    Abstract

    A number of studies have demonstrated the critical role of angiogenesis for successful wound repair in the surgical patient. Vascular disruption from tissue injury due to trauma or surgery leads to a hypoxic zone in the healing wound. In this dynamic process, angiogenesis is vital for the delivery of oxygen, nutrients, and growth factors necessary to initiate the synthetic processes of wound healing. Fibroblasts, invading the wound early in the healing process, are involved in extracellular matrix (ECM) deposition as well as wound contraction. However, the exact mechanisms by which important genes are regulated remain unknown. In order to examine these processes, we studied the effects of hypoxia on fibroblasts for the expression of VEGF, type IalphaI collagen, and matrix-metalloproteinase-3, three genes essential for the regulation of angiogenesis, ECM deposition, and ECM degradation in wound healing. Primary cell cultures of normal human dermal fibroblasts (NHDFs) were placed in hypoxia for varying periods of time. Northern blot hybridization was performed with [alpha32P]dCTP-labeled cDNA probes for VEGF, type IalphaI collagen, and MMP-3. The results demonstrated a time-dependent VEGF mRNA upregulation (470% of baseline) under hypoxia. Type IalphaI collagen increased (170% of baseline) at 24 h, but was then abruptly downregulated to 3.8% of baseline at 48 h. MMP-3 was incrementally downregulated to 2.2% of baseline at 48 h. These experiments focused on the effect of hypoxia on genes thought to play a role in wound repair. VEGF upregulation in the hypoxic microenvironment of the early wound may serve to stimulate angiogenesis. Type IalphaI collagen, though upregulated early on, was abruptly downregulated at 48 h. This downregulation may reflect the in vivo requirement for angiogenesis to deliver oxygen for successful hydroxylation and collagen synthesis in the wound. MMP-3, also downregulated at 48 h, may also implicate the need for angiogenesis. These data support the theory that hypoxia-driven angiogenesis is critical for ECM formation and remodeling in successful soft tissue repair. Furthermore, they may represent the role of hypoxia as an important regulator to efficiently balance these complex processes in the healing wound.

    View details for Web of Science ID 000081004400002

    View details for PubMedID 10357908

  • Human cartilage engineering: chondrocyte extraction, proliferation, and characterization for construct development. Annals of plastic surgery Saadeh, P. B., Brent, B., Mehrara, B. J., Steinbrech, D. S., Ting, V., Gittes, G. K., Longaker, M. T. 1999; 42 (5): 509-513

    Abstract

    To date, many efforts to engineer cartilage have focused on matrix construction with the goal of producing a durable construct as cartilage replaces the resorbing matrix. However, the importance of matrix construction is at least matched by the challenge of efficient chondrocyte extraction, culture expansion, and prevention of dedifferentiation. This challenge is underscored by the large number of chondrocytes needed for a clinically significant construct such as an ear. Because human rib provides a large, readily available source of hyaline cartilage, the authors evaluated human rib chondrocyte extraction and found that maximum viable cell yield occurred after a 6-hour digestion. They also evaluated human microtic auricular remnant chondrocyte extraction and identified fibroblast contamination as a shortcoming of this potential source of chondrocytes. Initially, rib chondrocytes proliferated in vitro with a doubling time of approximately 1 week. As the cells were passaged, proliferation decreased such that the cells stopped proliferating and adopted a large, spindle-shaped morphology by passage 6. Interestingly, no increase in proliferation was noted when rib chondrocytes were stimulated with transforming growth factor beta 1, bone morphogenetic protein 2, and basic fibroblast growth factor. The major obstacles to the use of autologous rib chondrocytes in matrix construction are the low cell yield from a small piece of rib and the limited proliferation that these cells will undergo in vitro. Further investigation of culture systems and mitogenic cytokines may help resolve these limitations.

