Marc Levenston

All Publications

• Co-Culture with Infrapatellar Fat Pad Differentially Stimulates Proteoglycan Synthesis and Accumulation in Cartilage and Meniscus Tissues. Connective tissue research Nishimuta, J. F., Bendernagel, M. F., Levenston, M. E. 2016: -?

  Abstract

  Although osteoarthritis is widely viewed as a disease of the whole joint, relatively few studies have focused on interactions among joint tissues in joint homeostasis and degeneration. In particular, few studies have examined the effects of the infrapatellar fat pad (IFP) on cartilaginous tissues. The aim of this study was to test the hypothesis that co-culture with healthy IFP would induce degradation of cartilage and meniscus tissues.Bovine articular cartilage, meniscus, and IFP were cultured isolated or as cartilage-fat or meniscus-fat co-cultures for up to 14 days. Conditioned media were assayed for sulfated glycosaminoglycan (sGAG) content, nitrite content, and matrix metalloproteinase (MMP) activity, and explants were assayed for sGAG and DNA contents.Co-cultures exhibited increased cumulative sGAG release and sGAG release rates for both cartilage and meniscus, and the cartilage (but not meniscus) exhibited a substantial synergistic effect of co-culture (sGAG release in co-culture was significantly greater than the summed release from isolated cartilage and fat). Fat co-culture did not significantly alter the sGAG content of either cartilage or meniscus explants, indicating that IFP co-culture stimulated net sGAG production by cartilage. Nitrite release was increased relative to isolated tissue controls in co-cultured meniscus, but not the cartilage, with no synergistic effect of co-culture. Interestingly, MMP-2 production was decreased by co-culture for both cartilage and meniscus.This study demonstrates that healthy IFP may modulate joint homeostasis by stimulating sGAG production in cartilage. Counter to our hypothesis, healthy IFP did not promote degradation of either cartilage or meniscus tissues.

  View details for PubMedID 27726455

• Bovine meniscal tissue exhibits age- and interleukin-1 dose-dependent degradation patterns and composition-function relationships JOURNAL OF ORTHOPAEDIC RESEARCH Ling, C. H., Lai, J. H., Wong, I. J., Levenston, M. E. 2016; 34 (5): 801-811

  Abstract

  Despite increasing evidence that meniscal degeneration is an early event in the development of knee osteoarthritis, relatively little is known regarding the sequence or functional implications of cytokine-induced meniscal degradation or how degradation varies with age. This study examined dose-dependent patterns of interleukin-1 (IL-1)-induced matrix degradation in explants from the radially middle regions of juvenile and adult bovine menisci. Tissue explants were cultured for 10 days in the presence of 0, 1.25, 5, or 20 ng/ml recombinant human IL-1α. Juvenile explants exhibited immediate and extensive sulfated glycosaminoglycan (sGAG) loss and subsequent collagen release beginning after 4-6 days, with relatively little IL-1 dose-dependence. Adult explants exhibited a more graded response to IL-1, with dose-dependent sGAG release and a lower fraction of sGAG released (but greater absolute release) than juvenile explants. In contrast to juvenile explants, adult explants exhibited minimal collagen release over the 10-day culture. Compressive and shear moduli reflected the changes in explant composition, with substantial decreases for both ages but a greater relative decrease in juvenile tissue. Dynamic moduli exhibited stronger dependence on explant sGAG content for juvenile tissue, likely reflecting concomitant changes to both proteoglycan and collagen tissue components. The patterns of tissue degradation suggest that, like in articular cartilage, meniscal proteoglycans may partially protect collagen from cell-mediated degeneration. A more detailed view of functional changes in meniscal tissue mechanics with degeneration will help to establish the relevance of in vitro culture models and will advance understanding of how meniscal degeneration contributes to overall joint changes in early stage osteoarthritis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:801-811, 2016.

