Fan Yang

All Publications

Articular cartilage regeneration by activated skeletal stem cells. Nature medicine Murphy, M. P., Koepke, L. S., Lopez, M. T., Tong, X., Ambrosi, T. H., Gulati, G. S., Marecic, O., Wang, Y., Ransom, R. C., Hoover, M. Y., Steininger, H., Zhao, L., Walkiewicz, M. P., Quarto, N., Levi, B., Wan, D. C., Weissman, I. L., Goodman, S. B., Yang, F., Longaker, M. T., Chan, C. K. 2020
Abstract

Osteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2-4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5-7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.

View details for DOI 10.1038/s41591-020-1013-2

View details for PubMedID 32807933
Mixed Composition Microribbon Hydrogels Induce Rapid and Synergistic Cartilage Regeneration by Mesenchymal Stem Cells in 3D via Paracrine Signaling Exchange ACS BIOMATERIALS SCIENCE & ENGINEERING Gegg, C., Tong, X., Yang, F. 2020; 6 (7): 4166–78
View details for DOI 10.1021/acsbiomaterials.0c00131

View details for Web of Science ID 000551355300041
Gelatin-Based Microribbon Hydrogels Support Robust MSC Osteogenesis across a Broad Range of Stiffness ACS BIOMATERIALS SCIENCE & ENGINEERING Conrad, B., Hayashi, C., Yang, F. 2020; 6 (6): 3454–63
View details for DOI 10.1021/acsbiomaterials.9b01792

View details for Web of Science ID 000541442600019
Nanoparticle-Mediated TGF-beta Release from Microribbon-Based Hydrogels Accelerates Stem Cell-Based Cartilage Formation In Vivo. Annals of biomedical engineering Barati, D., Gegg, C., Yang, F. 2020
Abstract

Conventional nanoporous hydrogels often lead to slow cartilage deposition by MSCs in 3D due to physical constraints and requirement for degradation. Our group has recently reported macroporous gelatin microribbon (muRB) hydrogels, which substantially accelerate MSC-based cartilage formation in vitro compared to conventional gelatin hydrogels. To facilitate translating the use of muRB-based scaffolds for supporting stem cell-based cartilage regeneration in vivo, there remains a need to develop a customize-designed drug delivery system that can be incorporated into muRB-based scaffolds. Towards this goal, here we report polydopamine-coated mesoporous silica nanoparticles (MSNs) that can be stably incorporated within the macroporous muRB scaffolds,and allow tunable release of transforming growth factor (TGF)-beta3. We hypothesize that increasing concentration of polydopamine coating on MSNs will slow down TGF- beta3 release, and TGF-beta3 release from polydopamine-coated MSNs can enhance MSC-based cartilage formation in vitro and in vivo. We demonstrate that TGF-beta3 released from MSNs enhance MSC-based cartilage regeneration in vitro to levels comparable to freshly added TGF-beta3 in the medium, as shown bybiochemical assays, mechanical testing, and histology. Furthermore, when implanted in vivo in a mouse subcutaneous model, onlythe group containing MSN-mediated TGF-beta3 release supported continuous cartilage formation, whereas control group without MSN showed loss of cartilage matrix and undesirable endochondral ossification. The modular design of MSN-mediated drug delivery can be customized for delivering multiple drugs with individually optimized release kinetics, and may be applicable to enhance regeneration of other tissue types.

View details for DOI 10.1007/s10439-020-02522-z

View details for PubMedID 32377980
Injectable and Crosslinkable PLGA-Based Microribbons as 3D Macroporous Stem Cell Niche. Small (Weinheim an der Bergstrasse, Germany) Barati, D., Watkins, K., Wang, Z., Yang, F. 2020: e1905820
Abstract

Poly(lactide-co-glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon-based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon-shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen-coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock-absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA muRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC-seeded PLGA muRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA muRBs as a promising injectable, macroporous, non-hydrogel-based scaffold for cell delivery and tissue regeneration applications.

View details for DOI 10.1002/smll.201905820

View details for PubMedID 32338432
Injectable and in situ crosslinkable gelatin microribbon hydrogels for stem cell delivery and bone regeneration in vivo. Theranostics Tang, Y., Tong, X., Conrad, B., Yang, F. 2020; 10 (13): 6035–47
Abstract

Rationale: Injectable matrices are highly desirable for stem cell delivery. Previous research has highlighted the benefit of scaffold macroporosity in enhancing stem cell survival and bone regeneration in vivo. However, there remains a lack of injectable and in situ crosslinkable macroporous matrices for stem cell delivery to achieve fast bone regeneration in immunocompetent animal model. The goal of this study is to develop an injectable gelatin-based muRB hydrogel supporting direct cell encapsulation that is available in clinics as macroporous matrices to enhance adipose-derived stromal cell (ASC) survival, engraftment and accelerate bone formation in craniofacial defect mouse. Methods: Injectable and in situ crosslinkable gelatin microribbon (muRB)-based macroporous hydrogels were developed by wet-spinning. Injectability was optimized by varying concentration of glutaraldehyde for intracrosslinking of muRB shape, and fibrinogen coating. The efficacy of injectable muRBs to support ASCs delivery and bone regeneration were further assessed in vivo using an immunocompetent mouse cranial defect model. ASCs survival was evaluated by bioluminescent imaging and bone regeneration was assessed by micro-CT. The degradation and biocompatibility were determined by histological analysis. Results: We first optimized injectability by varying concentration of glutaraldehyde used to fix gelatin muRBs. The injectable muRB formulation were subsequently coated with fibrinogen, which allows in situ crosslinking by thrombin. Fluorescence imaging and histology showed majority of muRBs degraded by the end of 3 weeks. Injectable muRBs supported comparable level of ASC proliferation and bone regeneration as implantable prefabricated muRB controls. Adding low dosage of BMP2 (100 ng per scaffold) with ASCs substantially accelerated the speed of mineralized bone regeneration, with 90% of the bone defect refilled by week 8. Immunostaining showed M1 (pro-inflammatory) macrophages were recruited to the defect at day 3, and was replaced by M2 (anti-inflammatory) macrophages by week 2. Adding muRBs or BMP2 did not alter macrophage response. Injectable RBs supported vascularization, and BMP-2 further enhanced vascularization. Conclusions: Our results demonstrated that RB-based scaffolds enhanced ASC survival and accelerated bone regeneration after injection into critical sized cranial defect mouse. Such injectable RB-based scaffold can provide a versatile biomaterial for delivering various stem cell types and enhancing tissue regeneration.

View details for DOI 10.7150/thno.41096

View details for PubMedID 32483436
Gradient hydrogels for screening stiffness effects on patient-derived glioblastoma xenograft cellfates in 3D. Journal of biomedical materials research. Part A Zhu, D., Trinh, P., Li, J., Grant, G., Yang, F. 2020
Abstract

Brain cancer is a devastating disease given its extreme invasiveness and intricate location. Glioblastoma multiforme (GBM) is one of the most common forms of brain cancer, andcancer progression is often correlated with significantly altered tissue stiffness. To elucidate the effect of matrix stiffness on GBM cell fates, previous research is largely limited to 2D studies using immortalized cell lines, which has limited physiological relevance. The objective of the study is to develop gradient hydrogels with brain-mimicking stiffness range as a 3Din vitroGBM model for screening of the effects of matrix stiffness on GBM. To increase the physiological relevance, patient-derived tumor xenograft (PDTX) GBM cells were used. Our gradient platform allows formation of cell-containing hydrogels with stiffness ranging from 40 Pa to 1300 Pa within a few minutes. By focusing on a brain-mimicking stiffness range, this gradient hydrogel platform is designed for investigating brain cancer. Increasing stiffness led to decreased GBMproliferation and less spreading, which is accompanied bydownregulation of matrix-metalloproteinases (MMPs). Using temozolomide(TMZ) as a model drug, we demonstrate that increasing stiffness led to higher drug resistance by PDTX GBM cells in 3D, suggesting matrix stiffness can directly modulate how GBM cells respond to drug treatment. While the current study focuses on stiffness gradient, the set upmay also be adapted for screening othercancer niche cues such as how biochemical ligand gradientmodulatesbrain cancer progression and drug responses using reduced materials and time. This article is protected by copyright. All rights reserved.

View details for DOI 10.1002/jbm.a.37093

View details for PubMedID 32862485
IL-4 Overexpressing Mesenchymal Stem Cells within Gelatin-Based Microribbon Hydrogels Enhance Bone Healing in a Murine Long Bone Critical-size Defect Model. Journal of biomedical materials research. Part A Ueno, M., Lo, C. W., Barati, D., Conrad, B., Lin, T., Kohno, Y., Utsunomiya, T., Zhang, N., Maruyama, M., Rhee, C., Huang, E., Romero-Lopez, M., Tong, X., Yao, Z., Zwingenberger, S., Yang, F., Goodman, S. B. 2020
Abstract

Mesenchymal stem cell (MSC)-based therapy is a promising strategy for bone repair. Furthermore, the innate immune system, and specifically macrophages, play a crucial role in the differentiation and activation of MSCs. The anti-inflammatory cytokine IL-4 converts pro-inflammatory M1 macrophages into a tissue regenerative M2 phenotype, which enhances MSC differentiation and function. We developed lentivirus-transduced IL-4 over-expressing MSCs (IL-4 MSCs) that continuously produce IL-4 and polarize macrophages toward an M2 phenotype. In the current study, we investigated the potential of IL-4 MSCs delivered using a macroporous gelatin-based microribbon (μRB) scaffold for healing of critical size long bone defects in Mice. IL-4 MSCs within μRBs enhanced M2 marker expression without inhibiting M1 marker expression in the early phase, and increased macrophage migration into the scaffold. Six weeks after establishing the bone defect, IL-4 MSCs within μRBs enhanced bone formation and helped bridge the long bone defect. IL-4 MSCs delivered using macroporous μRB scaffold is potentially a valuable strategy for the treatment of critical size long bone defects. This article is protected by copyright. All rights reserved.

View details for DOI 10.1002/jbm.a.36982

View details for PubMedID 32363683
A comparative study of brain tumor cells from different age and anatomical locations using 3D biomimetic hydrogels. Acta biomaterialia Wang, C., Sinha, S., Jiang, X., Fitch, S., Wilson, C., Caretti, V., Ponnuswami, A., Monje, M., Grant, G., Yang, F. 2020
Abstract

Brain tumors exhibit vast genotypic and phenotypic diversity depending on patient age and anatomical location. Hydrogels hold great promise as 3D in vitro models for studying brain tumor biology and drug screening, yet previous studies were limited to adult glioblastoma cells, and most studies used immortalized cell lines. Here we report a hydrogel platform that supports the proliferation and invasion of patient-derived brain tumor cell cultures (PDCs) isolated from different patient age groups and anatomical locations. Hydrogel stiffness was tuned by varying poly(ethylene-glycol) concentration. Cell adhesive peptide (CGRDS), hyaluronic acid, and MMP-cleavable crosslinkers were incorporated to facilitate cell adhesion and cell-mediated degradation. Three PDC lines were compared including adult glioblastoma cells (aGBM), pediatric glioblastoma cells (pGBM), and diffuse pontine intrinsic glioma (DIPG). A commonly used immortalized adult glioblastoma cell line U87 was included as a control. PDCs displayed stiffness-dependent behavior, with 40 Pa hydrogel promoting faster tumor proliferation and invasion. Adult GBM cells exhibited faster proliferation than pediatric GBM, and DIPG showed slowest proliferation. These results suggest both patient age and tumor location affects brain tumor behaviors. Adult GBM PDCs also exhibited very different cell proliferation and morphology from U87. The hydrogel reported here can provide a useful tool for future studies to better understand how age and anatomical locations impacts brain tumor progression using 3D in vitro models.

View details for DOI 10.1016/j.actbio.2020.09.007

View details for PubMedID 32911104
Matrix stiffness modulates patient-derived glioblastoma cell fates in 3D hydrogels. Tissue engineering. Part A Wang, C., Sinha, S., Jiang, X., Murphy, L., Fitch, S., Wilson, C., Grant, G., Yang, F. 2020
Abstract

Cancer progression is known to be accompanied by changes in tissue stiffness. Previous studies have primarily employed immortalized cell lines and 2D hydrogel substrates, which do not recapitulate the 3D tumor niche. How matrix stiffness affects patient-derived cancer cell fate in 3D remains unclear. Here we report a MMP-degradable poly(ethylene-glycol)-based hydrogel platform with brain-mimicking biochemical cues and tunable stiffness (40 to 26,600 Pa) for 3D culture of patient-derived glioblastoma xenograft (PDTX GBM) cells. Our results demonstrate that decreasing hydrogel stiffness enhanced PDTX GBM cell proliferation, and hydrogels with stiffnesses 240 Pa and below supported robust PDTX GBM cell spreading in 3D. PDTX GBM cells encapsulated in hydrogels demonstrated higher drug resistance than 2D control, and increasing hydrogel stiffness further enhanced drug resistance. Such 3D hydrogel platforms may provide a valuable tool for mechanistic studies of the role of niche cues in modulating cancer progression for different cancer types.

View details for DOI 10.1089/ten.TEA.2020.0110

View details for PubMedID 32731804
Gradient hydrogels for optimizing niche cues to enhance cell-based cartilage regeneration. Tissue engineering. Part A Liu, E., Zhu, D., Diaz, E. C., Tong, X., Yang, F. 2020
Abstract

Hydrogels have been widely used for cell delivery to enhance cell-based therapies for cartilage tissue regeneration. To better support cartilage deposition, it is imperative to determine hydrogel formulation with physical and biochemical cues that are optimized for different cell populations. Previous attempts to identify optimized hydrogels rely mostly on testing hydrogel formulations with discrete properties, which are time consuming and require large amounts of cells and materials. Gradient hydrogels encompass a range of continuous changes in niche properties, therefore offering a promising solution for screening a wide range of cell-niche interactions using less materials and time. However, harnessing gradient hydrogels to assess how matrix stiffness modulates cartilage formation by different cell types in vivo have never been investigated before. The goal of this study is to fabricate gradient hydrogels for screening the effects of varying hydrogel stiffness on cartilage formation by mesenchymal stem cells (MSCs) and chondrocytes respectively, two mostly commonly used cell populations for cartilage regeneration. We fabricated stiffness gradient hydrogels with tunable dimensions that support homogeneous cell encapsulation. Using gradient hydrogels with tunable stiffness range, we found MSCs and chondrocytes exhibit opposite trend in cartilage deposition in response to stiffness changes in vitro. Specifically, MSCs require soft hydrogels with Young's modulus less than 5 kPa to support faster cartilage deposition, as shown by type II collagen and sGAG staining. In contrast, chondrocytes produce cartilage more effectively in stiffer matrix (> 20 kPa). We chose optimal ranges of stiffness for each cell population for further testing in vivo using a mouse subcutaneous model. Our results further validated soft matrix (Young's modulus < 5 kPa) is better in supporting MSC-based cartilage deposition in 3D, whereas stiffer matrix (Young's modulus >20 kPa) is more desirable for supporting chondrocytes-based cartilage deposition. Our results show the importance of optimizing niche cues in a cell-type specific manner and validate the potential of using gradient hydrogels for optimizing niche cues to support cartilage regeneration in vitro and in vivo.

View details for DOI 10.1089/ten.TEA.2020.0158

View details for PubMedID 32940136
Gelatin-Based Microribbon Hydrogels Guided Mesenchymal Stem Cells to Undergo Endochondral Ossification In Vivo with Bone-Mimicking Mechanical Strength REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE Conrad, B., Hayashi, C., Yang, F. 2019
View details for DOI 10.1007/s40883-019-00138-x

View details for Web of Science ID 000556951700001
Spatially Patterned Microribbon-based Hydrogels Induce Zonally-Organized Cartilage Regeneration by Stem Cells in 3D. Acta biomaterialia Gegg, C., Yang, F. 2019
Abstract

Regenerating cartilage with biomimetic zonal organization, which is critical for tissue function, remains a great challenge. The objective of this study was to evaluate the potential of spatially-patterned, multi-compositional, macroporous, extracellular matrix-based microribbon (muRB) muRB scaffolds to regenerate cartilage with biochemical, mechanical, and morphological zonal organization by mesenchymal stem cells (MSCs) compared to conventional multi-layer nanoporous hydrogels. MSCs were seeded in either trilayer microribbon (muRB) or hydrogel (HG) scaffolds that were composed of layered biomaterial compositions that had been chosen for their ability to differentiate MSCs into chondrocytes with zonal properties. To mimic the aligned collagen morphology in the superficial layer of native cartilage, an additional experimental group added MSC-laden aligned muRBs to the surface of the superficial layer of a muRB trilayer. Tuning muRB alignment and compositions in different zones led to zonal-specific responses of MSCs to create neocartilage with zonal biochemical, morphological, and mechanical properties, while trilayer HGs led to minimal cartilaginous deposition overall. Trilayer muRBs created neocartilage exhibiting significant increases in compressive modulus (up to 456 kPa) and > 4-fold increase in sGAG production from superficial to deep zones. Aligned gelatin RBs in the superficial zone further enhanced biomimetic mimicry of the produced neocartilage by leading to robust collagen deposition and superficial zone protein production.