    View details for PubMedID 10340859

  • Expression of high-affinity receptors for TGF-beta during rat cranial suture fusion ANNALS OF PLASTIC SURGERY Mehrara, B. J., Steinbrech, D. S., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 1999; 42 (5): 502-508

    Abstract

    The etiology of craniosynostosis is unknown. The elucidation of the biological pathways responsible for this disorder has been hampered by an inability to evaluate cranial sutures before, during, and after cranial suture fusion. The programmed fusion of the rat posterofrontal (PF) suture postnatally provides an excellent model to study the molecular events that occur during cranial suture fusion. Previous experiments have implicated transforming growth factor beta (TGF-beta) growth factors in the regulation of PF suture fusion. The purpose of these experiments was to localize the expression of high-affinity receptors for these growth factors during cranial suture fusion. Four rats were sacrificed on postnatal days 8, 12, 17, and 40 (N = 16). The PF and sagittal sutures were harvested and prepared for immunohistochemical localization of TGF-beta receptor 1 and receptor 2 (Tbeta-RI, Tbeta-RII) protein. Results indicate that immunostaining for Tbeta-RI and Tbeta-RII is markedly increased in the dura mater and osteoblasts of the sutural margin of the PF suture during active suture fusion (on postnatal days 12, 17, and 40) compared with the osteoblasts and dura mater underlying the patent sagittal suture. These results, in combination with the authors' previous findings as well as studies supporting a role for TGF-beta molecules in the regulation of osteogenesis, implicate TGF-beta signaling in the regulation of suture fusion. The possible mechanisms of ligand-receptor interaction are discussed.

    View details for Web of Science ID 000080223600011

    View details for PubMedID 10340858

  • Chondrocyte extraction, proliferation, and characterization for construct development ANNALS OF PLASTIC SURGERY Saadeh, P. B., Brent, B., Mehrara, B. J., Steinbrech, D. S., Ting, V., Gittes, G. K., Longaker, M. T. 1999; 42 (5): 509-513
  • Gene expression of insulin-like growth factors I and II in rat membranous osteotomy healing ANNALS OF PLASTIC SURGERY Steinbrech, D. S., Mehrara, B. J., Rowe, N. M., Dudziak, M. E., Saadeh, P. B., Gittes, G. K., Longaker, M. T. 1999; 42 (5): 481-487

    Abstract

    Poorly healing mandibular osteotomies can be a difficult problem in reconstructive surgery. Many therapies have been attempted to augment the healing of mandibular fractures, defects, or osteotomies, but these methods have substantial drawbacks or have been ineffective. The difficulty in treating poorly healing bony defects has led to the exploration of gene therapy as a possible approach to supplement or accelerate mandibular fracture healing. To understand at what point the introduction of a suitable gene candidate might be of benefit in mandibular healing, it is imperative to examine the temporal expression of bone growth factors in a model of membranous bone healing. Insulinlike growth factors (IGFs) I and II are two such bone growth factor candidates because of their known potent in vitro as well as in vivo effects on bone formation. In this study the authors demonstrate the temporal pattern of IGF I and IGF II gene expression during mandibular osteotomy healing using a rat model. Their data reveal that IGF I and IGF II were elevated 7 days after a mandibular osteotomy that was held in external fixation. The upregulation of IGF I and IGF II during mandibular bone healing underscores the importance of these growth factors in bone repair. Gene therapy utilizing recombinant viral constructs containing IGFs I and II may be of benefit during mandibular bone healing in an effort to augment clinical scenarios of poor or retarded bony repair.

    View details for Web of Science ID 000080223600006

    View details for PubMedID 10340855

  • Rat mandibular distraction osteogenesis: II. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression PLASTIC AND RECONSTRUCTIVE SURGERY Mehrara, B. J., Rowe, N. M., Steinbrech, D. S., Dudziak, M. E., Saadeh, P. B., McCarthy, J. G., Gittes, G. K., Longaker, M. T. 1999; 103 (2): 536-547

    Abstract

    Distraction osteogenesis is a powerful technique capable of generating viable osseous tissue by the gradual separation of osteotomized bone edges. Although the histologic and ultrastructural changes associated with this process have been extensively delineated, the molecular events governing these changes remain essentially unknown. We have devised a rat model of mandibular distraction osteogenesis that facilitates molecular analysis of this process. Such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. In this study, we have evaluated the expression of transforming growth factor beta-1, a major regulator of osteogenesis during endochondral bone formation and development, and osteocalcin, an abundant noncollagenous extracellular matrix protein implicated in the regulation of mineralization and bone turnover. The right hemimandible of 36 adult male rats was osteotomized, and a customized distraction device was applied. Animals were allowed to recover and, after a 3-day latency period, were distracted at a rate of 0.25 mm twice daily for 6 days followed by a 2- or 4-week consolidation period. Distraction regenerate was harvested after the latency period, days 2, 4, or 6 of distraction, and after 2 or 4 weeks of consolidation and processed for Northern analysis (n = 4 at each time point) and immunohistochemical localization of TGF-beta1 (n = 2 at each time point). Six sham-operated animals (i.e., skin incision without osteotomy) were also killed (immediately postoperatively), and the mandibles were harvested and prepared in a similar fashion. Equal loading and transfer of RNA for Northern analysis was ensured by stripping and probing membranes with a probe against GAPDH (a housekeeping gene). Our results demonstrate that the spatial and temporal patterns of TGF-beta1 mRNA expression and protein production coincide with osteoblast migration, differentiation, and extracellular matrix synthesis. In addition, we demonstrate that TGF-beta1 production may be an important regulator of vasculogenesis during mandibular distraction osteogenesis. Finally, we have shown that osteocalcin gene expression coincides temporally with mineralization during rat mandibular distraction osteogenesis.