  View details for DOI 10.1002/jor.23096

  View details for Web of Science ID 000375602200009

  View details for PubMedID 26519862

• Meniscus is more susceptible than cartilage to catabolic and anti-anabolic effects of adipokines OSTEOARTHRITIS AND CARTILAGE Nishimuta, J. F., Levenston, M. E. 2015; 23 (9): 1551-1562

  View details for DOI 10.1016/j.joca.2015.04.014

  View details for Web of Science ID 000360655000014

• Quantitative tracking of passage and 3D culture effects on chondrocyte and fibrochondrocyte gene expression. Journal of tissue engineering and regenerative medicine Son, M., Levenston, M. E. 2015

  Abstract

  Success in cartilage and fibrocartilage tissue engineering relies heavily on using an appropriate cell source. Many different cell sources have been identified, including primary and stem cells, along with experimental strategies to obtain the required number of cells or to induce chondrogenesis. However, no definitive method exists to quantitatively evaluate the similarity of the resulting cell phenotypes to those of the native cells between candidate strategies. In this study, we develop an integrative approach to enable such evaluations by deriving, from gene expression profiles, two quantitative metrics representing the nearest location within the range of native cell phenotypes and the deviation from it. As an example application to evaluating potential cell sources for cartilage or meniscus tissue engineering, we examine phenotypic changes of juvenile and adult articular chondrocytes and fibrochondrocytes across multiple passages and subsequent 3D culture. A substantial change was observed in cell phenotype due to the isolation process itself, followed by a clear progression toward the outer meniscal cell phenotype with passage. The new metrics also indicated that 3D culture moderately reduced the passage-induced deviation from the native meniscal phenotypes for juvenile chondrocytes and adult fibrochondrocytes, which was not obvious through examination of individual gene expressions. However, brief 3D culture alone did not move any of the cells towards an inner meniscal phenotype, the most relevant target for meniscal tissue engineering. This integrative approach of examining and combining multiple gene expressions can be used to evaluate various other tissue-engineering strategies to direct cells toward the desired phenotype. Copyright © 2015 John Wiley & Sons, Ltd.

  View details for DOI 10.1002/term.2022

  View details for PubMedID 25824488

• FACT VERSUS ARTIFACT: AVOIDING ERRONEOUS ESTIMATES OF SULFATED GLYCOSAMINOGLYCAN CONTENT USING THE DIMETHYLMETHYLENE BLUE COLORIMETRIC ASSAY FOR TISSUE-ENGINEERED CONSTRUCTS EUROPEAN CELLS & MATERIALS Zheng, C. H., Levenston, M. E. 2015; 29: 224-236

  Abstract

  The 1,9-dimethylmethylene blue (DMMB) assay is widely used to quantify sulfated glycosaminoglycan (sGAG) contents of engineered tissues, culture media, tissue samples and bodily fluids, but the assay is subject to interference from polyanions such as hyaluronic acid (HA), DNA and RNA. We examined whether specific combinations of dye pH and absorbance wavelength could minimize non-sGAG artifacts without compromising DMMB assay sensitivity. HA and DNA solutions generated substantial signal at pH 3 but not at pH 1.5. Reducing dye pH did not significantly alter sGAG measurements for normal cartilage and meniscus tissues, but eliminated anomalously high apparent sGAG contents for enzymatically isolated chondrocytes, adipose-derived stem cell (ADSC)-agarose constructs and ADSC pellets. In a cartilage tissue-engineering case study, pH 3 dye indicated high apparent sGAG readings throughout culture in both basal and chondrogenic media, with a marked decline between day 14 and 21 for chondrogenic constructs. The pH 1.5 dye, however, indicated minimal sGAG accumulation in basal medium and stable sGAG content throughout culture in chondrogenic medium. As it is often difficult to know a priori whether all groups in a study will have sGAG contents high enough to overwhelm artifacts, we recommend modifying the standard DMMB assay to reduce the risk of spurious findings in tissue engineering and clinical research. Specifically, we recommend shifting to a pH 1.5 DMMB dye and basing quantification on the absorbance difference between 525 nm (µ peak) and 595 nm (β peak) to compensate for the moderate loss of sensitivity associated with reducing the dye pH.