View details for DOI 10.1016/j.actbio.2019.10.025

View details for PubMedID 31634627
Optimizing 3D Co-culture Models to Enhance Synergy Between Adipose-Derived Stem Cells and Chondrocytes for Cartilage Tissue Regeneration REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE Rogan, H., Yang, F. 2019; 5 (3): 270–79
View details for DOI 10.1007/s40883-019-00105-6

View details for Web of Science ID 000485306800004
The effects of ROCK inhibition on mesenchymal stem cell chondrogenesis are culture model dependent. Tissue engineering. Part A Gegg, C. A., Yang, F. 2019
Abstract

Rho-associated protein kinase (ROCK) signaling correlates with cell shape, with decreased cell spreading accompanied by decreased ROCK activity. However, how cell shape and ROCK activity impact chondrogenesis of mesenchymal stem cells (MSCs) remains inconclusive. Here we examine the effects of ROCK inhibition on human MSC chondrogenesis in four different culture models including 3D microribbon (muRB) scaffolds, 2D hydrogel substrates, 3D hydrogels, and pellet. For each culture model involving biomaterials, four polymers were compared including gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol. ROCK inhibition decreased MSC chondrogenesis in muRB model, enhanced chondrogenesis in pellet, and had minimal effect in 2D or 3D hydrogel models. Furthermore, we demonstrate that MSC chondrogenesis cannot be predicted using ROCK signaling alone. While varying biomaterial composition can impact the amount or phenotype of resulting cartilage, varying biomaterials did not change the chondrogenic response to ROCK inhibition within each culture model. Regardless of culture model or ROCK expression, increased cartilage formation was always accompanied by enhanced N-cadherin expression and production, suggesting N-cadherin is a robust marker to select culture conditions that promote chondrogenesis. Together, the results from this study may be used to enhance MSC-based cartilage regeneration in different culture models.

View details for DOI 10.1089/ten.TEA.2019.0068

View details for PubMedID 31411113
Comparing Single Cell Versus Pellet Encapsulation of Mesenchymal Stem Cells in Three-Dimensional Hydrogels for Cartilage Regeneration TISSUE ENGINEERING PART A Rogan, H., Ilagan, F., Yang, F. 2019
View details for DOI 10.1089/ten.tea.2018.0289

View details for Web of Science ID 000466513000001
Hydrogels with enhanced protein conjugation efficiency reveal stiffness-induced YAP localization in stem cells depends on biochemical cues BIOMATERIALS Lee, S., Stanton, A. E., Tong, X., Yang, F. 2019; 202: 26–34
View details for DOI 10.1016/j.biomaterials.2019.02.021

View details for Web of Science ID 000463304400003
Mimicking brain tumor-vasculature microanatomical architecture via co-culture of brain tumor and endothelial cells in 3D hydrogels BIOMATERIALS Wang, C., Li, J., Sinha, S., Peterson, A., Grant, G. A., Yang, F. 2019; 202: 35–44
View details for DOI 10.1016/j.biomaterials.2019.02.024

View details for Web of Science ID 000463304400004
Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages TISSUE ENGINEERING PART A Lin, T., Kohno, Y., Huang, J., Romero-Lopez, M., Maruyama, M., Ueno, M., Pajarinen, J., Nathan, K., Yao, Z., Yang, F., Wu, J. Y., Goodman, S. B. 2019
View details for DOI 10.1089/ten.tea.2018.0292

View details for Web of Science ID 000462509800001
Biochemical Ligand Density Regulates Yes-Associated Protein Translocation in Stem Cells through Cytoskeletal Tension and Integrins ACS APPLIED MATERIALS & INTERFACES Stanton, A. E., Tong, X., Lee, S., Yang, F. 2019; 11 (9): 8849–57
Abstract

Different tissue types are characterized by varying stiffness and biochemical ligands. Increasing substrate stiffness has been shown to trigger Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet the role of ligand density in modulating mechanotransduction and stem cell fate remains largely unexplored. Using polyacrylamide hydrogels coated with fibronectin as a model platform, we showed that stiffness-induced YAP translocation occurs only at intermediate ligand densities. At low or high ligand densities, YAP localization is dominated by ligand density independent of substrate stiffness. We further showed that ligand density-induced YAP translocation requires cytoskeleton tension and αVβ3-integrin binding. Finally, we demonstrate that increasing ligand density alone can enhance osteogenic differentiation regardless of matrix stiffness. Together, the findings from the present study establish ligand density as an important parameter for modulating stem cell mechanotransduction and differentiation, which is mediated by integrin clustering, focal adhesion, and cytoskeletal tension.

View details for DOI 10.1021/acsami.8b21270

View details for Web of Science ID 000460996900018

View details for PubMedID 30789697
Mimicking brain tumor-vasculature microanatomical architecture via co-culture of brain tumor and endothelial cells in 3D hydrogels. Biomaterials Wang, C., Li, J., Sinha, S., Peterson, A., Grant, G. A., Yang, F. 2019; 202: 35–44
Abstract

Glioblastoma (GBM) is an aggressive malignant brain tumor with median survival of 12 months and 5-year survival rate less than 5%. GBM is highly vascularized, and the interactions between tumor and endothelial cells play an important role in driving tumor growth. To study tumor-endothelial interactions, the gold standard co-culture model is transwell culture, which fails to recapitulate the biochemical or physical cues found in tumor niche. Recently, we reported the development of poly(ethylene-glycol)-based hydrogels as 3D niche that supported GBM proliferation and invasion. To further mimic the microanatomical architecture of tumor-endothelial interactions in vivo, here we developed a hydrogel-based co-culture model that mimics the spatial organization of tumor and endothelial cells. To increase the physiological relevance, patient-derived GBM cells and mouse brain endothelial cells were used as model cell types. Using hydrolytically-degradable alginate fibers as porogens, endothelial cells were deployed and patterned into vessel-like structures in 3D hydrogels with high cell viability and retention of endothelial phenotype. Co-culture led to a significant increase in GBM cell proliferation and decrease in endothelial cell expression of cell adhesion proteins. In summary, we have developed a novel 3D co-culture model that mimics the in vivo spatial organization of brain tumor and endothelial cells. Such model may provide a valuable tool for future mechanistic studies to elucidate the effects of tumor-endothelial interactions on tumor progression in a more physiologically-relevant manner.

View details for PubMedID 30836243
Hydrogels with enhanced protein conjugation efficiency reveal stiffness-induced YAP localization in stem cells depends on biochemical cues. Biomaterials Lee, S., Stanton, A. E., Tong, X., Yang, F. 2019; 202: 26–34
Abstract

Polyacrylamide hydrogels have been widely used in stem cell mechanotransduction studies. Conventional conjugation methods of biochemical cues to polyacrylamide hydrogels suffer from low conjugation efficiency, which leads to poor attachment of human pluripotent stem cells (hPSCs) on soft substrates. In addition, while it is well-established that stiffness-dependent regulation of stem cell fate requires cytoskeletal tension, and is mediated through nuclear translocation of transcription regulator, Yes-associated protein (YAP), the role of biochemical cues in stiffness-dependent YAP regulation remains largely unknown. Here we report a method that enhances the conjugation efficiency of biochemical cues on polyacrylamide hydrogels compared to conventional methods. This modified method enables robust hPSC attachment, proliferation and maintenance of pluripotency across varying substrate stiffness (3 kPa-38 kPa). Using this hydrogel platform, we demonstrate that varying the types of biochemical cues (Matrigel, laminin, GAG-peptide) or density of Matrigel can alter stiffness-dependent YAP localization in hPSCs. In particular, we show that stiffness-dependent YAP localization is overridden at low or high density of Matrigel. Furthermore, human mesenchymal stem cells display stiffness-dependent YAP localization only at intermediate fibronectin density. The hydrogel platform with enhanced conjugation efficiency of biochemical cues provides a powerful tool for uncovering the role of biochemical cues in regulating mechanotransduction of various stem cell types.

View details for PubMedID 30826537
Comparing Single Cell vs. Pellet Encapsulation of MSCs in 3D Hydrogels for Cartilage Regeneration. Tissue engineering. Part A Rogan, H., Ilagan, F., Yang, F. 2019
Abstract

While the gold standard for inducing MSCs chondrogenesis utilizes pellet culture, most tissue engineering strategies for cartilage regeneration encapsulate MSCs as single cells, partially due to the technical challenge to homogeneously encapsulate cell pellets in 3D hydrogels. It remains unclear whether encapsulating MSCs as single cell suspension or cell aggregates in 3D hydrogels would enhance MSC-based cartilage formation. Here we determined the optimal size of MSC micropellets (muPellet) that can be homogeneously encapsulated in hydrogels with high cell viability is 100 cells/pellet. Using optimized muPellet size, MSCs were encapsulated either as single cell suspension or muPellets in four soft hydrogel formulation with stiffness ranging 3-6 kPa. Regardless of hydrogel formulations, single cell encapsulation resulted in more neocartilage deposition with improved mechanical functions over Pellet encapsulation. For single cell encapsulation, PEG hydrogels containing chondroitin sulfate led to the most cartilage matrix deposition, with compressive modulus reaching 211 kPa after only 21 days, a range approaching the stiffness of native cartilage. The findings from this study offer valuable insights on guiding optimal method design for MSCs and hydrogel-based cartilage regeneration. The optimized muPellet encapsulation method may be broadly applicable to encapsulate other stem cell types or cancer cells as aggregates in hydrogels.

View details for PubMedID 30672386
Preconditioned or IL4-Secreting Mesenchymal Stem Cells Enhanced Osteogenesis at Different Stages. Tissue engineering. Part A Lin, T., Kohno, Y., Huang, J., Romero-Lopez, M., Maruyama, M., Ueno, M., Pajarinen, J., Nathan, K., Yao, Z., Yang, F., Wu, J., Goodman, S. B. 2019
Abstract

Chronic inflammation-associated bone diseases involve continuous destruction and impaired regeneration of bone. Mesenchymal stem cell (MSC)-based therapy has great potential to modulate inflammatory responses and enhance tissue regeneration. We previously showed that lipopolysaccharide [LPS] plus TNF preconditioned MSCs or genetically modified inflammation-sensing (driven by NFB activation) IL4-secreting MSCs enhanced immunomodulation of macrophages to the more desired tissue repaired M2 type. In the current study, the paracrine regulation of therapeutic MSCs on the pro-inflammatory response and osteogenesis of macrophage-MSC co-cultures (representing endogenous cells) was examined using an in vitro transwell system. In the co-cultures, IL4-secreting MSCs decreased TNF and iNOS expression, and increased Arginase 1 and CD206 expression in the presence of LPS-contaminated polyethylene particles. The preconditioned MSCs decreased TNF and CD206 expression in the bottom MSC-macrophage co-cultures in the presence of contaminated particles. In osteogenesis assays, IL4-secreting MSCs decreased ALP expression, but increased alizarin red staining in the presence of contaminated particles. The preconditioned MSCs increased ALP and osteocalcin expression, and had no significant effect on alizarin red staining. These results suggest that potential treatments using preconditioned MSCs at an earlier stage, or IL4-secreting MSCs at a later stage could enhance bone regeneration in inflammatory conditions including periprosthetic osteolysis.

View details for PubMedID 30652628
Extracellular matrix type modulates mechanotransduction of stem cells. Acta biomaterialia Stanton, A. E., Tong, X., Yang, F. 2019
Abstract

Extracellular matrix (ECM) is comprised of different types of proteins, which change in composition and ratios during morphogenesis and disease progression. ECM proteins provide cell adhesion and impart mechanical cues to the cells. Increasing substrate stiffness has been shown to induce Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet these mechanistic studies used fibronectin only as the biochemical cue. How varying the types of ECM modulates mechanotransduction of stem cells remains largely unknown. Using polyacrylamide hydrogels with tunable stiffness as substrates, here we conjugated four major ECM proteins commonly used for cell adhesion: fibronectin, collagen I, collagen IV and laminin, and assessed the effects of varying ECM type and density on YAP translocation in human mesenchymal stem cells (hMSCs). For all four ECM types, increasing ECM ligand density alone can induce YAP nuclear translocation without changing substrate stiffness. The ligand threshold for such biochemical ligand-induced YAP translocation differs across ECM types. While stiffness-dependent YAP translocation can be induced by all four ECM types, each ECM requires a different optimized ligand density for this to occur. Using antibody blocking, we further identified integrin subunits specifically involved in mechanotransduction of different ECM types. Finally, we demonstrated that altering ECM type further modulates hMSC osteogenesis without changing substrate stiffness. These findings highlight the important role of ECM type in modulating mechanotransduction and differentiation of stem cells, and call for future mechanistic studies to further elucidate the role of changes in ECM compositions in mediating mechanotransduction during morphogenesis and disease progression. STATEMENT OF SIGNIFICANCE: Our study addresses a critical gap of knowledge in mechanobiology. Increasing substrate stiffness has been shown to induce nuclear YAP translocation, yet only on fibronectin-coated substrates. However, extracellular matrix (ECM) is comprised of different protein types. How varying the type of ECM modulates stem cell mechanotransduction remains largely unknown. We here reveal that the choice of ECM type can directly modulate stem cell mechanotransduction, filling this critical gap. This work has broad impacts in mechanobiology and biomaterials, as it provides the first evidence that varying ECM type can impact YAP translocation independent of substrate stiffness, opening doors for a more rational biomaterials design tuning ECM properties to control cell fate for promoting normal development and for preventing disease progression.

View details for DOI 10.1016/j.actbio.2019.06.048

View details for PubMedID 31255664
Varying solvent type modulates collagen coating and stem cell mechanotransduction on hydrogel substrates. APL bioengineering Stanton, A. E., Tong, X., Yang, F. 2019; 3 (3): 036108
Abstract

Type I collagen is the most abundant extracellular matrix protein in the human body and is commonly used as a biochemical ligand for hydrogel substrates to support cell adhesion in mechanotransduction studies. Previous protocols for conjugating collagen I have used different solvents; yet, how varying solvent pH and composition impacts the efficiency and distribution of these collagen I coatings remains unknown. Here, we examine the effect of varying solvent pH and type on the efficiency and distribution of collagen I coatings on polyacrylamide hydrogels. We further evaluate the effects of varying solvent on mechanotransduction of human mesenchymal stem cells (MSCs) by characterizing cell spreading and localization of Yes-Associated Protein (YAP), a key transcriptional regulator of mechanotransduction. Increasing solvent pH to 5.2 and above increased the heterogeneity of coating with collagen bundle formation. Collagen I coating highly depends on the solvent type, with acetic acid leading to the highest conjugation efficiency and most homogeneous coating. Compared to HEPES or phosphate-buffered saline buffer, acetic acid-dissolved collagen I coatings substantially enhance MSC adhesion and spreading on both glass and polyacrylamide hydrogel substrates. When acetic acid was used for collagen coatings, even the low collagen concentration (1 μg/ml) induced robust MSC spreading and nuclear YAP localization on both soft (3 kPa) and stiff (38 kPa) substrates. Depending on the solvent type, stiffness-dependent nuclear YAP translocation occurs at a different collagen concentration. Together, the results from this study validate the solvent type as an important parameter to consider when using collagen I as the biochemical ligand to support cell adhesion.

View details for DOI 10.1063/1.5111762

View details for PubMedID 31592041

View details for PubMedCentralID PMC6768796
Tissue-engineered 3D Models for Elucidating Primary and Metastatic Bone Cancer Progression. Acta biomaterialia González Díaz, E. C., Sinha, S., Avedian, R. S., Yang, F. 2019
Abstract

Malignant bone tumors are aggressive neoplasms which arise from bone tissue or as a result of metastasis. The most prevalent types of cancer, such as breast, prostate, and lung cancer, all preferentially metastasize to bone, yet the role of the bone niche in promoting cancer progression remains poorly understood. Tissue engineering has the potential to bridge this knowledge gap by providing 3D in vitro systems that can be specifically designed to mimic key properties of the bone niche in a more physiologically relevant context than standard 2D culture. Elucidating the crucial components of the bone niche that recruit metastatic cells, support tumor growth, and promote cancer-induced destruction of bone tissue would support efforts for preventing and treating these devastating malignancies. In this review, we summarize recent efforts focused on developing in vitro 3D models of primary bone cancer and bone metastasis using tissue engineering approaches. Such 3D in vitro models can enable the identification of effective therapeutic targets and facilitate high-throughput drug screening to effectively treat bone cancers. STATEMENT OF SIGNIFICANCE: Biomaterials-based 3D culture have been traditionally used for tissue regeneration. Recent research harnessed biomaterials to create 3D in vitro cancer models, with demonstrated advantages over conventional 2D culture in recapitulating tumor progression and drug response in vivo. However, previous work has been largely limited to modeling soft tissue cancer, such as breast cancer and brain cancer. Unlike soft tissues, bone is characterized with high stiffness and mineral content. Primary bone cancer affects mostly children with poor treatment outcomes, and bone is the most common site of cancer metastasis. Here we summarize emerging efforts on engineering 3D bone cancer models using tissue engineering approaches, and future directions needed to further advance this relatively new research area.

View details for DOI 10.1016/j.actbio.2019.08.020

View details for PubMedID 31419564
Microribbon-hydrogel composite scaffold accelerates cartilage regeneration in vivo with enhanced mechanical properties using mixed stem cells and chondrocytes. Biomaterials Rogan, H., Ilagan, F., Tong, X., Chu, C. R., Yang, F. 2019; 228: 119579
Abstract

Juvenile chondrocytes are robust in regenerating articular cartilage, but their clinical application is hindered by donor scarcity. Stem cells offer an abundant autologous cell source but are limited by slow cartilage deposition with poor mechanical properties. Using 3D co-culture models, mixing stem cells and chondrocytes can induce synergistic cartilage regeneration. However, the resulting cartilage tissue still suffers from poor mechanical properties after prolonged culture. Here we report a microribbon/hydrogel composite scaffold that supports synergistic interactions using co-culture of adipose-derived stem cells (ADSCs) and neonatal chondrocytes (NChons). The composite scaffold is comprised of a macroporous, gelatin microribbon (μRB) scaffolds filled with degradable nanoporous chondroitin sulfate (CS) hydrogel. We identified an optimal CS concentration (6%) that best supported co-culture synergy in vitro. Furthermore, 7 days of TGF-β3 exposure was sufficient to induce catalyzed cartilage formation. When implanted in vivo, μRB/CS composite scaffold supported over a 40-fold increase in compressive moduli of cartilage produced by mixed ADSCs/NChons to ~330 kPa, which surpassed even the quality of cartilage produced by 100% NChons. Together, these results validate μRB/CS composite as a promising scaffold for cartilage regeneration using mixed populations of stem cells and chondrocytes.