    View details for Web of Science ID 000078331800026

    View details for PubMedID 9950542

  • Proliferative hemangiomas: Analysis of cytokine gene expression and angiogenesis PLASTIC AND RECONSTRUCTIVE SURGERY Chang, J., Most, D., Bresnick, S., Mehrara, B., Steinbrech, D. S., Reinisch, J., Longaker, M. T., Turk, A. E. 1999; 103 (1): 1-9

    Abstract

    Hemangiomas are benign vascular tumors of childhood that can lead to disfigurement and/or life-threatening consequences. The pathogenesis of hemangioma formation is likely to involve increased angiogenesis. Basic fibroblast growth factor and vascular endothelial growth factor are cytokines that stimulate angiogenesis in multiple in vivo and in vitro models. Proliferative hemangiomas have been found to have elevated levels of basic fibroblast growth factor and vascular endothelial growth factor protein, but the gene expression of these cytokines in human specimens has not been previously studied. We examined the gene expression and spatial distribution of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA in proliferative versus involuted human hemangioma specimens using nonisotopic in situ hybridization techniques. Thirteen hemangioma specimens were harvested during initial surgical excision. In situ hybridization was performed on frozen sections of both proliferative and involuted hemangioma specimens using genetically engineered antisense probes specific for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA. Controls were an interleukin-6 sense sequence and a transforming growth factor-beta 1 antisense sequence. A large number of cells within the specimens of proliferative hemangiomas revealed localized gene expression of basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (626 +/- 129 and 1660 +/- 371 cells/mm2, respectively). The majority of the cells were endothelial in origin. In contrast, involuted hemangioma specimens revealed significantly lower numbers of cells staining positive for basic fibroblast growth factor and vascular endothelial growth factor messenger RNA (44 +/- 11 and 431 +/- 76 cells/mm2, respectively; p < 0.05). Transforming growth factor-beta 1 messenger RNA was slightly more expressed by involuted hemangiomas (117 +/- 30 cells/mm2). There were very low levels of transforming growth factor-beta 1 gene expression from proliferative hemangiomas (37 +/- 24 cells/mm2; p < 0.02). These data demonstrate that (1) in situ hybridization allows identification and relative quantitation of cells expressing messenger RNA for specific growth factors in human hemangioma specimens; (2) basic fibroblast growth factor and vascular endothelial growth factor messenger RNA are up-regulated in proliferative hemangiomas; and (3) transforming growth factor-beta 1 messenger RNA remains low in both proliferative and involuted hemangiomas. Because basic fibroblast growth factor and vascular endothelial growth factor messenger RNA have been implicated in the pathobiology of human hemangioma formation, biochemical modulation of these angiogenic cytokines may eventually help inhibit proliferation and promote regression of hemangiomas.

    View details for Web of Science ID 000077707200001

    View details for PubMedID 9915157

  • Rat mandibular distraction osteogenesis: Part I. Histologic and radiographic analysis PLASTIC AND RECONSTRUCTIVE SURGERY Rowe, N. M., Mehrara, B. J., Dudziak, M. E., Steinbreck, D. S., Mackool, R. J., Gittes, G. K., McCarthy, J. G., Longaker, M. T. 1998; 102 (6): 2022-2032