  View details for Web of Science ID 000357101500013

  View details for PubMedID 25890595

• Mechanisms of osteoarthritis in the knee: MR imaging appearance. Journal of magnetic resonance imaging Shapiro, L. M., McWalter, E. J., Son, M., Levenston, M., Hargreaves, B. A., Gold, G. E. 2014; 39 (6): 1346-1356

  Abstract

  Osteoarthritis has grown to become a widely prevalent disease that has major implications in both individual and public health. Although originally considered to be a degenerative disease driven by "wear and tear" of the articular cartilage, recent evidence has led to a consensus that osteoarthritis pathophysiology should be perceived in the context of the entire joint and multiple tissues. MRI is becoming an increasingly more important modality for imaging osteoarthritis, due to its excellent soft tissue contrast and ability to acquire morphological and biochemical data. This review will describe the pathophysiology of osteoarthritis as it is associated with various tissue types, highlight several promising MR imaging techniques for osteoarthritis and illustrate the expected appearance of osteoarthritis with each technique.

  View details for DOI 10.1002/jmri.24562

  View details for PubMedID 24677706

• Regional variation in T1? and T2 times in osteoarthritic human menisci: correlation with mechanical properties and matrix composition. Osteoarthritis and cartilage Son, M., Goodman, S. B., Chen, W., Hargreaves, B. A., Gold, G. E., Levenston, M. E. 2013; 21 (6): 796-805

  Abstract

  Changes in T1ρ and T2 magnetic resonance relaxation times have been associated with articular cartilage degeneration, but similar relationships for meniscal tissue have not been extensively investigated. This work examined relationships between T1ρ and T2 measurements and biochemical and mechanical properties across regions of degenerate human menisci.Average T1ρ and T2 relaxation times were determined for nine regions each of seven medial and 13 lateral menisci from 14 total knee replacement patients. Sulfated glycosaminoglycan (sGAG), collagen and water contents were measured for each region. Biomechanical measurements of equilibrium compressive, dynamic compressive and dynamic shear moduli were made for anterior, central and posterior regions.T1ρ and T2 times showed similar regional patterns, with longer relaxation times in the (radially) middle region compared to the inner and outer regions. Pooled over all regions, T1ρ and T2 times showed strong correlations both with one another and with water content. Correlations with biochemical content varied depending on normalization to wet or dry mass, and both imaging parameters showed stronger correlations with collagen compared to sGAG content. Mechanical properties displayed moderate inverse correlations with increasing T1ρ and T2 times and water content.Both T1ρ and T2 relaxation times correlated strongly with water content and moderately with mechanical properties in osteoarthritic menisci, but not as strongly with sGAG or collagen contents alone. While the ability of magnetic resonance imaging (MRI) to detect early osteoarthritic changes remains the subject of investigation, these results suggest that T1ρ and T2 relaxation times have limited ability to detect compositional variations in degenerate menisci.

  View details for DOI 10.1016/j.joca.2013.03.002

  View details for PubMedID 23499673

• Application of advanced magnetic resonance imaging techniques in evaluation of the lower extremity. Radiologic clinics of North America Braun, H. J., Dragoo, J. L., Hargreaves, B. A., Levenston, M. E., Gold, G. E. 2013; 51 (3): 529-545

  View details for DOI 10.1016/j.rcl.2012.12.001

  View details for PubMedID 23622097

• Application of advanced magnetic resonance imaging techniques in evaluation of the lower extremity. Radiologic clinics of North America Braun, H. J., Dragoo, J. L., Hargreaves, B. A., Levenston, M. E., Gold, G. E. 2013; 51 (3): 529-545

  Abstract

  This article reviews current magnetic resonance imaging (MR imaging) techniques for imaging the lower extremity, focusing on imaging of the knee, ankle, and hip joints. Recent advancements in MR imaging include imaging at 7 T, using multiple receiver channels, T2* imaging, and metal suppression techniques, allowing more detailed visualization of complex anatomy, evaluation of morphologic changes within articular cartilage, and imaging around orthopedic hardware.