View details for DOI 10.1016/j.biomaterials.2019.119579

View details for PubMedID 31698227
Direct reprogramming of mouse fibroblasts into functional osteoblasts by defined factors Zhu, H., Conrad, B., Yang, F., Wu, J. WILEY. 2018: 94
View details for Web of Science ID 000450475400286
Biochemical and Mechanical Gradients Synergize To Enhance Cartilage Zonal Organization in 3D ACS BIOMATERIALS SCIENCE & ENGINEERING Zhu, D., Trinh, P., Liu, E., Yang, F. 2018; 4 (10): 3561–69
View details for DOI 10.1021/acsbiomaterials.8b00775

View details for Web of Science ID 000447118600011
Gelatin-based microribbon hydrogels accelerate cartilage formation by mesenchymal stem cells in 3D. Tissue engineering. Part A Conrad, B., Han, L., Yang, F. 2018
Abstract

Hydrogels are attractive matrices for cell-based cartilage tissue regeneration given their injectability and ability to fill defects with irregular shapes. However, most hydrogels developed to date often lack cell scale macroporosity, which restrains the encapsulated cells, leading to delayed new extracellular matrix deposition restricted to pericellular regions. Further, tissue engineered cartilage using conventional hydrogels generally suffers from poor mechanical property and fails to restore the load-bearing property of articular cartilage. The goal of this study was to evaluate the potential of macroporous gelatin-based microribbon (RB) hydrogels as novel 3D matrices for accelerating chondrogenesis and new cartilage formation by human mesenchymal stem cells (MSCs) in 3D with improved mechanical properties. Unlike conventional hydrogels, these RB hydrogels are inherently macroporous and exhibit cartilage-mimicking shock-absorbing mechanical property. After 21 days of culture, MSC-seeded RB scaffolds exhibit a 20-fold increase in compressive modulus to 225 kPa, a range that is approaching the level of native cartilage. In contrast, HGs only resulted in a modest increase in compressive modulus of 65 kPa. Compared to conventional hydrogels, macroporous RB scaffolds significantly increased the total amount of neocartilage produced by MSCs in 3D, with improved interconnectivity and mechanical strength. Together, these results validate gelatin-based muRBs as promising scaffolds for enhancing and accelerating MSC-based cartilage regeneration and may be used to enhance cartilage regeneration using other cell types as well.

View details for PubMedID 29926770
Recent Progress in Developing Injectable Matrices for Enhancing Cell Delivery and Tissue Regeneration ADVANCED HEALTHCARE MATERIALS Tong, X., Yang, F. 2018; 7 (7): e1701065
Abstract

Biomaterials are key factors in regenerative medicine. Matrices used for cell delivery are especially important, as they provide support to transplanted cells that is essential for promoting cell survival, retention, and desirable phenotypes. Injectable matrices have become promising and attractive due to their minimum invasiveness and ease of use. Conventional injectable matrices mostly use hydrogel precursor solutions that form solid, cell-laden hydrogel scaffolds in situ. However, these materials are associated with challenges in biocompatibility, shear-induced cell death, lack of control over cellular phenotype, lack of macroporosity and remodeling, and relatively weak mechanical strength. This Progress Report provides a brief overview of recent progress in developing injectable matrices to overcome the limitations of conventional in situ hydrogels. Biocompatible chemistry and shear-thinning hydrogels have been introduced to promote cell survival and retention. Emerging investigations of the effects of matrix properties on cellular function in 3D provide important guidelines for promoting desirable cellular phenotypes. Moreover, several novel approaches are combining injectability with macroporosity to achieve macroporous, injectable matrices for cell delivery.

View details for PubMedID 29280328
Targeting Tumor Hypoxia Using Nanoparticle-engineered CXCR4-overexpressing Adipose-derived Stem Cells THERANOSTICS Jiang, X., Wang, C., Fitch, S., Yang, F. 2018; 8 (5): 1350–60
Abstract

Hypoxia, a hallmark of malignant tumors, often correlates with increasing tumor aggressiveness and poor treatment outcomes. Due to a lack of vasculature, effective drug delivery to hypoxic tumor regions remains challenging. Signaling through the chemokine SDF-1α and its receptor CXCR4 plays a critical role in the homing of stem cells to ischemia for potential use as drug-delivery vehicles. To harness this mechanism for targeting tumor hypoxia, we developed polymeric nanoparticle-induced CXCR4-overexpressing human adipose-derived stem cells (hADSCs). Using glioblastoma multiforme (GBM) as a model tumor, we evaluated the ability of CXCR4-overexpressing hADSCs to target tumor hypoxia in vitro using a 2D migration assay and a 3D collagen hydrogel model. Compared to untransfected hADSCs, CXCR4-overexpressing hADSCs showed enhanced migration in response to hypoxia and penetrated the hypoxic core within tumor spheres. When injected in the contralateral brain in a mouse intracranial GBM xenograft, CXCR4-overexpressing hADSCs exhibited long-range migration toward GBM and preferentially penetrated the hypoxic tumor core. Intravenous injection also led to effective targeting of tumor hypoxia in a subcutaneous tumor model. Together, these results validate polymeric nanoparticle-induced CXCR4-overexpressing hADSCs as a potent cellular vehicle for targeting tumor hypoxia, which may be broadly useful for enhancing drug delivery to various cancer types.

View details for PubMedID 29507625
A comparative study of chondroitin sulfate and heparan sulfate for directing three-dimensional chondrogenesis of mesenchymal stem cells STEM CELL RESEARCH & THERAPY Wang, T., Yang, F. 2017; 8: 284
Abstract

Mesenchymal stem cells (MSCs) hold great promise for cartilage repair given their relative abundance, ease of isolation, and chondrogenic potential. To enhance MSC chondrogenesis, extracellular matrix components can be incorporated into three-dimensional (3D) scaffolds as an artificial cell niche. Chondroitin sulfate (CS)-containing hydrogels have been shown to support 3D chondrogenesis, but the effects of varying CS concentration and hydrogel stiffness on 3D MSC chondrogenesis remains elusive. Heparan sulfate (HS) is commonly used as a growth factor reservoir due to its ability to sequester growth factors; however, how it compares to CS in supporting 3D MSC chondrogenesis remains unknown.We fabricated photocrosslinkable hydrogels containing physiologically relevant concentrations (0-10%) of CS or HS with two stiffnesses (~7.5 kPa and ~ 36 kPa) as a 3D niche for MSC chondrogenesis.CS is a more potent factor in enhancing MSC chondrogenesis, especially in soft hydrogels (~ 7.5 kPa). A moderate dosage of CS (5%) led to the highest amount of neocartilage deposition. Stiff hydrogels (~ 36 kPa) generally inhibited neocartilage formation regardless of the biochemical cues.Taken together, the results from this study demonstrated that CS-containing hydrogels at low mechanical stiffness can provide a promising scaffold for enhancing MSC-based cartilage tissue regeneration.

View details for PubMedID 29258589
Bioacoustic-enabled patterning of human iPSC-derived cardiomyocytes into 3D cardiac tissue BIOMATERIALS Serpooshan, V., Chen, P., Wu, H., Lee, S., Sharma, A., Hu, D. A., Venkatraman, S., Ganesan, A. V., Usta, O. B., Yarmush, M., Yang, F., Wu, J. C., Demirci, U., Wu, S. M. 2017; 131: 47-57
Abstract

The creation of physiologically-relevant human cardiac tissue with defined cell structure and function is essential for a wide variety of therapeutic, diagnostic, and drug screening applications. Here we report a new scalable method using Faraday waves to enable rapid aggregation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) into predefined 3D constructs. At packing densities that approximate native myocardium (10(8)-10(9) cells/ml), these hiPSC-CM-derived 3D tissues demonstrate significantly improved cell viability, metabolic activity, and intercellular connection when compared to constructs with random cell distribution. Moreover, the patterned hiPSC-CMs within the constructs exhibit significantly greater levels of contractile stress, beat frequency, and contraction-relaxation rates, suggesting their improved maturation. Our results demonstrate a novel application of Faraday waves to create stem cell-derived 3D cardiac tissue that resembles the cellular architecture of a native heart tissue for diverse basic research and clinical applications.

View details for DOI 10.1016/j.biomaterials.2017.03.037

View details for PubMedID 28376365
Contractile force generation by 3D hiPSC-derived cardiac tissues is enhanced by rapid establishment of cellular interconnection in matrix with muscle-mimicking stiffness BIOMATERIALS Lee, S., Serpooshan, V., Tong, X., Venkatraman, S., Lee, M., Lee, J., Chirikian, O., Wu, J. C., Wu, S. M., Yang, F. 2017; 131: 111-120
Abstract

Engineering 3D human cardiac tissues is of great importance for therapeutic and pharmaceutical applications. As cardiac tissue substitutes, extracellular matrix-derived hydrogels have been widely explored. However, they exhibit premature degradation and their stiffness is often orders of magnitude lower than that of native cardiac tissue. There are no reports on establishing interconnected cardiomyocytes in 3D hydrogels at physiologically-relevant cell density and matrix stiffness. Here we bioengineer human cardiac microtissues by encapsulating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in chemically-crosslinked gelatin hydrogels (1.25 × 10(8)/mL) with tunable stiffness and degradation. In comparison to the cells in high stiffness (16 kPa)/slow degrading hydrogels, hiPSC-CMs in low stiffness (2 kPa)/fast degrading and intermediate stiffness (9 kPa)/intermediate degrading hydrogels exhibit increased intercellular network formation, α-actinin and connexin-43 expression, and contraction velocity. Only the 9 kPa microtissues exhibit organized sarcomeric structure and significantly increased contractile stress. This demonstrates that muscle-mimicking stiffness together with robust cellular interconnection contributes to enhancement in sarcomeric organization and contractile function of the engineered cardiac tissue. This study highlights the importance of intercellular connectivity, physiologically-relevant cell density, and matrix stiffness to best support 3D cardiac tissue engineering.

View details for DOI 10.1016/j.biomaterials.2017.03.039

View details for PubMedID 28384492
Mimicking Cartilage Tissue Zonal Organization by Engineering Tissue-scale Gradient Hydrogels as 3D Cell Niche. Tissue engineering. Part A Zhu, D., Tong, X., Trinh, P., Yang, F. 2017
Abstract

Zonal organization plays an important role in cartilage structure and function, whereas most tissue-engineering strategies developed to date have only allowed the regeneration of cartilage with homogeneous biochemical and mechanical cues. To better restore tissue structure and function, there is a strong need to engineer materials with biomimetic gradient niche cues that recapitulate native tissue organization. To address this critical unmet need, here we report a method for rapid formation of tissue-scale gradient hydrogels as a 3D cell niche with tunable biochemical and physical properties. When encapsulated in stiffness gradient hydrogels, both chondrocytes and mesenchymal stem cells demonstrated zonal-specific response and extracellular deposition that mimics zonal organization of articular cartilage. Blocking cell mechanosensing using blebbistatin abolished the zonal response of chondrocytes in 3D hydrogels with a stiffness gradient. Such tissue scale gradient hydrogels can provide a 3D artificial cell niche to enable tissue engineering of various tissue types with zonal organizations or tissue interfaces.

View details for DOI 10.1089/ten.TEA.2016.0453

View details for PubMedID 28385124
Elastin-like protein-hyaluronic acid (ELP-HA) hydrogels with decoupled mechanical and biochemical cues for cartilage regeneration. Biomaterials Zhu, D., Wang, H., Trinh, P., Heilshorn, S. C., Yang, F. 2017
Abstract

Hyaluronic acid (HA) is a major component of cartilage extracellular matrix and is an attractive material for use as 3D injectable matrices for cartilage regeneration. While previous studies have shown the promise of HA-based hydrogels to support cell-based cartilage formation, varying HA concentration generally led to simultaneous changes in both biochemical cues and stiffness. How cells respond to the change of biochemical content of HA remains largely unknown. Here we report an adaptable elastin-like protein-hyaluronic acid (ELP-HA) hydrogel platform using dynamic covalent chemistry, which allows variation of HA concentration without affecting matrix stiffness. ELP-HA hydrogels were created through dynamic hydrazone bonds via the reaction between hydrazine-modified ELP (ELP-HYD) and aldehyde-modified HA (HA-ALD). By tuning the stoichiometric ratio of aldehyde groups to hydrazine groups while maintaining ELP-HYD concentration constant, hydrogels with variable HA concentration (1.5%, 3%, or 5%) (w/v) were fabricated with comparable stiffness. To evaluate the effects of HA concentration on cell-based cartilage regeneration, chondrocytes were encapsulated within ELP-HA hydrogels with varying HA concentration. Increasing HA concentration led to a dose-dependent increase in cartilage-marker gene expression and enhanced sGAG deposition while minimizing undesirable fibrocartilage phenotype. The use of adaptable protein hydrogels formed via dynamic covalent chemistry may be broadly applicable as 3D scaffolds with decoupled niche properties to guide other desirable cell fates and tissue repair.

View details for DOI 10.1016/j.biomaterials.2017.02.010

View details for PubMedID 28268018
Effect of matrix metalloproteinase-mediated matrix degradation on glioblastoma cell behavior in 3D PEG-based hydrogels JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Wang, C., Tong, X., Jiang, X., Yang, F. 2017; 105 (3): 770-778
Abstract

Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor with median survival of 12 months. To improve clinical outcomes, it is critical to develop in vitro models that support GBM proliferation and invasion for deciphering tumor progression and screening drug candidates. A key hallmark of GBM cells is their extreme invasiveness, a process mediated by matrix metalloproteinase (MMP)-mediated degradation of the extracellular matrix. We recently reported the development of a MMP-degradable, poly(ethylene-glycol)-based hydrogel platform for culturing GBM cells. In the present study, we modulated the percentage of MMP-degradable crosslinks in 3D hydrogels to analyze the effects of MMP-degradability on GBM fates. Using an immortalized GBM cell line (U87) as a model cell type, our results showed that MMP-degradability was not required for supporting GBM proliferation. All hydrogel formulations supported robust GBM proliferation, up to 10 fold after 14 days. However, MMP-degradability was essential for facilitating tumor spreading, and 50% MMP-degradable hydrogels were sufficient to enable both robust tumor cell proliferation and spreading in 3D. The findings of this study highlight the importance of modulating MMP-degradability in engineering 3D in vitro brain cancer models and may be applied for engineering in vitro models for other cancer types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2016.

View details for DOI 10.1002/jbm.a.35947

View details for Web of Science ID 000393957700009
Mutant CCL2 protein coating mitigates wear particle-induced bone loss in a murine continuous polyethylene infusion model BIOMATERIALS Nabeshima, A., Pajarinen, J., Lin, T., Jiang, X., Gibon, E., Cordova, L. A., Loi, F., Lu, L., Jamsen, E., Egashira, K., Yang, F., Yao, Z., Goodman, S. B. 2017; 117: 1-9
Abstract

Wear particle-induced osteolysis limits the long-term survivorship of total joint replacement (TJR). Monocyte/macrophages are the key cells of this adverse reaction. Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) is the most important chemokine regulating trafficking of monocyte/macrophages in particle-induced inflammation. 7ND recombinant protein is a mutant of CCL2 that inhibits CCL2 signaling. We have recently developed a layer-by-layer (LBL) coating platform on implant surfaces that can release biologically active 7ND. In this study, we investigated the effect of 7ND on wear particle-induced bone loss using the murine continuous polyethylene (PE) particle infusion model with 7ND coating of a titanium rod as a local drug delivery device. PE particles were infused into hollow titanium rods with or without 7ND coating implanted in the distal femur for 4 weeks. Specific groups were also injected with RAW 264.7 as the reporter macrophages. Wear particle-induced bone loss and the effects of 7ND were evaluated by microCT, immunohistochemical staining, and bioluminescence imaging. Local delivery of 7ND using the LBL coating decreased systemic macrophage recruitment, the number of osteoclasts and wear particle-induced bone loss. The development of a novel orthopaedic implant coating with anti-CCL2 protein may be a promising strategy to mitigate peri-prosthetic osteolysis.

View details for DOI 10.1016/j.biomaterials.2016.11.039

View details for PubMedID 27918885
Detection of Stem Cell Transplant Rejection with Ferumoxytol MR Imaging: Correlation of MR Imaging Findings with Those at Intravital Microscopy. Radiology Daldrup-Link, H. E., Chan, C., Lenkov, O., Taghavigarmestani, S., Nazekati, T., Nejadnik, H., Chapelin, F., Khurana, A., Tong, X., Yang, F., Pisani, L., Longaker, M., Gambhir, S. S. 2017: 161139-?
Abstract

Purpose To determine whether endogenous labeling of macrophages with clinically applicable nanoparticles enables noninvasive detection of innate immune responses to stem cell transplants with magnetic resonance (MR) imaging. Materials and Methods Work with human stem cells was approved by the institutional review board and the stem cell research oversight committee, and animal experiments were approved by the administrative panel on laboratory animal care. Nine immunocompetent Sprague-Dawley rats received intravenous injection of ferumoxytol, and 18 Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice received rhodamine-conjugated ferumoxytol. Then, 48 hours later, immune-matched or mismatched stem cells were implanted into osteochondral defects of the knee joints of experimental rats and calvarial defects of Jax mice. All animals underwent serial MR imaging and intravital microscopy (IVM) up to 4 weeks after surgery. Macrophages of Jax C57BL/6-Tg (Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6) 2Bck/J mice express enhanced green fluorescent protein (GFP), which enables in vivo correlation of ferumoxytol enhancement at MR imaging with macrophage quantities at IVM. All quantitative data were compared between experimental groups by using a mixed linear model and t tests. Results Immune-mismatched stem cell implants demonstrated stronger ferumoxytol enhancement than did matched stem cell implants. At 4 weeks, T2 values of mismatched implants were significantly lower than those of matched implants in osteochondral defects of female rats (mean, 10.72 msec for human stem cells and 11.55 msec for male rat stem cells vs 15.45 msec for sex-matched rat stem cells; P = .02 and P = .04, respectively) and calvarial defects of recipient mice (mean, 21.7 msec vs 27.1 msec, respectively; P = .0444). This corresponded to increased recruitment of enhanced GFP- and rhodamine-ferumoxytol-positive macrophages into stem cell transplants, as visualized with IVM and histopathologic examination. Conclusion Endogenous labeling of macrophages with ferumoxytol enables noninvasive detection of innate immune responses to stem cell transplants with MR imaging. (©) RSNA, 2017 Online supplemental material is available for this article.