    Abstract

    The application of distraction osteogenesis to craniofacial surgery has altered the approach and treatment of congenital and acquired craniofacial defects. Although the histologic and ultrastructural changes associated with distraction osteogenesis have been described extensively, relatively little is known about the molecular regulation of this process. The elucidation of the molecular mechanisms of distraction osteogenesis has important clinical implications because it may facilitate the use of recombinant proteins or gene therapy to accelerate bone regeneration. Molecular analysis of distraction osteogenesis has been hindered by the use of large animal models in which only limited genetic information is available. In this study, a rat model of mandibular distraction osteogenesis is described. This report includes a pilot study (n = 50) to develop an appropriate distraction device and to determine the optimal placement of the osteotomy. The study subsequently included 80 animals, 35 of which were distracted at a rate of 0.25 mm per day for 6 days (1.5 mm total) and 35 that were distracted at a rate of 0.25 mm twice per day (3.0 mm total). These animals were killed at various time points (after latency and during the distraction and consolidation periods) and displayed histologic and radiographic findings of membranous bone distraction osteogenesis that were consistent with those in large ,animal and clinical models. In addition, five animals each were acutely lengthened 1.5 mm and 3.0 mm and demonstrated a fibrous nonunion. Furthermore, the utility of this model is demonstrated in the analysis of the molecular mechanisms of distraction osteogenesis by applying the polymerase chain reaction to total cellular RNA isolated from normal and distracted rat mandibles. In conclusion, it is believed that the rat model of distraction osteogenesis has significant advantages over traditional models, including decreased costs and facilitation of molecular analysis.

    View details for Web of Science ID 000076924100033

    View details for PubMedID 9811000

  • Molecular studies in flexor tendon wound healing: The role of basic fibroblast growth factor gene expression JOURNAL OF HAND SURGERY-AMERICAN VOLUME Chang, J., Most, D., Thunder, R., Mehrara, B., Longaker, M. T., Lineaweaver, W. C. 1998; 23A (6): 1052-1058

    Abstract

    Basic fibroblast growth factor (bFGF) is a cytokine that plays a fundamental role in angiogenesis. This study examines bFGF messenger RNA (mRNA) expression in a rabbit flexor tendon wound healing model. Thirty-four New Zealand white rabbit forepaws underwent transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals and analyzed by in situ hybridization and immunohistochemistry. Few tenocytes and tendon sheath cells expressed bFGF mRNA in unwounded tendons. In contrast, tendons subjected to transection and repair exhibited an increased signal for bFGF mRNA in both resident tenocytes concentrated along the epitenon and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal tenocytes and tendon sheath cells are capable of bFGF production, (2) bFGF mRNA is upregulated in the tendon wound environment, and (3) the upregulation of this angiogenic cytokine occurs in tenocytes as well as in tendon sheath fibroblasts and inflammatory cells.

    View details for Web of Science ID 000077120700015

  • Scarless healing - The fetal wound CLINICS IN PLASTIC SURGERY Mackool, R. J., Gittes, G. K., Longaker, M. T. 1998; 25 (3): 357-?

    Abstract

    Fetal wounds heal without a scar early in gestation, and may hold the key to scarless repair. Several important concepts central to the fetal wound-healing response have been determined. The fetal fibroblast modulates the wound-healing response through collagen deposition, extracellular matrix deposition, and growth factor secretion. Fetal repair is both gestational-age and wound-size dependent, with a transition from scarless to scarring repair occurring during fetal life. Fetal fibroblasts manifest a decreased ability to induce dermal appendage formation from fetal epithelium at the same time that scarring in the fetus begins, suggesting that epithelial-mesenchymal interactions play an important role in scarless fetal repair. The fetal immune response during wound healing differs from the adult response, with a primarily mononuclear cell infiltrate and decreased activity and presence of polymorphonuclear leukocytes, whereas the cytokine profile of the fetal wound differs markedly from that of the adult wound. Patterning genes (homeobox genes) involved in organogenesis may prove integral to fetal healing, and are emerging as an active area of research. Once the biology of fetal wound healing is fully determined, attempts to manipulate the adult wound undoubtedly will progress rapidly, and scarless repair may become a clinical reality in children and adults.

    View details for Web of Science ID 000075153600004

    View details for PubMedID 9696898

Conference Proceedings


  • Regenerative medicine: a surgeon's perspective Longaker, M. T. W B SAUNDERS CO-ELSEVIER INC. 2010: 11-18

    Abstract

    More than 200 million incisions are made in the world each year on children and adults. They all end up with a scar unless there is an unusual situation where we are operating on an early gestation fetus. The question is, "why do we not regenerate?" and "why do we always heal with either a 'normal amount of scarring' or, approximately 15% of the time, with a pathologic amount of scarring (hypertrophic scar or keloid)?"