  View details for DOI 10.1016/j.rcl.2012.12.001

  View details for PubMedID 23622097

• Modulation of Mesenchymal Stem Cell Shape in Enzyme-Sensitive Hydrogels Is Decoupled from Upregulation of Fibroblast Markers Under Cyclic Tension TISSUE ENGINEERING PART A Yang, P. J., Levenston, M. E., Temenoff, J. S. 2012; 18 (21-22): 2365-2375

  Abstract

  Our laboratory has developed a tensile culture bioreactor as a system for understanding mesenchymal stem cell (MSC) differentiation toward a tendon/ligament fibroblast phenotype in response to cyclic tensile strain. In this study, we investigated whether increased degradability of the biomaterial carrier would induce changes in MSC morphology and subsequent upregulation of tendon/fibroblast markers under tensile strain. Degradability of a synthetic poly(ethylene glycol) hydrogel was introduced by incorporating either fast- or slow-degrading matrix metalloproteinase (MMP)-sensitive peptide sequences into the polymer backbone. Although a decline in cellularity was observed over culture in all sample groups, at 14 days, MSCs were significantly more spread in fast-cleaving gels (84%±8%) compared with slow-cleaving gels (59%±4%). Cyclic tensile strain upregulated tendon/ligament fibroblast-related genes, such as collagen III (3.8-fold vs. 2.1-fold in fast-degrading gels) and tenascin-C (2.5-fold vs. 1.7-fold in fast-degrading gels). However, few differences were observed in gene expression between different gel types. Immunostaining demonstrated increased collagen III deposition in dynamically strained gels at day 14, as well as increased collagen I and tenascin-C deposition at day 14 in all groups. Results suggest that cell spreading may not be a major factor controlling MSC response to cyclic strain in this system over 14 days. However, these findings provide key parameters for the design of future biomaterial carriers and strain regimens to prime stem cells to a tendon/ligament phenotype prior to release and use in vivo.

  View details for DOI 10.1089/ten.tea.2011.0727

  View details for Web of Science ID 000310752900018

  View details for PubMedID 22703182

• Variations in chondrogenesis of human bone marrow-derived mesenchymal stem cells in fibrin/alginate blended hydrogels ACTA BIOMATERIALIA Ma, K., Titan, A. L., Stafford, M., Zheng, C. H., Levenston, M. E. 2012; 8 (10): 3754-3764

  Abstract

  Fibrin and alginate hydrogels have been widely used to support chondrogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs) for articular cartilage and fibrocartilage tissue engineering, with each material offering distinct advantages and disadvantages. Attempting to produce a gel scaffold exhibiting beneficial characteristics of both materials, we fabricated fibrin/alginate blended hydrogels at various blend ratios and evaluated the gel morphology, mechanical properties and their support for BM-MSC chondrogenesis. Results show that when the fibrin/alginate ratio decreased, the fibrin architecture transitioned from uniform to interconnected fibrous and finally to disconnected islands against an alginate background, with opposing trends in the alginate architecture. Fibrin maintained gel extensibility and promoted cell proliferation, while alginate improved the gel biostability and better supported glycosaminoglycan and collagen II production and chondrogenic gene expression. Blended gels had physical and biological characteristics intermediate between fibrin and alginate. Of the blends examined, FA 40:8 (40 mg ml(-1) fibrinogen blended with 8 mg ml(-1) alginate) was found to be the most appropriate group for future studies on tension-driven BM-MSC fibrochondrogenesis. As BM-MSC differentiation appeared to vary between fibrin and alginate regions of blended scaffolds, this study also highlighted the potential to develop spatially heterogeneous tissues through manipulating the heterogeneity of scaffold composition.

  View details for DOI 10.1016/j.actbio.2012.06.028

  View details for Web of Science ID 000309301400022

  View details for PubMedID 22750738

• Self-assembling nanoparticles for intra-articular delivery of anti-inflammatory proteins BIOMATERIALS Whitmire, R. E., Wilson, D. S., Singh, A., Levenston, M. E., Murthy, N., Garcia, A. J. 2012; 33 (30): 7665-7675