View details for DOI 10.1148/radiol.2017161139

View details for PubMedID 28128708
Pharmacological rescue of diabetic skeletal stem cell niches. Science translational medicine Tevlin, R., Seo, E. Y., Marecic, O., McArdle, A., Tong, X., Zimdahl, B., Malkovskiy, A., Sinha, R., Gulati, G., Li, X., Wearda, T., Morganti, R., Lopez, M., Ransom, R. C., Duldulao, C. R., Rodrigues, M., Nguyen, A., Januszyk, M., Maan, Z., Paik, K., Yapa, K., Rajadas, J., Wan, D. C., Gurtner, G. C., Snyder, M., Beachy, P. A., Yang, F., Goodman, S. B., Weissman, I. L., Chan, C. K., Longaker, M. T. 2017; 9 (372)
Abstract

Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

View details for DOI 10.1126/scitranslmed.aag2809

View details for PubMedID 28077677
Covalently adaptable elastin-like protein - hyaluronic acid (ELP - HA) hybrid hydrogels with secondary thermoresponsive crosslinking for injectable stem cell delivery. Advanced functional materials Wang, H., Zhu, D., Paul, A., Cai, L., Enejder, A., Yang, F., Heilshorn, S. C. 2017; 27 (28)
Abstract

Shear-thinning, self-healing hydrogels are promising vehicles for therapeutic cargo delivery due to their ability to be injected using minimally invasive surgical procedures. We present an injectable hydrogel using a novel combination of dynamic covalent crosslinking with thermoresponsive engineered proteins. Ex situ at room temperature, rapid gelation occurs through dynamic covalent hydrazone bonds by simply mixing two components: hydrazine-modified elastin-like protein (ELP) and aldehyde-modified hyaluronic acid. This hydrogel provides significant mechanical protection to encapsulated human mesenchymal stem cells during syringe needle injection and rapidly recovers after injection to retain the cells homogeneously within a 3D environment. In situ, the ELP undergoes a thermal phase transition, as confirmed by Coherent anti-Stokes Raman scattering microscopy observation of dense ELP thermal aggregates. The formation of the secondary network reinforces the hydrogel and results in a 10-fold slower erosion rate compared to a control hydrogel without secondary thermal crosslinking. This improved structural integrity enables cell culture for three weeks post injection, and encapsulated cells maintain their ability to differentiate into multiple lineages, including chondrogenic, adipogenic, and osteogenic cell types. Together, these data demonstrate the promising potential of ELP-HA hydrogels for injectable stem cell transplantation and tissue regeneration.

View details for DOI 10.1002/adfm.201605609

View details for PubMedID 33041740

View details for PubMedCentralID PMC7546546
Modulating stem cell-chondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels JOURNAL OF MATERIALS CHEMISTRY B Wang, T., Lai, J. H., Han, L., Tong, X., Yang, F. 2016; 4 (47): 7641-7650
View details for DOI 10.1039/c6tb01583b

View details for Web of Science ID 000391777800014
Nanoparticle engineered TRAIL-overexpressing adipose-derived stem cells target and eradicate glioblastoma via intracranial delivery PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jiang, X., Fitch, S., Wang, C., Wilson, C., Li, J., Grant, G. A., Yang, F. 2016; 113 (48): 13857-13862
Abstract

Glioblastoma multiforme (GBM) is one of the most intractable of human cancers, principally because of the highly infiltrative nature of these neoplasms. Tracking and eradicating infiltrating GBM cells and tumor microsatellites is of utmost importance for the treatment of this devastating disease, yet effective strategies remain elusive. Here we report polymeric nanoparticle-engineered human adipose-derived stem cells (hADSCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as drug-delivery vehicles for targeting and eradicating GBM cells in vivo. Our results showed that polymeric nanoparticle-mediated transfection led to robust up-regulation of TRAIL in hADSCs, and that TRAIL-expressing hADSCs induced tumor-specific apoptosis. When transplanted in a mouse intracranial xenograft model of patient-derived glioblastoma cells, hADSCs exhibited long-range directional migration and infiltration toward GBM tumor. Importantly, TRAIL-overexpressing hADSCs inhibited GBM growth, extended survival, and reduced the occurrence of microsatellites. Repetitive injection of TRAIL-overexpressing hADSCs significantly prolonged animal survival compared with single injection of these cells. Taken together, our data suggest that nanoparticle-engineered TRAIL-expressing hADSCs exhibit the therapeutically relevant behavior of "seek-and-destroy" tumortropic migration and could be a promising therapeutic approach to improve the treatment outcomes of patients with malignant brain tumors.

View details for DOI 10.1073/pnas.1615396113

View details for Web of Science ID 000388835700085

View details for PubMedID 27849590

View details for PubMedCentralID PMC5137687
Effect of Matrix Metalloproteinase-Mediated Matrix Degradation on Glioblastoma Cell Behavior in 3D PEG-based Hydrogels. Journal of biomedical materials research. Part A Wang, C., Tong, X., Jiang, X., Yang, F. 2016
Abstract

Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor with median survival of 12 months. To improve clinical outcomes, it is critical to develop in vitro models that support GBM proliferation and invasion for deciphering tumor progression and screening drug candidates. A key hallmark of GBM cells is their extreme invasiveness, a process mediated by matrix metalloproteinase (MMP)-mediated degradation of the extracellular matrix. We recently reported the development of a MMP-degradable, poly(ethylene-glycol)-based hydrogel platform for culturing GBM cells. In the present study, we modulated the percentage of MMP-degradable crosslinks in 3D hydrogels to analyze the effects of MMP-degradability on GBM fates. Using an immortalized GBM cell line (U87) as a model cell type, our results showed that MMP-degradability was not required for supporting GBM proliferation. All hydrogel formulations supported robust GBM proliferation, up to 10 fold after 14 days. However, MMP-degradability was essential for facilitating tumor spreading, and 50% MMP-degradable hydrogels were sufficient to enable both robust tumor cell proliferation and spreading in 3D. The findings of this study highlight the importance of modulating MMP-degradability in engineering 3D in vitro brain cancer models and may be applied for engineering in vitro models for other cancer types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2016.

View details for DOI 10.1002/jbm.a.35947

View details for PubMedID 27770562
Effects of Hydrogel Stiffness and Extracellular Compositions on Modulating Cartilage Regeneration by Mixed Populations of Stem Cells and Chondrocytes In Vivo. Tissue engineering. Part A Wang, T., Lai, J. H., Yang, F. 2016: -?
Abstract

Cell-based therapies offer great promise for repairing cartilage. Previous strategies often involved using a single cell population such as stem cells or chondrocytes. A mixed cell population may offer an alternative strategy for cartilage regeneration while overcoming donor scarcity. We have recently reported that adipose-derived stem cells (ADSCs) can catalyze neocartilage formation by neonatal chondrocytes (NChons) when mixed co-cultured in 3D hydrogels in vitro. However, it remains unknown how the biochemical and mechanical cues of hydrogels modulate cartilage formation by mixed cell populations in vivo. The present study seeks to answer this question by co-encapsulating ADSCs and NChons in 3D hydrogels with tunable stiffness (∼1-33 kPa) and biochemical cues, and evaluating cartilage formation in vivo using a mouse subcutaneous model. Three extracellular matrix molecules were examined, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Our results showed that the type of biochemical cue played a dominant role in modulating neocartilage deposition. CS and HA enhanced type II collagen deposition, a desirable phenotype for articular cartilage. In contrast, HS promoted fibrocartilage phenotype with the upregulation of type I collagen and failed to retain newly deposited matrix. Hydrogels with stiffnesses of ∼7-33 kPa led to a comparable degree of neocartilage formation, and a minimal initial stiffness was required to retain hydrogel integrity over time. Results from this study highlight the important role of matrix cues in directing neocartilage formation, and they offer valuable insights in guiding optimal scaffold design for cartilage regeneration by using mixed cell populations.

View details for PubMedID 27676200
The effect of local IL-4 delivery or CCL2 blockade on implant fixation and bone structural properties in a mouse model of wear particle induced osteolysis. Journal of biomedical materials research. Part A Sato, T., Pajarinen, J., Behn, A., Jiang, X., Lin, T., Loi, F., Yao, Z., Egashira, K., Yang, F., Goodman, S. B. 2016; 104 (9): 2255-2262
Abstract

Modulation of macrophage polarization and prevention of CCL2-induced macrophage chemotaxis are emerging strategies to reduce wear particle induced osteolysis and aseptic total joint replacement loosening. In this study, the effect of continuous IL-4 delivery or bioactive implant coating that constitutively releases a protein inhibitor of CCL2 signaling (7ND) on particle induced osteolysis were studied in the murine continuous femoral intramedullary particle infusion model. Polyethylene particles with or without IL-4 were infused into mouse distal femurs implanted with hollow titanium rods using subcutaneous infusion pumps. In another experimental group, particles were infused into the femur through a 7ND coated rod. After four weeks, fixation of the implant was assessed using a pullout test. The volume of trabecular bone and the geometry of the local cortical bone were assessed by µCT and the corresponding structural properties of the cortical bone determined by torsional testing. Continuous IL-4 delivery led to increased trabecular bone volume as well as enhanced local bone geometry and structural properties, while 7ND implant coating did not have effect on these parameters. The results suggest that local IL-4 treatment is a promising strategy to mitigate wear particle induced osteolysis. This article is protected by copyright. All rights reserved.

View details for DOI 10.1002/jbm.a.35759

View details for PubMedID 27114284
Sliding Hydrogels with Mobile Molecular Ligands and Crosslinks as 3D Stem Cell Niche. Advanced materials Tong, X., Yang, F. 2016; 28 (33): 7257-7263
Abstract

The development of a sliding hydrogel with mobile crosslinks and biochemical ligands as a 3D stem cell niche is reported. The molecular mobility of this sliding hydrogel allows stem cells to reorganize the surrounding ligands and change their morphology in 3D. Without changing matrix stiffness, sliding hydrogels support efficient stem cell differentiation toward multiple lineages including adipogenesis, chondrogenesis, and osteogenesis.

View details for DOI 10.1002/adma.201601484

View details for PubMedID 27305637
Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Keeney, M., Chung, M. T., Zielins, E. R., Paik, K. J., McArdle, A., Morrison, S. D., Ransom, R. C., Barbhaiya, N., Atashroo, D., Jacobson, G., Zare, R. N., Longaker, M. T., Wan, D. C., Yang, F. 2016; 104 (8): 2099-2107
Abstract

Scaffold-mediated gene delivery holds great promise for tissue regeneration. However, previous attempts to induce bone regeneration using scaffold-mediated non-viral gene delivery rarely resulted in satisfactory healing. We report a novel platform with sustained release of minicircle DNA (MC) from PLGA scaffolds to accelerate bone repair. MC was encapsulated inside PLGA scaffolds using supercritical CO2 , which showed prolonged release of MC. Skull-derived osteoblasts transfected with BMP-2 MC in vitro result in higher osteocalcin gene expression and mineralized bone formation. When implanted in a critical-size mouse calvarial defect, scaffolds containing luciferase MC lead to robust in situ protein production up to at least 60 days. Scaffold-mediated BMP-2 MC delivery leads to substantially accelerated bone repair as early as two weeks, which continues to progress over 12 weeks. This platform represents an efficient, long-term nonviral gene delivery system, and may be applicable for enhancing repair of a broad range of tissues types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2099-2107, 2016.

View details for DOI 10.1002/jbm.a.35735

View details for Web of Science ID 000379736500025

View details for PubMedID 27059085
Winner of the Young Investigator Award of the Society for Biomaterials at the 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Microribbon-based hydrogels accelerate stem cell-based bone regeneration in a mouse critical-size cranial defect model JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Han, L., Conrad, B., Chung, M. T., Deveza, L., Jiang, X., Wang, A., Butte, M. J., Longaker, M. T., Wan, D., Yang, F. 2016; 104 (6): 1321-1331
Abstract

Stem cell-based therapies hold great promise for enhancing tissue regeneration. However, the majority of cells die shortly after transplantation, which greatly diminishes the efficacy of stem cell-based therapies. Poor cell engraftment and survival remain a major bottleneck to fully exploiting the power of stem cells for regenerative medicine. Biomaterials such as hydrogels can serve as artificial matrices to protect cells during delivery and guide desirable cell fates. However, conventional hydrogels often lack macroporosity, which restricts cell proliferation and delays matrix deposition. Here we report the use of injectable, macroporous microribbon (μRB) hydrogels as stem cell carriers for bone repair, which supports direct cell encapsulation into a macroporous scaffold with rapid spreading. When transplanted in a critical-sized, mouse cranial defect model, μRB-based hydrogels significantly enhanced the survival of transplanted adipose-derived stromal cells (ADSCs) (81%) and enabled up to three-fold cell proliferation after 7 days. In contrast, conventional hydrogels only led to 27% cell survival, which continued to decrease over time. MicroCT imaging showed μRBs enhanced and accelerated mineralized bone repair compared to hydrogels (61% vs. 34% by week 6), and stem cells were required for bone repair to occur. These results suggest that paracrine signaling of transplanted stem cells are responsible for the observed bone repair, and enhancing cell survival and proliferation using μRBs further promoted the paracrine-signaling effects of ADSCs for stimulating endogenous bone repair. We envision μRB-based scaffolds can be broadly useful as a novel scaffold for enhancing stem cell survival and regeneration of other tissue types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1321-1331, 2016.

View details for DOI 10.1002/jbm.a.35715

View details for Web of Science ID 000375117200001

View details for PubMedID 26991141

View details for PubMedCentralID PMC5142823
Winner of the Young Investigator Award of the Society for Biomaterials (USA) for 2016, 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Aligned microribbon-like hydrogels for guiding three-dimensional smooth muscle tissue regeneration JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Lee, S., Tong, X., Han, L., Behn, A., Yang, F. 2016; 104 (5): 1064-1071
Abstract

Smooth muscle tissue is characterized by aligned structures, which is critical for its contractile functions. Smooth muscle injury is common and can be caused by various diseases and degenerative processes, and there remains a strong need to develop effective therapies for smooth muscle tissue regeneration with restored structures. To guide cell alignment, previously cells were cultured on 2D nano/microgrooved substrates, but such method is limited to fabricating 2D aligned cell sheets only. Alternatively, aligned electrospun nanofiber has been employed as 3D scaffold for cell alignment, but cells can only be seeded post fabrication, and nanoporosity of electrospun fiber meshes often leads to poor cell distribution. To overcome these limitations, we report aligned gelatin-based microribbons (µRBs) as macroporous hydrogels for guiding smooth muscle alignment in 3D. We developed aligned µRB-like hydrogels using wet spinning, which allows easy fabrication of tissue-scale (cm) macroporous matrices with alignment cues and supports direct cell encapsulation. The macroporosity within µRB-based hydrogels facilitated cell proliferation, new matrix deposition, and nutrient diffusion. In aligned µRB scaffold, smooth muscle cells showed high viability, rapid adhesion, and alignment following µRB direction. Aligned µRB scaffolds supported retention of smooth muscle contractile phenotype, and accelerated uniaxial deposition of new matrix (collagen I/IV) along the µRB. In contrast, cells encapsulated in conventional gelatin hydrogels remained round with matrix deposition limited to pericellular regions only. We envision such aligned µRB scaffold can be broadly applicable in growing other anisotropic tissues including tendon, nerves and blood vessel. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1064-1071, 2016.

View details for DOI 10.1002/jbm.a.35662

View details for PubMedID 26799256
Hydrogels with Dual Gradients of Mechanical and Biochemical Cues for Deciphering Cell-Niche Interactions ACS BIOMATERIALS-SCIENCE & ENGINEERING Tong, X., Jiang, J., Zhu, D., Yang, F. 2016; 2 (5): 845-852
View details for DOI 10.1021/acsbiomaterials.6b00074

View details for Web of Science ID 000375893600016
Effects of the poly(ethylene glycol) hydrogel crosslinking mechanism on protein release. Biomaterials science Lee, S., Tong, X., Yang, F. 2016; 4 (3): 405-411
Abstract

Poly(ethylene glycol) (PEG) hydrogels are widely used to deliver therapeutic biomolecules, due to high hydrophilicity, tunable physicochemical properties, and anti-fouling properties. Although different hydrogel crosslinking mechanisms are known to result in distinct network structures, it is still unknown how these various mechanisms influence biomolecule release. Here we compared the effects of chain-growth and step-growth polymerization for hydrogel crosslinking on the efficiency of protein release and diffusivity. For chain-growth-polymerized PEG hydrogels, while decreasing PEG concentration increased both the protein release efficiency and diffusivity, it was unexpected to find out that increasing PEG molecular weight did not significantly change either parameter. In contrast, for step-growth-polymerized PEG hydrogels, both decreasing PEG concentration and increasing PEG molecular weight resulted in an increase in the protein release efficiency and diffusivity. For step-growth-polymerized hydrogels, the protein release efficiency and diffusivity were further decreased by increasing crosslink functionality (4-arm to 8-arm) of the chosen monomer. Altogether, our results demonstrate that the crosslinking mechanism has a differential effect on controlling protein release, and this study provides valuable information for the rational design of hydrogels for sophisticated drug delivery.