    View details for DOI 10.1016/j.jpedsurg.2009.10.004

    View details for Web of Science ID 000274393800002

    View details for PubMedID 20105574

  • Highlights of the proceedings from the 10th International Congress of the International Society of Craniofacial Surgery. Lorenz, H. P., Longaker, M. T. 2004: 533-537

    View details for PubMedID 15111825

  • Differential expression of transforming growth factor-beta receptors in a rabbit zone II flexor tendon wound healing model Ngo, M., Pham, H., Longaker, M. T., Chang, J. LIPPINCOTT WILLIAMS & WILKINS. 2001: 1260-1267

    Abstract

    Flexor tendon repair in zone II is complicated by adhesions that impair normal postoperative gliding. Transforming growth factor-beta (TGF-beta) is a family of growth factors that has been implicated in scar formation. The TGF-beta family of proteins binds to three distinct classes of membrane receptors, termed RI, RII, and RIII. In this study, we analyzed the temporal and spatial distribution of TGF-beta receptor isoforms (RI, RII, and RIII) in a rabbit zone II flexor tendon wound healing model.Twenty-eight adult New Zealand White rabbit forepaws underwent isolation of the middle digit flexor digitorum profundus tendon in zone II. The tendons underwent transection in zone II and immediate repair. The tendons were harvested at increasing time points: 1, 3, 7, 14, 28, and 56 days postoperatively (n = 4 at each time point). The control flexor tendons were harvested without transection and repair (n = 4). Immunohistochemical analysis was used to detect the expression patterns for TGF-beta receptors RI, RII, and RIII. Immunohistochemical staining of the transected and repaired tendons demonstrated up-regulation of TGF-beta RI, RII, and RIII protein levels. TGF-beta receptor production in the experimental group (transection and repair) was concentrated in the epitenon and along the repair site. Furthermore, the TGF-beta receptor expression levels peaked at day 14 and decreased by day 56 postoperatively. In contrast, minimal receptor expression was observed in the untransected and unrepaired control tendons. These data provide evidence that (1) TGF-beta receptors are up-regulated after injury and repair; (2) peak levels of TGF-beta receptor expression occurred at day 14 and decreased by day 56 after wounding and repair; and (3) both the tendon sheath and epitenon have the highest receptor expression, and both may play critical roles in flexor tendon wound healing. Understanding the up-regulation of TGF-beta isoforms and the up-regulation of their corresponding receptors during flexor tendon wound healing provides new targets for biomolecular modulation of postoperative scar formation.

    View details for Web of Science ID 000171428900025

    View details for PubMedID 11604629

  • Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts Chin, G. S., Liu, W., Peled, Z., Lee, T. Y., Steinbrech, D. S., HSU, M., Longaker, M. T. LIPPINCOTT WILLIAMS & WILKINS. 2001: 423-429

    Abstract

    Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands. Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.

    View details for Web of Science ID 000170096900022

    View details for PubMedID 11496185

  • Studies in flexor tendon wound healing: Neutralizing antibody to TGF-beta 1 increases postoperative range of motion Chang, J., Thunder, R., Most, D., Longaker, M. T., Lineaweaver, W. C. LIPPINCOTT WILLIAMS & WILKINS. 2000: 148-155