  Abstract

  Intra-articular delivery of therapeutics to modulate osteoarthritis (OA) is challenging. Delivery of interleukin-1 receptor antagonist (IL-1Ra), the natural protein inhibitor of IL-1, to modulate IL-1-based inflammation through gene therapy or bolus protein injections has emerged as a promising therapy for OA. However, these approaches suffer from rapid clearance and reduced potency over time. Nano/microparticles represent a promising strategy for overcoming the shortcomings of intra-articular drug delivery. However, these delivery vehicles are limited for delivery of protein therapeutics due to their hydrophobic character, low drug loading efficiency, and harsh chemical conditions during particle processing. We designed a new block copolymer that assembles into submicron-scale particles and provides for covalently tethering proteins to the particle surface for controlled intra-articular protein delivery. This block copolymer self-assembles into 300 nm-diameter particles with a protein tethering moiety for surface covalent conjugation of IL-1Ra protein. This copolymer particle system efficiently bound IL-1Ra and maintained protein bioactivity in vitro. Furthermore, particle-tethered IL-1Ra bound specifically to target synoviocyte cells via surface IL-1 receptors. Importantly, IL-1Ra nanoparticles inhibited IL-1-mediated signaling to equivalent levels as soluble IL-1Ra. Finally, the ability of nanoparticles to retain IL-1Ra in the rat stifle joint was evaluated by in vivo imaging over 14 days. IL-1Ra-tethered nanoparticles significantly increased the retention time of IL-1Ra in the rat stifle joint over 14 days with enhanced IL-1Ra half-life (3.01 days) compared to that of soluble IL-1Ra (0.96 days) and without inducing degenerative changes in cartilage structure or composition.

  View details for DOI 10.1016/j.biomaterials.2012.06.101

  View details for Web of Science ID 000308524000035

  View details for PubMedID 22818981

• Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus JOURNAL OF ANATOMY Vanderploeg, E. J., Wilson, C. G., Imler, S. M., Ling, C. H., Levenston, M. E. 2012; 221 (2): 174-186

  Abstract

  A deeper understanding of the composition and organization of extracellular matrix molecules in native, healthy meniscus tissue is required to fully appreciate the degeneration that occurs in joint disease and the intricate environment in which an engineered meniscal graft would need to function. In this study, regional variations in the tissue-level and pericellular distributions of collagen types I, II and VI and the proteoglycans aggrecan, biglycan and decorin were examined in the juvenile bovine meniscus. The collagen networks were extensively, but not completely, colocalized, with tissue-level organization that varied with radial position across the meniscus. Type VI collagen exhibited close association with large bundles composed of type I and II collagen and, in contrast to type I and II collagen, was further concentrated in the pericellular matrix. Aggrecan was detected throughout the inner region of the meniscus but was restricted to the pericellular matrix and sheaths of collagen bundles in the middle and outer regions. The small proteoglycans biglycan and decorin exhibited regional variations in staining intensity but were consistently localized in the intra- and/or peri-cellular compartments. These results provide insight into the complex hierarchy of extracellular matrix organization in the meniscus and provide a framework for better understanding meniscal degeneration and disease progression and evaluating potential repair and regeneration strategies.

  View details for DOI 10.1111/j.1469-7580.2012.01523.x

  View details for Web of Science ID 000305897300008

  View details for PubMedID 22703476

• Quantitative imaging of cartilage and bone morphology, reactive oxygen species, and vascularization in a rodent model of osteoarthritis ARTHRITIS AND RHEUMATISM Xie, L., Lin, A. S., Kundu, K., Levenston, M. E., Murthy, N., Guldberg, R. E. 2012; 64 (6): 1899-1908

  Abstract

  To assess temporal changes in cartilage and bone morphology, reactive oxygen species (ROS), and vascularization in rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA), using advanced imaging methodologies.Right knees of 8-week-old male Wistar rats were injected with 1 mg MIA in 50 μl saline and left knees were injected with 50 μl saline as controls. After 1, 2, and 3 weeks (n = 5 at each time point), changes in cartilage morphology and composition were quantified using equilibrium partitioning of an ionic contrast agent microfocal computed tomography (μCT), and changes in subchondral and trabecular bone were assessed by standard μCT. ROS were characterized by in vivo fluorescence imaging at 1, 11, and 21 days (n = 5 at each time point). Three weeks following fluorescence imaging, alterations in knee joint vascularity were quantified with μCT after perfusion of a vascular contrast agent.Femoral cartilage volume, thickness, and proteoglycan content were significantly decreased in MIA-injected knees compared with control knees, accompanied by loss of trabecular bone and erosion of subchondral bone surface. ROS quantities were significantly increased 1 day after MIA injection and subsequently decreased gradually, having returned to normal by 21 days. Vascularity in whole knees and distal femora was significantly increased at 21 days after MIA injection.Contrast-enhanced μCT and fluorescence imaging were combined to characterize articular cartilage, subchondral bone, vascularization, and ROS, providing unprecedented 3-dimensional joint imaging and quantification in multiple tissues during OA progression. These advanced imaging techniques have the potential to become standardized methods for comprehensive evaluation of articular joint degeneration and evaluation of therapeutic efficacy.