View details for DOI 10.1039/c5bm00256g

View details for PubMedID 26539660
Polymer-DNA Nanoparticle-Induced CXCR4 Overexpression Improves Stem Cell Engraftment and Tissue Regeneration in a Mouse Hindlimb Ischemia Model THERANOSTICS Deveza, L., Choi, J., Lee, J., Huang, N., Cooke, J., Yang, F. 2016; 6 (8): 1176-1189
Abstract

Peripheral arterial disease affects nearly 202 million individuals worldwide, sometimes leading to non-healing ulcers or limb amputations in severe cases. Genetically modified stem cells offer potential advantages for therapeutically inducing angiogenesis via augmented paracrine release mechanisms and tuned dynamic responses to environmental stimuli at disease sites. Here, we report the application of nanoparticle-induced CXCR4-overexpressing stem cells in a mouse hindlimb ischemia model. We found that CXCR4 overexpression improved stem cell survival, modulated inflammation in situ, and accelerated blood reperfusion. These effects, unexpectedly, led to complete limb salvage and skeletal muscle repair, markedly outperforming the efficacy of the conventional angiogenic factor control, VEGF. Importantly, assessment of CXCR4-overexpressing stem cells in vitro revealed that CXCR4 overexpression induced changes in paracrine signaling of stem cells, promoting a therapeutically desirable pro-angiogenic and anti-inflammatory phenotype. These results suggest that nanoparticle-induced CXCR4 overexpression may promote favorable phenotypic changes and therapeutic efficacy of stem cells in response to the ischemic environment.

View details for DOI 10.7150/thno.12866

View details for Web of Science ID 000378529400009

View details for PubMedID 27279910

View details for PubMedCentralID PMC4893644
Local delivery of mutant CCL2 protein-reduced orthopaedic implant wear particle-induced osteolysis and inflammation in vivo. Journal of orthopaedic research Jiang, X., Sato, T., Yao, Z., Keeney, M., Pajarinen, J., Lin, T., Loi, F., Egashira, K., Goodman, S., Yang, F. 2016; 34 (1): 58-64
Abstract

Total joint replacement (TJR) has been widely used as a standard treatment for late-stage arthritis. One challenge for long-term efficacy of TJR is the generation of ultra-high molecular weight polyethylene wear particles from the implant surface that activates an inflammatory cascade that may lead to bone loss, prosthetic loosening and eventual failure of the procedure. Here we investigate the efficacy of local administration of mutant CCL2 proteins, such as 7ND, on reducing wear particle-induced inflammation and osteolysis in vivo using a mouse calvarial model. Mice were treated with local injection of 7ND or phosphate buffered saline (PBS) every other day for up to 14 days. Wear particle-induced osteolysis and the effects of 7ND treatment were evaluated using micro-CT, histology and immunofluorescence staining. Compared with the PBS control, 7ND treatment significantly decreased wear particle-induced osteolysis, which led to a higher bone volume fraction and bone mineral density. Furthermore, immunofluorescence staining showed 7ND treatment decreased the number of recruited inflammatory cells and osteoclasts. Together, our results support the feasibility of local delivery of 7ND for mitigating wear particle-induced inflammation and osteolysis, which may offer a promising strategy for extending the life time of TJRs. This article is protected by copyright. All rights reserved.

View details for DOI 10.1002/jor.22977

View details for PubMedID 26174978
Modulating stem cell-chondrocyte interactions for cartilage repair using combinatorial extracellular matrix-containing hydrogels. Journal of materials chemistry. B Wang, T., Lai, J. H., Han, L. H., Tong, X., Yang, F. 2016; 4 (47): 7641–50
Abstract

Stem cells can contribute to cartilage repair either directly through chondrogenic differentiation or indirectly through paracrine signaling. Using a 3D co-culture model, we have recently reported that adipose-derived stem cells (ADSCs) can catalyze cartilage formation by neonatal chondrocytes (NChons) when mixed co-cultured in biomimetic hydrogels. However, how matrix cues influence such catalyzed cartilage formation remains unknown. To answer this question, ADSCs and NChons were co-encapsulated in 39 combinatorial hydrogel compositions with decoupled biochemical and mechanical properties. Methacrylated extracellular matrix (ECM) molecules including chondroitin sulfate, hyaluronic acid and heparan sulfate were incorporated at varying concentrations (0.5%, 1.25%, 2.5% and 5%) (w/v). Mechanical testing confirmed that hydrogel stiffness was largely decoupled from ECM cues (15 kPa, 40 kPa and 100 kPa). The biochemical assay and histology results showed that the type of ECM cue played a dominant role in modulating catalyzed cartilage formation, while varying hydrogel stiffness and doses of ECM led to more modest changes. Both chondroitin sulfate and hyaluronic acid led to robust articular cartilage matrix deposition, as shown by the intense staining of aggrecan and type II collagen. In soft hydrogels (15 kPa), chondroitin sulfate led to the highest amount of sulfated glycosaminoglycan deposition and increased compressive moduli. In contrast, heparan sulfate promoted type I collagen deposition, an undesirable fibrocartilage phenotype, and increasing heparan sulfate decreased cell proliferation and ECM deposition. Findings from the present study may guide the optimal scaffold design to maximize the synergistic cartilage formation using mixed cell populations.

View details for DOI 10.1039/c6tb01583b

View details for PubMedID 32263820
Long-Term Controlled Protein Release from Poly(Ethylene Glycol) Hydrogels by Modulating Mesh Size and Degradation MACROMOLECULAR BIOSCIENCE Tong, X., Lee, S., Bararpour, L., Yang, F. 2015; 15 (12): 1679-1686
Abstract

Poly(ethylene glycol) (PEG)-based hydrogels are popular biomaterials for protein delivery to guide desirable cellular fates and tissue repair. However, long-term protein release from PEG-based hydrogels remains challenging. Here, we report a PEG-based hydrogel platform for long term protein release, which allows efficient loading of proteins via physical entrapment. Tuning hydrogel degradation led to increase in hydrogel mesh size and gradual release of protein over 60 days of with retained bioactivity. Importantly, this platform does not require the chemical modification of loaded proteins, and may serve as a versatile tool for long-term delivery of a wide range of proteins for drug-delivery and tissue-engineering applications.

View details for DOI 10.1002/mabi.201500245

View details for Web of Science ID 000368456500007

View details for PubMedCentralID PMC5127624
Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells FASEB JOURNAL Lee, J., Taylor, S. E., Smeriglio, P., Lai, J., Maloney, W. J., Yang, F., Bhutani, N. 2015; 29 (8): 3399-3410
Abstract

Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening.-Lee, J., Taylor, S. E. B., Smeriglio, P., Lai, J., Maloney, W. J., Yang, F., Bhutani, N. Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells.

View details for DOI 10.1096/fj.14-269720

View details for Web of Science ID 000358796900027

View details for PubMedID 25911615
Gene delivery of osteoinductive signals to a human fetal osteoblast cell line induces cell death in a dose-dependent manner. Drug delivery and translational research Ramasubramanian, A., Jeeawoody, S., Yang, F. 2015; 5 (2): 160-167
Abstract

Gene delivery provides a powerful tool for regulating tissue regeneration by activating or inhibiting specific genes associated with targeted signaling pathways. Up-regulating bone morphogenetic protein-2 (BMP-2) or silencing GNAS and Noggin gene expression in stem cells has been shown to enhance osteogenic differentiation and bone tissue formation. However, few studies have examined how such gene delivery would influence other differentiated cell types residing in the bone. In this study, we examined the effects of DNA delivery of BMP-2 and siRNA delivery of GNAS or Noggin on a widely used human fetal osteoblast cell line (hFOB1.19) using biomaterials-mediated gene delivery. Our results showed that both GNAS and Noggin siRNA delivery increased cell death in hFOB1.19 in a dose-dependent manner. In particular, groups treated with the highest doses of BMP-2, siGNAS or siNoggin showed a more than 50 % decline in cell proliferation and a 90 % decline in cell viability compared to untransfected and sham DNA/siRNA-transfected controls. TUNEL staining showed that BMP-2, siGNAS or siNoggin induced cell apoptosis in hFOBs. In contrast, cells transfected using sham DNA or siRNA showed no noticeable cell death or apoptosis. These results elucidate the nuanced responses of progenitor and immortalized cell populations to the delivery of exogenous osteoinductive genes. In particular, they highlight the differences between immortalized and primary cell lines and underscore the importance of targeted gene delivery mechanisms in the regeneration of injured bone tissue.

View details for DOI 10.1007/s13346-013-0163-x

View details for PubMedID 25787741
Improved Approach for Chondrogenic Differentiation of Human Induced Pluripotent Stem Cells STEM CELL REVIEWS AND REPORTS Nejadnik, H., Diecke, S., Lenkov, O. D., Chapelin, F., Donig, J., Tong, X., Derugin, N., Chan, R. C., Gaur, A., Yang, F., Wu, J. C., Daldrup-Link, H. E. 2015; 11 (2): 242-253
Abstract

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However, current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation, which is required to generate endodermal, ectodermal, and mesodermal cell lineages. We report a new, straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs, which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1, GAG, and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats, magnetic resonance imaging studies showed gradual engraftment, and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs, which could improve cartilage regeneration outcomes in arthritic joints.

View details for DOI 10.1007/s12015-014-9581-5

View details for Web of Science ID 000353149700004

View details for PubMedID 25578634
Interaction Between Osteoarthritic Chondrocytes and Adipose-Derived Stem Cells Is Dependent on Cell Distribution in Three-Dimension and Transforming Growth Factor-ß3 Induction. Tissue engineering. Part A Lai, J. H., Rogan, H., Kajiyama, G., Goodman, S. B., Smith, R. L., Maloney, W., Yang, F. 2015; 21 (5-6): 992-1002
Abstract

Stem cells hold great promise for treating cartilage degenerative diseases such as osteoarthritis (OA). The efficacy of stem cell-based therapy for cartilage repair is highly dependent on their interactions with local cells in the joint. This study aims at evaluating the interactions between osteoarthritic chondrocytes (OACs) and adipose-derived stem cells (ADSCs) using three dimensional (3D) biomimetic hydrogels. To examine the effects of cell distribution on such interactions, ADSCs and OACs were co-cultured in 3D using three co-culture models: conditioned medium (CM), bi-layered, and mixed co-culture with varying cell ratios. Furthermore, the effect of transforming growth factor (TGF)-β3 supplementation on ADSC-OAC interactions and the resulting cartilage formation was examined. Outcomes were analyzed using quantitative gene expression, cell proliferation, cartilage matrix production, and histology. TGF-β3 supplementation led to a substantial increase in cartilage matrix depositions in all groups, but had differential effects on OAC-ADSC interactions in different co-culture models. In the absence of TGF-β3, CM or bi-layered co-culture had negligible effects on gene expression or cartilage formation. With TGF-β3 supplementation, CM and bi-layered co-culture inhibited cartilage formation by both ADSCs and OACs. In contrast, a mixed co-culture with moderate OAC ratios (25% and 50%) resulted in synergistic interactions with enhanced cartilage matrix deposition and reduced catabolic marker expression. Our results suggested that the interaction between OACs and ADSCs is highly dependent on cell distribution in 3D and soluble factors, which should be taken into consideration when designing stem cell-based therapy for treating OA patients.

View details for DOI 10.1089/ten.TEA.2014.0244

View details for PubMedID 25315023
Microfluidic Synthesis of Biodegradable Polyethylene-Glycol Microspheres for Controlled Delivery of Proteins and DNA Nanoparticles ACS BIOMATERIALS-SCIENCE & ENGINEERING Deveza, L., Ashoken, J., Castaneda, G., Tong, X., Keeney, M., Han, L., Yang, F. 2015; 1 (3): 157-165
View details for DOI 10.1021/ab500051v

View details for Web of Science ID 000369347200004
Collagen VI Enhances Cartilage Tissue Generation by Stimulating Chondrocyte Proliferation. Tissue engineering. Part A Smeriglio, P., Dhulipala, L., Lai, J. H., Goodman, S. B., Dragoo, J. L., Smith, R. L., Maloney, W. J., Yang, F., Bhutani, N. 2015; 21 (3-4): 840-849
Abstract

Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.

View details for DOI 10.1089/ten.TEA.2014.0375

View details for PubMedID 25257043
Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels. Tissue engineering. Part A Smeriglio, P., Lai, J. H., Dhulipala, L., Behn, A. W., Goodman, S. B., Smith, R. L., Maloney, W. J., Yang, F., Bhutani, N. 2015; 21 (1-2): 147-155
Abstract

Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, as well as embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old and diseased chondrocytes, here we examined cartilage formation by juvenile, adult and OA chondrocytes in 3D biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and GAG accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.

View details for DOI 10.1089/ten.TEA.2014.0070

View details for PubMedID 25054343
Long-Term Controlled Protein Release from Poly(Ethylene Glycol) Hydrogels by Modulating Mesh Size and Degradation. Macromolecular bioscience Tong, X., Lee, S., Bararpour, L., Yang, F. 2015; 15 (12): 1679–86
Abstract

Poly(ethylene glycol) (PEG)-based hydrogels are popular biomaterials for protein delivery to guide desirable cellular fates and tissue repair. However, long-term protein release from PEG-based hydrogels remains challenging. Here, we report a PEG-based hydrogel platform for long term protein release, which allows efficient loading of proteins via physical entrapment. Tuning hydrogel degradation led to increase in hydrogel mesh size and gradual release of protein over 60 days of with retained bioactivity. Importantly, this platform does not require the chemical modification of loaded proteins, and may serve as a versatile tool for long-term delivery of a wide range of proteins for drug-delivery and tissue-engineering applications.

View details for PubMedID 26259711
3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation. Journal of visualized experiments : JoVE Smeriglio, P., Lai, J. H., Yang, F., Bhutani, N. 2015
Abstract

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

View details for DOI 10.3791/53085

View details for PubMedID 26484414

View details for PubMedCentralID PMC4692641
The effects of varying poly(ethylene glycol) hydrogel crosslinking density and the crosslinking mechanism on protein accumulation in three-dimensional hydrogels ACTA BIOMATERIALIA Lee, S., Tong, X., Yang, F. 2014; 10 (10): 4167-4174
Abstract

Matrix stiffness has been shown to play an important role in modulating various cell fate processes such as differentiation and cell cycle. Given that the stiffness can be easily tuned by varying the crosslinking density, poly(ethylene glycol) (PEG) hydrogels have been widely used as an artificial cell niche. However, little is known about how changes in the hydrogel crosslinking density may affect the accumulation of exogenous growth factors within 3-D hydrogel scaffolds formed by different crosslinking mechanisms. To address such shortcomings, we measured protein diffusivity and accumulation within PEG hydrogels with varying PEG molecular weight, concentration and crosslinking mechanism. We found that protein accumulation increased substantially above a critical mesh size, which was distinct from the protein diffusivity trend, highlighting the importance of using protein accumulation as a parameter to better predict the cell fates in addition to protein diffusivity, a parameter commonly reported by researchers studying protein diffusion in hydrogels. Furthermore, we found that chain-growth-polymerized gels allowed more protein accumulation than step-growth-polymerized gels, which may be the result of network heterogeneity. The strategy used here can help quantify the effects of varying the hydrogel crosslinking density and crosslinking mechanism on protein diffusion in different types of hydrogel. Such tools could be broadly useful for interpreting cellular responses in hydrogels of varying stiffness for various tissue engineering applications.

View details for DOI 10.1016/j.actbio.2014.05.023

View details for Web of Science ID 000342523800011
The effects of varying poly(ethylene glycol) hydrogel crosslinking density and the crosslinking mechanism on protein accumulation in three-dimensional hydrogels. Acta biomaterialia Lee, S., Tong, X., Yang, F. 2014; 10 (10): 4167-4174
Abstract

Matrix stiffness has been shown to play an important role in modulating various cell fate processes such as differentiation and cell cycle. Given that the stiffness can be easily tuned by varying the crosslinking density, poly(ethylene glycol) (PEG) hydrogels have been widely used as an artificial cell niche. However, little is known about how changes in the hydrogel crosslinking density may affect the accumulation of exogenous growth factors within 3-D hydrogel scaffolds formed by different crosslinking mechanisms. To address such shortcomings, we measured protein diffusivity and accumulation within PEG hydrogels with varying PEG molecular weight, concentration and crosslinking mechanism. We found that protein accumulation increased substantially above a critical mesh size, which was distinct from the protein diffusivity trend, highlighting the importance of using protein accumulation as a parameter to better predict the cell fates in addition to protein diffusivity, a parameter commonly reported by researchers studying protein diffusion in hydrogels. Furthermore, we found that chain-growth-polymerized gels allowed more protein accumulation than step-growth-polymerized gels, which may be the result of network heterogeneity. The strategy used here can help quantify the effects of varying the hydrogel crosslinking density and crosslinking mechanism on protein diffusion in different types of hydrogel. Such tools could be broadly useful for interpreting cellular responses in hydrogels of varying stiffness for various tissue engineering applications.