    Abstract

    The postoperative outcome of hand flexor tendon repair remains limited by tendon adhesions that prevent normal range of motion. Recent studies using in situ hybridization techniques have implicated transforming growth factor beta-1 (TGF-beta1) in both intrinsic and extrinsic mechanisms of repair. TGF-beta1 is a growth factor that plays multiple roles in wound healing and has also been implicated in the pathogenesis of excessive scar formation. The purpose of this study was to examine the effect of neutralizing antibody to TGF-beta1 in a rabbit zone II flexor tendon wound-healing model. Twenty-two adult New Zealand White rabbits underwent complete transection of the middle digit flexor digitorum profundus tendon in zone II. The tendons were immediately repaired and received intraoperative infiltration of one of the following substances: (1) control phosphate-buffered saline; (2) 50 microg neutralizing antibody to TGF-beta1; (3) 50 microg each of neutralizing antibody to TGF-beta1 and to TGF-beta2. Eight rabbits that had not been operated on underwent analysis for determination of normal flexion range of motion at their proximal and distal interphalangeal joints, using a 1.2-N axial load applied to the flexor digitorum profundus tendon. All rabbits that had been operated on were placed in casts for 8 weeks to allow maximal tendon adhesion and were then killed to determine their flexion range of motion. Statistical analysis was performed using the Student's unpaired t test. When a 1.2-N load was used on rabbit forepaws that had not been operated on, normal combined flexion range of motion at the proximal and distal interphalangeal joints was 93+/-6 degrees. Previous immobilization in casts did not reduce the range of motion in these forepaws (93+/-4 degrees). In the experimental groups, complete transection and repair of the flexor digitorum profundus tendon with infiltration of control phosphate-buffered saline solution resulted in significantly decreased range of motion between the proximal and distal phalanges [15+/-6 degrees (n = 8)]. However, in the tendon repairs infiltrated with neutralizing antibody to TGF-beta1, flexion range of motion increased to 32+/-9 degrees (n = 7; p = 0.002). Interestingly, a combination of neutralizing antibody to TGF-beta1 and that to TGF-beta2 did not improve postoperative range of motion [18+/-4 degrees (n = 7; p = 0.234)]. These data demonstrate that (1) the rabbit flexor tendon repair model is useful for quantifying tendon scar formation on the basis of degrees of flexion between proximal and distal phalanges; (2) intraoperative infiltration of neutralizing antibody to TGF-beta1 improves flexor tendon excursion; and (3) simultaneous infiltration of neutralizing antibody to TGF-beta2 nullifies this effect. Because TGF-beta1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that intraoperative biochemical modulation of TGF-beta1 levels limits flexor tendon adhesion formation.

    View details for Web of Science ID 000084513700025

    View details for PubMedID 10626983

  • Gene expression of transforming growth factor beta-1 in rabbit zone II flexor tendon wound healing: Evidence for dual mechanisms of repair Chang, J., Most, D., Stelnicki, E., Siebert, J. W., Longaker, M. T., Hui, K., Lineaweaver, W. C. LIPPINCOTT WILLIAMS & WILKINS. 1997: 937-944

    Abstract

    The postoperative outcome of hand flexor tendon repair can be complicated by adhesions between the repair site and surrounding tissue. To date, the biology of hand flexor tendon wound healing remains controversial--both intrinsic (resident tenocyte) and extrinsic (tendon sheath fibroblast and inflammatory cell) processes may contribute to repair. Transforming growth factor beta-1 is a cytokine that plays multiple roles in wound healing but is also implicated in the pathogenesis of excessive scar formation. This study examines the activation of transforming growth factor beta-1 mRNA in a rabbit zone II flexor tendon wound-healing model. Forty New Zealand White rabbit forepaws underwent complete transection and repair of the middle digit flexor digitorum profundus tendon in zone II. Tendons were harvested at increasing time intervals (1, 3, 7, 14, 28, and 56 days) and analyzed by in situ hybridization and immunohistochemistry to determine the expression patterns of transforming growth factor beta-1. A small number of tenocytes exhibited expression of transforming growth factor beta-1 mRNA at baseline in nonwounded control tendon specimens. The surrounding tendon sheath in these control specimens also revealed low numbers of fibroblasts and inflammatory cells expressing transforming growth factor beta-1 mRNA. In contrast, flexor tendons subjected to transection and repair exhibited increased signal for transforming growth factor beta-1 mRNA in both resident tenocytes and infiltrating fibroblasts and inflammatory cells from the tendon sheath. These data demonstrate that (1) normal unwounded tenocytes and tendon sheath cells are capable of transforming growth factor beta-1 production, (2) this cytokine is activated in the tendon wound environment, as evidenced by mRNA upregulation, and (3) the upregulation of this cytokine in both "intrinsic" tenocytes and "extrinsic" tendon sheath fibroblasts and inflammatory cells supports dual mechanisms for tendon repair. Because transforming growth factor beta-1 is thought to contribute to the pathogenesis of excessive scar formation, the findings presented here suggest that perioperative biochemical modulation of transforming growth factor beta-1 levels may help limit flexor tendon adhesion formation.

    View details for Web of Science ID A1997XW23900016

    View details for PubMedID 9290662

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