View details for DOI 10.1016/j.actbio.2014.05.023

View details for PubMedID 24887284
Co-release of cells and polymeric nanoparticles from sacrificial microfibers enhances nonviral gene delivery inside 3D hydrogels. Tissue engineering. Part C, Methods Madl, C. M., Keeney, M., Li, X., Han, L., Yang, F. 2014; 20 (10): 798-805
Abstract

Hydrogels can promote desirable cellular phenotype by mimicking tissue-like stiffness or serving as a gene delivery depot. However, nonviral gene delivery inside three-dimensional (3D) hydrogels remains a great challenge, and increasing hydrogel stiffness generally results in further decrease in gene delivery efficiency. Here we report a method to enhance nonviral gene delivery efficiency inside 3D hydrogels across a broad range of stiffness using sacrificial microfibers for co-releasing cells and polymeric nanoparticles (NPs). We fabricated hydrolytically degradable alginate as sacrificial microfibers, and optimized the degradation profile of alginate by varying the degree of oxidization. Scanning electron microscopy confirmed degradation of alginate microfibers inside hydrogels, leaving behind microchannel-like structures within 3D hydrogels. Sacrificial microfibers also serve as a delivery vehicle for co-releasing encapsulated cells and NPs, allowing cell attachment and spreading within the microchannel surface upon microfiber degradation. To examine the effects of sacrificial microfibers on nonviral gene delivery inside 3D hydrogels, alginate microfibers containing human embryonic kidney 293 cells and polymeric NPs were encapsulated within 3D hydrogel scaffolds with varying stiffness (9, 58, and 197 kPa). Compared with cells encapsulated in bulk hydrogels, we observed up to 15-fold increase in gene delivery efficiency using sacrificial microfibers, and gene delivery efficiency increased as hydrogel stiffness increased. The platform reported herein provides a strategy for enhancing nonviral gene delivery inside 3D hydrogels across a broad range of stiffness, and may aid tissue regeneration by engaging both mechanotransduction and nonviral gene delivery.

View details for DOI 10.1089/ten.TEC.2013.0669

View details for PubMedID 24483329
Co-Release of Cells and Polymeric Nanoparticles from Sacrificial Microfibers Enhances Nonviral Gene Delivery Inside 3D Hydrogels TISSUE ENGINEERING PART C-METHODS Madl, C. M., Keeney, M., Li, X., Han, L., Yang, F. 2014; 20 (10): 798-805
Abstract

Hydrogels can promote desirable cellular phenotype by mimicking tissue-like stiffness or serving as a gene delivery depot. However, nonviral gene delivery inside three-dimensional (3D) hydrogels remains a great challenge, and increasing hydrogel stiffness generally results in further decrease in gene delivery efficiency. Here we report a method to enhance nonviral gene delivery efficiency inside 3D hydrogels across a broad range of stiffness using sacrificial microfibers for co-releasing cells and polymeric nanoparticles (NPs). We fabricated hydrolytically degradable alginate as sacrificial microfibers, and optimized the degradation profile of alginate by varying the degree of oxidization. Scanning electron microscopy confirmed degradation of alginate microfibers inside hydrogels, leaving behind microchannel-like structures within 3D hydrogels. Sacrificial microfibers also serve as a delivery vehicle for co-releasing encapsulated cells and NPs, allowing cell attachment and spreading within the microchannel surface upon microfiber degradation. To examine the effects of sacrificial microfibers on nonviral gene delivery inside 3D hydrogels, alginate microfibers containing human embryonic kidney 293 cells and polymeric NPs were encapsulated within 3D hydrogel scaffolds with varying stiffness (9, 58, and 197 kPa). Compared with cells encapsulated in bulk hydrogels, we observed up to 15-fold increase in gene delivery efficiency using sacrificial microfibers, and gene delivery efficiency increased as hydrogel stiffness increased. The platform reported herein provides a strategy for enhancing nonviral gene delivery inside 3D hydrogels across a broad range of stiffness, and may aid tissue regeneration by engaging both mechanotransduction and nonviral gene delivery.

View details for DOI 10.1089/ten.tec.2013.0669

View details for Web of Science ID 000342562700004
Mutant monocyte chemoattractant protein 1 protein attenuates migration of and inflammatory cytokine release by macrophages exposed to orthopedic implant wear particles. Journal of biomedical materials research. Part A Yao, Z., Keeney, M., Lin, T., Pajarinen, J., Barcay, K., Waters, H., Egashira, K., Yang, F., Goodman, S. 2014; 102 (9): 3291-3297
Abstract

Wear particles generated from total joint replacements can stimulate macrophages to release chemokines, such as monocyte chemoattractant protein 1 (MCP-1), which is the most important chemokine regulating systemic and local cell trafficking and infiltration of monocyte/macrophages in chronic inflammation. One possible strategy to curtail the adverse events associated with wear particles is to mitigate migration and activation of monocyte/macrophages. The purpose of this study is to modulate the adverse effects of particulate biomaterials and inflammatory stimuli such as endotoxin by interfering with the biological effects of the chemokine MCP-1. In the current study, the function of MCP-1 was inhibited by the mutant MCP-1 protein called 7ND, which blocks its receptor, the C-C chemokine receptor type 2 (CCR2) on macrophages. Addition of 7ND decreased MCP-1-induced migration of THP-1 cells in cell migration experiments in a dose-dependent manner. Conditioned media from murine macrophages exposed to clinically relevant polymethylmethacrylate (PMMA) particles with/without endotoxin [lipopolysaccharide (LPS)] had a chemotactic effect on human macrophages, which was decreased dramatically by 7ND. 7ND demonstrated no adverse effects on the viability of macrophages, and the capability of mesenchymal stem cells (MSCs) to form bone at the doses tested. Finally, proinflammatory cytokine production was mitigated when macrophages were exposed to PMMA particles with/without LPS in the presence of 7ND. Our studies confirm that the MCP-1 mutant protein 7ND can decrease macrophage migration and inflammatory cytokine release without adverse effects at the doses tested. Local delivery of 7ND at the implant site may provide a therapeutic strategy to diminish particle-associated periprosthetic inflammation and osteolysis. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

View details for DOI 10.1002/jbm.a.34981

View details for PubMedID 24123855
Chondrogenic differentiation of adipose-derived stromal cells in combinatorial hydrogels containing cartilage matrix proteins with decoupled mechanical stiffness. Tissue engineering. Part A Wang, T., Lai, J. H., Han, L., Tong, X., Yang, F. 2014; 20 (15-16): 2131-2139
Abstract

Adipose-derived stromal cells (ADSCs) are attractive autologous cell sources for cartilage repair given their relative abundance and ease of isolation. Previous studies have demonstrated the potential of extracellular matrix (ECM) molecules as three-dimensional (3D) scaffolds for promoting chondrogenesis. However, few studies have compared the effects of varying types or doses of ECM molecules on chondrogenesis of ADSCs in 3D. Furthermore, increasing ECM molecule concentrations often result in simultaneous changes in the matrix stiffness, which makes it difficult to elucidate the relative contribution of biochemical cues or matrix stiffness on stem cell fate. Here we report the development of an ECM-containing hydrogel platform with largely decoupled biochemical and mechanical cues by modulating the degree of methacrylation of ECM molecules. Specifically, we incorporated three types of ECM molecules that are commonly found in the cartilage matrix, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). To elucidate the effects of interactive biochemical and mechanical signaling on chondrogenesis, ADSCs were encapsulated in 39 combinatorial hydrogel compositions with independently tunable ECM types (CS, HA, and HS), concentrations (0.5%, 1.25%, 2.5%, and 5% [w/v]), and matrix stiffness (3, 30, and 90 kPa). Our results show that the effect of ECM composition on chondrogenesis is dependent on the matrix stiffness of hydrogels, suggesting that matrix stiffness and biochemical cues interact in a nonlinear manner to regulate chondrogenesis of ADSCs in 3D. In soft hydrogels (∼3kPa), increasing HA concentrations resulted in substantial upregulation of aggrecan and collagen type II expression in a dose-dependent manner. This trend was reversed in HA-containing hydrogels with higher stiffness (∼90 kPa). The platform reported herein could provide a useful tool for elucidating how ECM biochemical cues and matrix stiffness interact together to regulate stem cell fate, and for rapidly optimizing ECM-containing scaffolds to support stem cell differentiation and tissue regeneration.

View details for DOI 10.1089/ten.tea.2013.0531

View details for PubMedID 24707837
Suppression of wear-particle-induced pro-inflammatory cytokine and chemokine production in macrophages via NF-?B decoy oligodeoxynucleotide: A preliminary report. Acta biomaterialia Lin, T., Yao, Z., Sato, T., Keeney, M., Li, C., Pajarinen, J., Yang, F., Egashira, K., Goodman, S. B. 2014; 10 (8): 3747-3755
Abstract

Total joint replacement (TJR) is a very cost-effective surgery for end-stage arthritis. One important goal is to decrease the revision rate especially because TJR has been extended to younger patients. Continuous production of ultra-high molecular weight polyethylene (UHMWPE) wear particles induces macrophage infiltration and chronic inflammation, which can lead to peri-prosthetic osteolysis. Targeting individual pro-inflammatory cytokines directly has not reversed the osteolytic process in clinical trials, due to compensatory upregulation of other pro-inflammatory factors. We hypothesized that targeting the important transcription factor NF-κB could mitigate the inflammatory response to wear particles, potentially diminishing osteolysis. In the current study, we suppressed NF-κB activity in mouse RAW264.7 and human THP1 macrophage cell lines, as well as primary mouse and human macrophages, via competitive binding with double strand decoy oligodeoxynucleotide (ODN) containing an NF-κB binding element. We found that macrophage exposure to UHMWPE particles induced multiple pro-inflammatory cytokine and chemokine expression including TNF-α, MCP1, MIP1α and others. Importantly, the decoy ODN significantly suppressed the induced cytokine and chemokine expression in both murine and human macrophages, and resulted in suppression of macrophage recruitment. The strategic use of decoy NF-κB ODN, delivered locally, could potentially diminish particle-induced peri-prosthetic osteolysis.

View details for DOI 10.1016/j.actbio.2014.04.034

View details for PubMedID 24814879
Bioengineered 3D Brain Tumor Model To Elucidate the Effects of Matrix Stiffness on Glioblastoma Cell Behavior Using PEG-Based Hydrogels. Molecular pharmaceutics Wang, C., Tong, X., Yang, F. 2014; 11 (7): 2115-2125
Abstract

Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor with a median survival of 12-15 months, and the mechanisms underlying GBM tumor progression remain largely elusive. Given the importance of tumor niche signaling in driving GBM progression, there is a strong need to develop in vitro models to facilitate analysis of brain tumor cell-niche interactions in a physiologically relevant and controllable manner. Here we report the development of a bioengineered 3D brain tumor model to help elucidate the effects of matrix stiffness on GBM cell fate using poly(ethylene-glycol) (PEG)-based hydrogels with brain-mimicking biochemical and mechanical properties. We have chosen PEG given its bioinert nature and tunable physical property, and the resulting hydrogels allow tunable matrix stiffness without changing the biochemical contents. To facilitate cell proliferation and migration, CRGDS and a MMP-cleavable peptide were chemically incorporated. Hyaluronic acid (HA) was also incorporated to mimic the concentration in the brain extracellular matrix. Using U87 cells as a model GBM cell line, we demonstrate that such biomimetic hydrogels support U87 cell growth, spreading, and migration in 3D over the course of 3 weeks in culture. Gene expression analyses showed U87 cells actively deposited extracellular matrix and continued to upregulate matrix remodeling genes. To examine the effects of matrix stiffness on GBM cell fate in 3D, we encapsulated U87 cells in soft (1 kPa) or stiff (26 kPa) hydrogels, which respectively mimics the matrix stiffness of normal brain or GBM tumor tissues. Our results suggest that changes in matrix stiffness induce differential GBM cell proliferation, morphology, and migration modes in 3D. Increasing matrix stiffness led to delayed U87 cell proliferation inside hydrogels, but cells formed denser spheroids with extended cell protrusions. Cells cultured in stiff hydrogels also showed upregulation of HA synthase 1 and matrix metalloproteinase-1 (MMP-1), while simultaneously downregulating HA synthase 2 and MMP-9. This suggests that varying matrix stiffness can induce differential ECM deposition and remodeling by employing different HA synthases or MMPs. Furthermore, increasing matrix stiffness led to simultaneous upregulation of Hras, RhoA, and ROCK1, suggesting a potential link between the mechanosensing pathways and the observed differential cell responses to changes in matrix stiffness. The bioengineered 3D hydrogel platform reported here may provide a useful 3D in vitro brain tumor model for elucidating the mechanisms underlying GBM progression, as well as for evaluating the efficacy of potential drug candidates for treating GBM.

View details for DOI 10.1021/mp5000828

View details for PubMedID 24712441
Bioengineered 3D Brain Tumor Model To Elucidate the Effects of Matrix Stiffness on Glioblastoma Cell Behavior Using PEG-Based Hydrogels MOLECULAR PHARMACEUTICS Wang, C., Tong, X., Yang, F. 2014; 11 (7): 2115-2125
Abstract

Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor with a median survival of 12-15 months, and the mechanisms underlying GBM tumor progression remain largely elusive. Given the importance of tumor niche signaling in driving GBM progression, there is a strong need to develop in vitro models to facilitate analysis of brain tumor cell-niche interactions in a physiologically relevant and controllable manner. Here we report the development of a bioengineered 3D brain tumor model to help elucidate the effects of matrix stiffness on GBM cell fate using poly(ethylene-glycol) (PEG)-based hydrogels with brain-mimicking biochemical and mechanical properties. We have chosen PEG given its bioinert nature and tunable physical property, and the resulting hydrogels allow tunable matrix stiffness without changing the biochemical contents. To facilitate cell proliferation and migration, CRGDS and a MMP-cleavable peptide were chemically incorporated. Hyaluronic acid (HA) was also incorporated to mimic the concentration in the brain extracellular matrix. Using U87 cells as a model GBM cell line, we demonstrate that such biomimetic hydrogels support U87 cell growth, spreading, and migration in 3D over the course of 3 weeks in culture. Gene expression analyses showed U87 cells actively deposited extracellular matrix and continued to upregulate matrix remodeling genes. To examine the effects of matrix stiffness on GBM cell fate in 3D, we encapsulated U87 cells in soft (1 kPa) or stiff (26 kPa) hydrogels, which respectively mimics the matrix stiffness of normal brain or GBM tumor tissues. Our results suggest that changes in matrix stiffness induce differential GBM cell proliferation, morphology, and migration modes in 3D. Increasing matrix stiffness led to delayed U87 cell proliferation inside hydrogels, but cells formed denser spheroids with extended cell protrusions. Cells cultured in stiff hydrogels also showed upregulation of HA synthase 1 and matrix metalloproteinase-1 (MMP-1), while simultaneously downregulating HA synthase 2 and MMP-9. This suggests that varying matrix stiffness can induce differential ECM deposition and remodeling by employing different HA synthases or MMPs. Furthermore, increasing matrix stiffness led to simultaneous upregulation of Hras, RhoA, and ROCK1, suggesting a potential link between the mechanosensing pathways and the observed differential cell responses to changes in matrix stiffness. The bioengineered 3D hydrogel platform reported here may provide a useful 3D in vitro brain tumor model for elucidating the mechanisms underlying GBM progression, as well as for evaluating the efficacy of potential drug candidates for treating GBM.

View details for DOI 10.1021/mp5000828

View details for Web of Science ID 000338748200020
Photo-crosslinkable PEG-Based Microribbons for Forming 3D Macroporous Scaffolds with Decoupled Niche Properties. Advanced materials Han, L., Tong, X., Yang, F. 2014; 26 (11): 1757-1762
Abstract

PEG-based microribbons are designed and fabricated as building blocks for constructing a 3D cell niche with independently tunable biochemical, mechanical, and topographical cues. This platform supports direct cell encapsulation, allows spatial patterning of biochemical cues, and may provide a valuable tool for facilitating the analyses of how interactive niche signaling regulates cell fate in three dimensions.

View details for DOI 10.1002/adma.201304805

View details for PubMedID 24347028
A Facile Method to Fabricate Hydrogels with Microchannel-Like Porosity for Tissue Engineering TISSUE ENGINEERING PART C-METHODS Hammer, J., Han, L., Tong, X., Yang, F. 2014; 20 (2): 169-176
Abstract

Hydrogels are widely used as three-dimensional (3D) tissue engineering scaffolds due to their tissue-like water content, as well as their tunable physical and chemical properties. Hydrogel-based scaffolds are generally associated with nanoscale porosity, whereas macroporosity is highly desirable to facilitate nutrient transfer, vascularization, cell proliferation and matrix deposition. Diverse techniques have been developed for introducing macroporosity into hydrogel-based scaffolds. However, most of these methods involve harsh fabrication conditions that are not cell friendly, result in spherical pore structure, and are not amenable for dynamic pore formation. Human tissues contain abundant microchannel-like structures, such as microvascular network and nerve bundles, yet fabricating hydrogels containing microchannel-like pore structures remains a great challenge. To overcome these limitations, here we aim to develop a facile, cell-friendly method for engineering hydrogels with microchannel-like porosity using stimuli-responsive microfibers as porogens. Microfibers with sizes ranging 150-200 μm were fabricated using a coaxial flow of alginate and calcium chloride solution. Microfibers containing human embryonic kidney (HEK) cells were encapsulated within a 3D gelatin hydrogel, and then exposed to ethylenediaminetetraacetic acid (EDTA) solution at varying doses and duration. Scanning electron microscopy confirmed effective dissolution of alginate microfibers after EDTA treatment, leaving well-defined, interconnected microchannel structures within the 3D hydrogels. Upon release from the alginate fibers, HEK cells showed high viability and enhanced colony formation along the luminal surfaces of the microchannels. In contrast, HEK cells in non-EDTA treated control exhibited isolated cells, which remained entrapped in alginate microfibers. Together, our results showed a facile, cell-friendly process for dynamic microchannel formation within hydrogels, which may simultaneously release cells in 3D hydrogels in a spatiotemporally controlled manner. This platform may be adapted to include other cell-friendly stimuli for porogen removal, such as Matrix metalloproteinase-sensitive peptides or photodegradable gels. While we used HEK cells in this study as proof of principle, the concept described in this study may also be used for releasing clinically relevant cell types, such as smooth muscle and endothelial cells that are useful for repairing tissues involving tubular structures.

View details for DOI 10.1089/ten.tec.2013.0176

View details for Web of Science ID 000330310700008

View details for PubMedID 23745610
Engineering interpenetrating network hydrogels as biomimetic cell niche with independently tunable biochemical and mechanical properties. Biomaterials Tong, X., Yang, F. 2014; 35 (6): 1807-1815
Abstract

Hydrogels have been widely used as artificial cell niche to mimic extracellular matrix with tunable properties. However, changing biochemical cues in hydrogels developed-to-date would often induce simultaneous changes in mechanical properties, which do not support mechanistic studies on stem cell-niche interactions. Here we report the development of a PEG-based interpenetrating network (IPN), which is composed of two polymer networks that can independently and simultaneously crosslink to form hydrogels in a cell-friendly manner. The resulting IPN hydrogel allows independently tunable biochemical and mechanical properties, as well as stable and more homogeneous presentation of biochemical ligands in 3D than currently available methods. We demonstrate the potential of our IPN platform for elucidating stem cell-niche interactions by modulating osteogenic differentiation of human adipose-derived stem cells. The versatility of such IPN hydrogels is further demonstrated using three distinct and widely used polymers to form the mechanical network while keeping the biochemical network constant.

View details for DOI 10.1016/j.biomaterials.2013.11.064

View details for PubMedID 24331710
Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels SCIENTIFIC REPORTS Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. 2013; 3
Abstract

Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

View details for DOI 10.1038/srep03553

View details for Web of Science ID 000328623200007

View details for PubMedID 24352100
Modulating polymer chemistry to enhance non-viral gene delivery inside hydrogels with tunable matrix stiffness. Biomaterials Keeney, M., Onyiah, S., Zhang, Z., Tong, X., Han, L., Yang, F. 2013; 34 (37): 9657-9665
Abstract

Non-viral gene delivery holds great promise for promoting tissue regeneration, and offers a potentially safer alternative than viral vectors. Great progress has been made to develop biodegradable polymeric vectors for non-viral gene delivery in 2D culture, which generally involves isolating and modifying cells in vitro, followed by subsequent transplantation in vivo. Scaffold-mediated gene delivery may eliminate the need for the multiple-step process in vitro, and allows sustained release of nucleic acids in situ. Hydrogels are widely used tissue engineering scaffolds given their tissue-like water content, injectability and tunable biochemical and biophysical properties. However, previous attempts on developing hydrogel-mediated non-viral gene delivery have generally resulted in low levels of transgene expression inside 3D hydrogels, and increasing hydrogel stiffness further decreased such transfection efficiency. Here we report the development of biodegradable polymeric vectors that led to efficient gene delivery inside poly(ethylene glycol) (PEG)-based hydrogels with tunable matrix stiffness. Photocrosslinkable gelatin was maintained constant in the hydrogel network to allow cell adhesion. We identified a lead biodegradable polymeric vector, E6, which resulted in increased polyplex stability, DNA protection and achieved sustained high levels of transgene expression inside 3D PEG-DMA hydrogels for at least 12 days. Furthermore, we demonstrated that E6-based polyplexes allowed efficient gene delivery inside hydrogels with tunable stiffness ranging from 2 to 175 kPa, with the peak transfection efficiency observed in hydrogels with intermediate stiffness (28 kPa). The reported hydrogel-mediated gene delivery platform using biodegradable polyplexes may serve as a local depot for sustained transgene expression in situ to enhance tissue engineering across broad tissue types.

View details for DOI 10.1016/j.biomaterials.2013.08.050

View details for PubMedID 24011715
Mutant MCP-1 protein delivery from layer-by-layer coatings on orthopedic implants to modulate inflammatory response. Biomaterials Keeney, M., Waters, H., Barcay, K., Jiang, X., Yao, Z., Pajarinen, J., Egashira, K., Goodman, S. B., Yang, F. 2013; 34 (38): 10287-10295
Abstract

Total joint replacement (TJR) is a common and effective surgical procedure for hip or knee joint reconstruction. However, the production of wear particles is inevitable for all TJRs, which activates macrophages and initiates an inflammatory cascade often resulting in bone loss, prosthetic loosening and eventual TJR failure. Macrophage Chemoattractant Protein-1 (MCP-1) is one of the most potent cytokines responsible for macrophage cell recruitment, and previous studies suggest that mutant MCP-1 proteins such as 7ND may be used as a decoy drug to block the receptor and reduce inflammatory cell recruitment. Here we report the development of a biodegradable, layer-by-layer (LBL) coating platform that allows efficient loading and controlled release of 7ND proteins from the surface of orthopedic implants using as few as 14 layers. Scanning electron microscopy and fluorescence imaging confirmed effective coating using the LBL procedure on titanium rods. 7ND protein loading concentration and release kinetics can be modulated by varying the polyelectrolytes of choice, the polymer chemistry, the pH of the polyelectrolyte solution, and the degradation rate of the LBL assembly. The released 7ND from LBL coating retained its bioactivity and effectively reduced macrophage migration towards MCP-1. Finally, the LBL coating remained intact following a femoral rod implantation procedure as determined by immunostaining of the 7ND coating. The LBL platform reported herein may be applied for in situ controlled release of 7ND protein from orthopedic implants, to reduce wear particle-induced inflammatory responses in an effort to prolong the lifetime of implants.

View details for DOI 10.1016/j.biomaterials.2013.09.028

View details for PubMedID 24075408
Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Keeney, M., Deveza, L., Yang, F. 2013
Abstract

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

View details for DOI 10.3791/50736

View details for Web of Science ID 000209228100046
Development of Poly(ß-amino ester)-Based Biodegradable Nanoparticles for Nonviral Delivery of Minicircle DNA. ACS nano Keeney, M., Ong, S., Padilla, A., Yao, Z., Goodman, S., Wu, J. C., Yang, F. 2013; 7 (8): 7241-7250
Abstract

Gene therapy provides a powerful tool for regulating cellular processes and tissue repair. Minicircle (MC) DNA are supercoiled DNA molecules free of bacterial plasmid backbone elements and have been reported to enhance prolonged gene expression compared to conventional plasmids. Despite the great promise of MC DNA for gene therapy, methods for safe and efficient MC DNA delivery remain lacking. To overcome this bottleneck, here we report the development of a poly(β-amino ester) (PBAE)-based, biodegradable nanoparticulate platform for efficient delivery of MC DNA driven by a Ubc promoter in vitro and in vivo. By synthesizing and screening a small library of 18 PBAE polymers with different backbone and end-group chemistry, we identified lead cationic PBAE structures that can complex with minicircle DNA to form nanoparticles, and delivery efficiency can be further modulated by tuning PBAE chemistry. Using human embryonic kidney 293 cells and mouse embryonic fibroblasts as model cell types, we identified a few PBAE polymers that allow efficient MC delivery at levels that are comparable or even surpassing Lipofectamine 2000. The biodegradable nature of PBAE-based nanoparticles facilitates in vivo applications and clinical translation. When injected via intraperitoneal route in vivo, MC alone resulted in high transgene expression, and a lead PBAE/MC nanoparticle formulation achieved a further 2-fold increase in protein expression compared to MC alone. Together, our results highlight the promise of PBAE-based nanoparticles as promising nonviral gene carriers for MC delivery, which may provide a valuable tool for broad applications of MC DNA-based gene therapy.

View details for DOI 10.1021/nn402657d

View details for PubMedID 23837668
Dynamic tissue engineering scaffolds with stimuli-responsive macroporosity formation BIOMATERIALS Han, L., Lai, J. H., Yu, S., Yang, F. 2013; 34 (17): 4251-4258
Abstract

Macropores in tissue engineering scaffolds provide space for vascularization, cell-proliferation and cellular interactions, and is crucial for successful tissue regeneration. Modulating the size and density of macropores may promote desirable cellular processes at different stages of tissue development. Most current techniques for fabricating macroporous scaffolds produce fixed macroporosity and do not allow the control of porosity during cell culture. Most macropore-forming techniques also involve non-physiological conditions, such that cells can only be seeded in a post-fabrication process, which often leads to low cell seeding efficiency and uneven cell distribution. Here we report a process to create dynamic hydrogels as tissue engineering scaffolds with tunable macroporosity using stimuli-responsive porogens of gelatin, alginate and hyaluronic acid, which degrade in response to specific stimuli including temperature, chelating and enzymatic digestion, respectively. SEM imaging confirmed sequential pore formation in response to sequential stimulations: 37 °C on day 0, EDTA on day 7, and hyaluronidase on day 14. Bovine chondrocytes were encapsulated in the Alg porogen, which served as cell-delivery vehicles, and changes in cell viability, proliferation and tissue formation during sequential stimuli treatments were evaluated. Our results showed effective cell release from Alg porogen with high cell viability and markedly increased cell proliferation and spreading throughout the 3D hydrogels. Dynamic pore formation also led to significantly enhanced type II and X collagen production by chondrocytes. This platform provides a valuable tool to create stimuli-responsive scaffolds with dynamic macroporosity for a broad range of tissue engineering applications, and may also be used for fundamental studies to examine cell responses to dynamic niche properties.

View details for DOI 10.1016/j.biomaterials.2013.02.051

View details for Web of Science ID 000317700400006

View details for PubMedID 23489920
The future of biologic coatings for orthopaedic implants BIOMATERIALS Goodman, S. B., Yao, Z., Keeney, M., Yang, F. 2013; 34 (13): 3174-3183
Abstract

Implants are widely used for orthopaedic applications such as fixing fractures, repairing non-unions, obtaining a joint arthrodesis, total joint arthroplasty, spinal reconstruction, and soft tissue anchorage. Previously, orthopaedic implants were designed simply as mechanical devices; the biological aspects of the implant were a byproduct of stable internal/external fixation of the device to the surrounding bone or soft tissue. More recently, biologic coatings have been incorporated into orthopaedic implants in order to modulate the surrounding biological environment. This opinion article reviews current and potential future use of biologic coatings for orthopaedic implants to facilitate osseointegration and mitigate possible adverse tissue responses including the foreign body reaction and implant infection. While many of these coatings are still in the preclinical testing stage, bioengineers, material scientists and surgeons continue to explore surface coatings as a means of improving clinical outcome of patients undergoing orthopaedic surgery.

View details for DOI 10.1016/j.biomaterials.2013.01.074

View details for PubMedID 23391496
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 PubMedID 23216074
Effects of Polymer End-Group Chemistry and Order of Deposition on Controlled Protein Delivery from Layer-by-Layer Assembly BIOMACROMOLECULES Keeney, M., Mathur, M., Cheng, E., Tong, X., Yang, F. 2013; 14 (3): 794-800
Abstract

Layer-by-layer (LBL) assembly is an attractive platform for controlled release of biologics given its mild fabrication process and versatility in coating substrates of any shape. Proteins can be incorporated into LBL coatings by sequentially depositing oppositely charged polyelectrolytes, which self-assemble into nanoscale films on medical devices or tissue engineering scaffolds. However, previously reported LBL platforms often require the use of a few hundred layers to avoid burst release, which hinders their broad translation due to the lengthy fabrication process, cost, and batch-to-batch variability. Here we report a biodegradable LBL platform composed of only 10 layers with tunable protein release kinetics, which is an order of magnitude less than previously reported LBL platforms. We performed a combinatorial study to examine the effects of polymer chemistry and order of deposition of poly(β-amino) esters on protein release kinetics under 81 LBL assembly conditions. Using the optimal "polyelectrolyte couples" for constructing the LBL film, basic fibroblast growth factor (bFGF) was released gradually over 14 days with retained biological activity to stimulate cell proliferation. The method reported herein is applicable for coating various substrates including metals, polymers, and ceramics and may be used for a broad range of biomedical and tissue engineering applications.

View details for DOI 10.1021/bm3018559

View details for Web of Science ID 000316044700024

View details for PubMedID 23360295
The effects of interactive mechanical and biochemical niche signaling on osteogenic differentiation of adipose-derived stem cells using combinatorial hydrogels ACTA BIOMATERIALIA Nii, M., Lai, J. H., Keeney, M., Han, L., Behn, A., Imanbayev, G., Yang, F. 2013; 9 (3): 5475-5483
Abstract

Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often leads to simultaneous changes in mechanical properties, which makes it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell-material interactions have emerged as a tool to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on two-dimensional environments. Here we developed three-dimensional combinatorial hydrogels with independent control of biochemical and mechanical properties to facilitate analysis of interactive biochemical and mechanical signaling on adipose-derived stem cell osteogenesis in three dimensions. Our results suggest that scaffold biochemical and mechanical signals synergize only at specific combinations to promote bone differentiation. Leading compositions were identified to have intermediate stiffness (∼55kPa) and low concentration of fibronectin (10μg ml(-1)), which led to an increase in osteocalcin gene expression of over 130-fold. Our results suggest that scaffolds with independently tunable niche cues could provide a powerful tool for conducting mechanistic studies to decipher how complex niche cues regulate stem cell fate in three dimensions, and facilitate rapid identification of optimal niche cues that promote desirable cellular processes or tissue regeneration.

View details for DOI 10.1016/j.actbio.2012.11.002

View details for Web of Science ID 000315536000007

View details for PubMedID 23153761
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 (β-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

View details for PubMedCentralID PMC3594010
Paracrine Release from Nonviral Engineered Adipose-Derived Stem Cells Promotes Endothelial Cell Survival and Migration In Vitro STEM CELLS AND DEVELOPMENT Deveza, L., Choi, J., Imanbayev, G., Yang, F. 2013; 22 (3): 483-491
Abstract

Stem cells hold great potential for therapeutic angiogenesis due to their ability to directly contribute to new vessel formation or secrete paracrine signals. Adipose-derived stem cells (ADSCs) are a particularly attractive autologous cell source for therapeutic angiogenesis due to their ease of isolation and relative abundance. Gene therapy may be used to further enhance the therapeutic efficacy of ADSCs by overexpressing desired therapeutic factors. Here, we developed vascular endothelial growth factor (VEGF)-overexpressing ADSCs utilizing poly(β-amino esters) (PBAEs), a hydrolytically biodegradable polymer, and examined the effects of paracrine release from nonviral modified ADSCs on the angiogenic potential of human umbilical vein endothelial cells (HUVECs) in vitro. PBAE polymeric vectors delivered DNA into ADSCs with high efficiency and low cytotoxicity, leading to an over 3-fold increase in VEGF production by ADSCs compared with Lipofectamine 2000. Paracrine release from PBAE/VEGF-transfected ADSCs enhanced HUVEC viability and decreased HUVEC apoptosis under hypoxia. Further, paracrine release from PBAE/VEGF-transfected ADSCs significantly enhanced HUVEC migration and tube formation, two critical cellular processes for effective angiogenesis. Our results demonstrate that genetically engineered ADSCs using biodegradable polymeric nanoparticles may provide a promising autologous cell source for therapeutic angiogenesis in treating cardiovascular diseases.

View details for DOI 10.1089/scd.2012.0201

View details for Web of Science ID 000313677000012

View details for PubMedID 22889246

View details for PubMedCentralID PMC3549626
Microribbon-Like Elastomers for Fabricating Macroporous and Highly Flexible Scaffolds that Support Cell Proliferation in 3D ADVANCED FUNCTIONAL MATERIALS Han, L., Yu, S., Wang, T., Behn, A. W., Yang, F. 2013; 23 (3): 346-358
View details for DOI 10.1002/adfm.201201212

View details for Web of Science ID 000313691200011
The Effects of Polymer End-group Chemistry and Order of Deposition on Controlled Protein Delivery from Layer-by-layer Assembly Biomacromolecules Keeney M, Mathur M, Cheng E, Yang F 2013; 14 (3): 794-800
Programming stem cells for therapeutic angiogenesis using biodegradable polymeric nanoparticles. Journal of visualized experiments : JoVE Keeney, M., Deveza, L., Yang, F. 2013
Abstract

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

View details for DOI 10.3791/50736

View details for PubMedID 24121540
Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels. Scientific reports Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. 2013; 3: 3553-?
Abstract

Cartilage loss is a leading cause of disability among adults and effective therapy remains elusive. Neonatal chondrocytes (NChons) are an attractive allogeneic cell source for cartilage repair, but their clinical translation has been hindered by scarce donor availability. Here we examine the potential for catalyzing cartilage tissue formation using a minimal number of NChons by co-culturing them with adipose-derived stem cells (ADSCs) in 3D hydrogels. Using three different co-culture models, we demonstrated that the effects of co-culture on cartilage tissue formation are dependent on the intercellular distance and cell distribution in 3D. Unexpectedly, increasing ADSC ratio in mixed co-culture led to increased synergy between NChons and ADSCs, and resulted in the formation of large neocartilage nodules. This work raises the potential of utilizing stem cells to catalyze tissue formation by neonatal chondrocytes via paracrine signaling, and highlights the importance of controlling cell distribution in 3D matrices to achieve optimal synergy.

View details for DOI 10.1038/srep03553

View details for PubMedID 24352100
Therapeutic angiogenesis using genetically engineered human endothelial cells JOURNAL OF CONTROLLED RELEASE Cho, S., Yang, F., Son, S. M., Park, H., Green, J. J., Bogatyrev, S., Mei, Y., Park, S., Langer, R., Anderson, D. G. 2012; 160 (3): 515-524
Abstract

Cell therapy holds promise as a method for the treatment of ischemic disease. However, one significant challenge to the efficacy of cell therapy is poor cell survival in vivo. Here we describe a non-viral, gene therapy approach to improve the survival and engraftment of cells transplanted into ischemic tissue. We have developed biodegradable poly(β-amino esters) (PBAE) nanoparticles as vehicles to genetically modify human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF). VEGF transfection using these nanoparticles significantly enhanced VEGF expression in HUVECs, compared with a commercially-available transfection reagent. Transfection resulted in the upregulation of survival factors, and improved viability under simulated ischemic conditions. In a mouse model of hindlimb ischemia, VEGF nanoparticle transfection promoted engraftment of HUVECs into mouse vasculature as well as survival of transplanted HUVECs in ischemic tissues, leading to improved angiogenesis and ischemic limb salvage. This study demonstrates that biodegradable polymer nanoparticles may provide a safe and effective method for genetic engineering of endothelial cells to enhance therapeutic angiogenesis.

View details for DOI 10.1016/j.jconrel.2012.03.006

View details for Web of Science ID 000305789300013

View details for PubMedID 22450331

View details for PubMedCentralID PMC3372613
Tissue Engineering: Focus on musculoskeletal system Biomaterials Science-an integrated clinical and engineering approach Keeney M, Han LH, Onyiah S, Yang F 2012
Nanomaterials for Engineering Cell Microenvironment and Gene delivery Tissue Engineering and Regenerative Medicine: A Nano Approach. CRC Press. Lai JH, Ramasubranian A, Jeeawoody S, Yang F 2012
Nonviral delivery of genetic medicine for therapeutic angiogenesis ADVANCED DRUG DELIVERY REVIEWS Park, H., Yang, F., Cho, S. 2012; 64 (1): 40-52
Abstract

Genetic medicines that induce angiogenesis represent a promising strategy for the treatment of ischemic diseases. Many types of nonviral delivery systems have been tested as therapeutic angiogenesis agents. However, their delivery efficiency, and consequently therapeutic efficacy, remains to be further improved, as few of these technologies are being used in clinical applications. This article reviews the diverse nonviral gene delivery approaches that have been applied to the field of therapeutic angiogenesis, including plasmids, cationic polymers/lipids, scaffolds, and stem cells. This article also reviews clinical trials employing nonviral gene therapy and discusses the limitations of current technologies. Finally, this article proposes a future strategy to efficiently develop delivery vehicles that might be feasible for clinically relevant nonviral gene therapy, such as high-throughput screening of combinatorial libraries of biomaterials.

View details for DOI 10.1016/j.addr.2011.09.005

View details for Web of Science ID 000302843300006

View details for PubMedID 21971337
Therapeutic Angiogenesis for Treating Cardiovascular Diseases THERANOSTICS Deveza, L., Choi, J., Yang, F. 2012; 2 (8): 801-814
Abstract

Cardiovascular disease is the leading cause of death worldwide and is often associated with partial or full occlusion of the blood vessel network in the affected organs. Restoring blood supply is critical for the successful treatment of cardiovascular diseases. Therapeutic angiogenesis provides a valuable tool for treating cardiovascular diseases by stimulating the growth of new blood vessels from pre-existing vessels. In this review, we discuss strategies developed for therapeutic angiogenesis using single or combinations of biological signals, cells and polymeric biomaterials. Compared to direct delivery of growth factors or cells alone, polymeric biomaterials provide a three-dimensional drug-releasing depot that is capable of facilitating temporally and spatially controlled release. Biomimetic signals can also be incorporated into polymeric scaffolds to allow environmentally-responsive or cell-triggered release of biological signals for targeted angiogenesis. Recent progress in exploiting genetically engineered stem cells and endogenous cell homing mechanisms for therapeutic angiogenesis is also discussed.

View details for DOI 10.7150/thno.4419

View details for Web of Science ID 000307648500006

View details for PubMedID 22916079

View details for PubMedCentralID PMC3425124
Non-viral Delivery of Inductive and Suppressive Genes to Adipose-Derived Stem Cells for Osteogenic Differentiation PHARMACEUTICAL RESEARCH Ramasubramanian, A., Shiigi, S., Lee, G. K., Yang, F. 2011; 28 (6): 1328-1337
Abstract

To assess the effects of co-delivering osteoinductive DNA and/or small interfering RNA in directing the osteogenic differentiation of human adipose-derived stem cells (hADSCs) using a combinatorial, non-viral gene delivery approach.hADSCs were transfected using combinations of the following genes: BMP2, siGNAS and siNoggin using poly(β-amino esters) or lipid-like molecules. A total of 15 groups were evaluated by varying DNA doses, timing of treatment, and combinations of signals. All groups were cultured in osteogenic medium for up to 37 days, and outcomes were measured using gene expression, biochemical assays, and histology.Biomaterials-mediated gene delivery led to a dose-dependent up-regulation of BMP2 and significant gene silencing of GNAS and Noggin in hADSCs. BMP2 alone slightly up-regulates osteogenic marker expression in hADSCs. In contrast, co-delivery of BMP2 and siGNAS or siNoggin significantly accelerates the hADSC differentiation towards osteogenic differentiation, with marked increase in bone marker expression and mineralization.We report a combinatorial platform for identifying synergistic interactions among multiple genetic signals associated with osteogenic differentiation of hADSCs. Our results suggest that inductive or suppressive genetic switches interact in a complex manner, and highlight the promise of combinatorial approaches towards rapidly identifying optimal signals for promoting desired stem cell differentiation.

View details for DOI 10.1007/s11095-011-0406-9

View details for Web of Science ID 000290804000009

View details for PubMedID 21424160
Preparation of Mineralized Nanofibers: Collagen Fibrils Containing Calcium Phosphate NANO LETTERS Maas, M., Guo, P., Keeney, M., Yang, F., Hsu, T. M., Fuller, G. G., Martin, C. R., Zare, R. N. 2011; 11 (3): 1383-1388
Abstract

We report a straightforward, bottom-up, scalable process for preparing mineralized nanofibers. Our procedure is based on flowing feed solution, containing both inorganic cations and polymeric molecules, through a nanoporous membrane into a receiver solution with anions, which leads to the formation of mineralized nanofibers at the exit of the pores. With this strategy, we were able to achieve size control of the nanofiber diameters. We illustrate this approach by producing collagen fibrils with calcium phosphate incorporated inside the fibrils. This structure, which resembles the basic constituent of bones, assembles itself without the addition of noncollagenous proteins or their polymeric substitutes. Rheological experiments demonstrated that the stiffness of gels derived from these fibrils is enhanced by mineralization. Growth experiments of human adipose derived stem cells on these gels showed the compatibility of the fibrils in a tissue-regeneration context.

View details for DOI 10.1021/nl200116d

View details for Web of Science ID 000288061500082

View details for PubMedID 21280646

View details for PubMedCentralID PMC3053435
Recent Progress in Cartilage Tissue Engineering Curr Opin Biotechnol Keeney M, Lai J, Yang F 2011; 22 (5): 734-740
Combinatorial Extracellular Matrices for Human Embryonic Stem Cell Differentiation in 3D BIOMACROMOLECULES Yang, F., Cho, S., Son, S. M., Hudson, S. P., Bogatyrev, S., Keung, L., Kohane, D. S., Langer, R., Anderson, D. G. 2010; 11 (8): 1909-1914
Abstract

Embryonic stem cells (ESCs) are promising cell sources for tissue engineering and regenerative medicine. Scaffolds for ESC-based tissue regeneration should provide not only structural support, but also signals capable of supporting appropriate cell differentiation and tissue development. Extracellular matrix (ECM) is a key component of the stem cell niche in vivo and can influence stem cell fate via mediating cell attachment and migration, presenting chemical and physical cues, as well as binding soluble factors. Here we investigated the effects of combinatorial extracellular matrix proteins on controlled human ESC (hESC) differentiation. Varying ECM compositions in 3D markedly affects cell behavior, and optimal compositions of ECM hydrogels are identified that facilitate specific-lineage differentiation of stem cells. To our knowledge, this is the first combinatorial analysis of ECM hydrogels for their effects on hESC differentiation in 3D. The 3D matrices described herein may provide a useful platform for studying the interactive ECM signaling in influencing stem cell differentiation.

View details for DOI 10.1021/bm100357t

View details for Web of Science ID 000280583400002

View details for PubMedID 20614932

View details for PubMedCentralID PMC2946176
Genetic Engineering of Human Stem Cells for Enhanced Angiogenesis Using Biodegradable Polymeric Nanoparticles. Proceedings of the National Academy of Sciences Yang F, Cho SW, Son SM, Bogatyrev S, Singh D, Green JJ, Mei Y, Park S, Bhang SH, Kim BS, Langer R, Anderson DG 2010; 107 (8): 3317-22
Small Molecule End Group of Linear Polymer Determines Cell-type Gene Delivery Efficacy Advanced Materials Sunshine J, Green JJ, Mahon K, Yang F, Langer R, Anderson DG 2009; 21: 1-5
Lipid-like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells. Advanced Functional Materials Cho SW, Goldberg M, Son SM, Xu Q, Yang F, Mei Y, Bogatyrev S, Langer R, Anderson DG 2009; 19 (19): 3112-3118
High-throughput Optimization of Stem Cell Microenvrionments Combinatorial Chemistry & High Throughput Screening Yang F, Mei Y, Langer R, Anderson DG 2009; 12 (6): 544-553
Gene Delivery to Human Adult and Embryonic cell-derived Stem Cells Using Biodegradable Nanoparticulate Polymeric Vectors Gene Therapy Yang F, Green JJ, Dinio T, Keung L, Cho SW, Park H, Langer R, Anderson DG 2009; 16 (4): 533-546
The study of abnormal bone development in the Apert syndrome Fgfr2(+/S252W) mouse using a 3D hydrogel culture model BONE Yang, F., Wang, Y., Zhang, Z., Hsu, B., Jabs, E. W., Elisseeff, J. H. 2008; 43 (1): 55-63
Abstract

Apert syndrome is caused by mutations in fibroblast growth factor receptor 2 (Fgfr2) and is characterized by craniosynostosis and other skeletal abnormalities. The Apert syndrome Fgfr2+/S252W mouse model exhibits perinatal lethality. A 3D hydrogel culture model, derived from tissue engineering strategies, was used to extend the study of the effect of the Fgfr2+/S252W mutation in differentiating osteoblasts postnatally. We isolated cells from the long bones of Apert Fgfr2+/S252W mice (n=6) and cells from the wild-type sibling mice (n=6) to be used as controls. During monolayer expansion, Fgfr2+/S252W cells demonstrated increased proliferation and ALP activity, as well as altered responses of these cellular functions in the presence of FGF ligands with different binding specificity (FGF2 or FGF10). To better mimic the in vivo disease development scenario, cells were also encapsulated in 3D hydrogels and their phenotype in 3D in vitro culture was compared to that of in vivo tissue specimens. After 4 weeks in 3D culture in osteogenic medium, Fgfr2+/S252W cells expressed 2.8-fold more collagen type I and 3.3-fold more osteocalcin than did wild-type controls (p<0.01). Meanwhile, Fgfr2+/S252W cells showed decreased bone matrix remodeling and expressed 87% less Metalloprotease-13 and 71% less Noggin (p<0.01). The S252W mutation also led to significantly higher production of collagen type I and II in 3D as shown by immunofluorescence staining. In situ hybridization and alizarin red S staining of postnatal day 0 (P0) mouse limb sections demonstrated significantly higher levels of osteopontin expression and mineralization in Fgfr2+/S252W mice. Complementary to in vivo findings, this 3D hydrogel culture system provides an effective in vitro venue to study the pathogenesis of Apert syndrome caused by the analogous mutation in humans.

View details for DOI 10.1016/j.bone.2008.02.008

View details for Web of Science ID 000257151100008

View details for PubMedID 18407821
Abnormal Tissue Development of Osteoblasts from an Apert Syndrome FGFR2+/S252W Mouse Model in 3D Hydrogels Bone Yang F, Wang YL, Zhang Z, Hsu B, Jabs EW, Elisseeff JH 2008; 43 (1): 55-63
Delivery of Small Interfering RNA for Inhibition of Endothelial Cell Apoptosis by Hypoxia and Serum Deprivation Biochemical and Biophysical Communications Cho SW, Hartle L, Son SM, Yang F, Goldberg M, Xu Q, Langer R, Anderson DG 2008; 376 (1): 158-163
Tissue Engineering: The Therapeutic Strategy of the 21st Century Nanotechnology and Tissue Engineering Yang F, Neeley WL, Moore MJ, Karp JM, Shukla A, Langer R 2008
Metabolic changes in mesenchymal stem cells in osteogenic medium measured by autofluorescence spectroscopy STEM CELLS Reyes, J. M., Fermanian, S., Yang, F., Zhou, S., Herretes, S., Murphy, D. B., Elisseeff, J. H., Chuck, R. S. 2006; 24 (5): 1213-1217
Abstract

The purpose of this study was to measure metabolic changes in mesenchymal stem cells (MSCs) placed in osteogenic medium by autofluorescence spectroscopy. MSCs were plated in stem cell-supporting or osteogenic medium and imaged. Shift from the basic growth environment to the inductive osteogenic environment was confirmed by reverse transcription-polymerase chain reaction. Reduced pyridine nucleotides were detected by exciting near 366 nm and measuring fluorescence at 450 nm, and oxidized flavoproteins were detected by exciting at 460 nm and measuring fluorescence at 540 nm. The ratio of these fluorescence measurements, reduction-oxidation (redox) fluorometry, is a noninvasive measure of the cellular metabolic state. The detected pyridine nucleotide to flavoprotein ratio decreased upon transitioning from the stem cell to the differentiated state, as well as with increasing cell density and cell-cell contact. MSC metabolism increased upon placement in differentiating medium and with increasing cell density and contact. Redox fluorometry is a feasible, noninvasive technique for distinguishing MSCs from further differentiated cells.

View details for DOI 10.1634/stemcells.2004-0324

View details for Web of Science ID 000240639200009

View details for PubMedID 16439616
Cartilage Tissue Engineering Biomedical Engineering Handbook, Tissue Engineering Section Yang F, Elisseeff JH 2006
The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells BIOMATERIALS Yang, F., Williams, C. G., Wang, D. A., LEE, H., Manson, P. N., Elisseeff, J. 2005; 26 (30): 5991-5998
Abstract

Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg-Gly-Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.

View details for DOI 10.1016/j.biomaterials.2005.03.018

View details for Web of Science ID 000230538700008

View details for PubMedID 15878198
Abnormalities in cartilage and bone development in the Apert syndrome FGFR2(+/S252W) mouse DEVELOPMENT Wang, Y. L., Xiao, R., Yang, F., Karim, B. O., Iacovelli, A. J., Cai, J. L., Lerner, C. P., Richtsmeier, J. T., Leszl, J. M., Hill, C. A., Yu, K., Ornitz, D. M., Elisseeff, J., Huso, D. L., Jabs, E. W. 2005; 132 (15): 3537-3548
Abstract

Apert syndrome is an autosomal dominant disorder characterized by malformations of the skull, limbs and viscera. Two-thirds of affected individuals have a S252W mutation in fibroblast growth factor receptor 2 (FGFR2). To study the pathogenesis of this condition, we generated a knock-in mouse model with this mutation. The Fgfr2(+/S252W) mutant mice have abnormalities of the skeleton, as well as of other organs including the brain, thymus, lungs, heart and intestines. In the mutant neurocranium, we found a midline sutural defect and craniosynostosis with abnormal osteoblastic proliferation and differentiation. We noted ectopic cartilage at the midline sagittal suture, and cartilage abnormalities in the basicranium, nasal turbinates and trachea. In addition, from the mutant long bones, in vitro cell cultures grown in osteogenic medium revealed chondrocytes, which were absent in the controls. Our results suggest that altered cartilage and bone development play a significant role in the pathogenesis of the Apert syndrome phenotype.

View details for DOI 10.1242/dev.01914

View details for Web of Science ID 000231627800019

View details for PubMedID 15975938
Advances in skeletal tissue engineering with hydrogels. Orthodontics & craniofacial research Elisseeff, J., Puleo, C., Yang, F., SHARMA, B. 2005; 8 (3): 150-161
Abstract

Tissue engineering has the potential to make a significant impact on improving tissue repair in the craniofacial system. The general strategy for tissue engineering includes seeding cells on a biomaterial scaffold. The number of scaffold and cell choices for tissue engineering systems is continually increasing and will be reviewed.Multilayered hydrogel systems were developed to coculture different cell types and develop osteochondral tissues for applications including the temporomandibular joint.Hydrogels are one form of scaffold that can be applied to cartilage and bone repair using fully differentiated cells, adult and embryonic stem cells.Case studies represent an overview of our laboratory's investigations.Bilayered scaffolds to promote tissue development and the formation of more complex osteochondral tissues were developed and proved to be effective.Tissue engineering provides a venue to investigate tissue development of mutant or diseased cells and potential therapeutics.

View details for PubMedID 16022717
Bioresponsive phosphoester hydrogels for bone tissue engineering TISSUE ENGINEERING Wang, D. A., Williams, C. G., Yang, F., Cher, N., LEE, H., Elisseeff, J. H. 2005; 11 (1-2): 201-213
Abstract

Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester- poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG-PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.

View details for Web of Science ID 000227513600019

View details for PubMedID 15738675
Enhancing the tissue-biomaterial interface: Tissue-initiated integration of biomaterials ADVANCED FUNCTIONAL MATERIALS Wang, D. A., Williams, C. G., Yang, F., Elisseeff, J. H. 2004; 14 (12): 1152-1159
View details for DOI 10.1002/adfm.200305018

View details for Web of Science ID 000226103500002

Associate Professor of Orthopaedic Surgery and of Bioengineering

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