  View details for DOI 10.1002/art.34370

  View details for Web of Science ID 000304522100023

  View details for PubMedID 22231023

• Response of cartilage and meniscus tissue explants to in vitro compressive overload OSTEOARTHRITIS AND CARTILAGE NISHIMUTA, J. F., Levenston, M. E. 2012; 20 (5): 422-429

  Abstract

  To examine the relative susceptibility of cartilage and meniscus tissues to mechanical injury by applying a single, controlled overload and observing cellular, biochemical, and mechanical changes.Cartilage and meniscus tissue explants in radial confinement were subjected to a range of injury by indenting to 40% strain at three different strain rates: 0.5%/s (slow), 5%/s (medium), or 50%/s (fast). Following injury, samples were cultured for either 1 or 9 days. Explants were assayed for cell metabolic activity, water content, and sulfated glycosaminoglycan (sGAG) content. Mechanical properties of explants were determined in torsional shear and unconfined compression. Conditioned medium was assayed for sGAG and lactate dehydrogenase (LDH) release.Peak injury force increased with strain rate but both tissues displayed little to no macroscopic damage. Cell metabolism was lowest in medium and fast groups on day 1. Cell lysis increased with peak injury force and loading rate in both tissues. In contrast, sGAG content and release did not significantly vary with loading rate. Additionally, mechanical properties did not significantly vary with loading rate in either tissue.By use of a custom confinement chamber, large peak forces were obtained without macroscopic destruction of the explants. At the loads achieved in this studied, cell damage was induced without detectable physical or compositional changes. These results indicate that sub-failure injury can induce biologic damage that may not be readily detected and could be an early event in osteoarthritis genesis.

  View details for DOI 10.1016/j.joca.2012.01.004

  View details for Web of Science ID 000303297100012

  View details for PubMedID 22289896

• Depth-Dependent Transverse Shear Properties of the Human Corneal Stroma INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE Petsche, S. J., Chernyak, D., Martiz, J., Levenston, M. E., Pinsky, P. M. 2012; 53 (2): 873-880

  Abstract

  To measure the transverse shear modulus of the human corneal stroma and its profile through the depth by mechanical testing, and to assess the validity of the hypothesis that the shear modulus will be greater in the anterior third due to increased interweaving of lamellae.Torsional rheometry was used to measure the transverse shear properties of 6 mm diameter buttons of matched human cadaver cornea pairs. One cornea from each pair was cut into thirds through the thickness with a femtosecond laser and each stromal third was tested individually. The remaining intact corneas were tested to measure full stroma shear modulus. The shear modulus from a 1% shear strain oscillatory test was measured at various levels of axial compression for all samples.After controlling for axial compression, the transverse shear moduli of isolated anterior layers were significantly higher than central and posterior layers. Mean modulus values at 0% axial strain were 7.71 ± 6.34 kPa in the anterior, 1.99 ± 0.45 kPa in the center, 1.31 ± 1.01 kPa in the posterior, and 9.48 ± 2.92 kPa for full thickness samples. A mean equilibrium compressive modulus of 38.7 ± 8.6 kPa at 0% axial strain was calculated from axial compression measured during the shear tests.Transverse shear moduli are two to three orders of magnitude lower than tensile moduli reported in the literature. The profile of shear moduli through the depth displayed a significant increase from posterior to anterior. This gradient supports the hypothesis and corresponds to the gradient of interwoven lamellae seen in imaging of stromal cross-sections.

  View details for DOI 10.1167/iovs.11-8611

  View details for Web of Science ID 000302788600046

  View details for PubMedID 22205608

• DISCRIMINATION OF MENISCAL CELL PHENOTYPES USING GENE EXPRESSION PROFILES EUROPEAN CELLS & MATERIALS Son, M., Levenston, M. E. 2012; 23: 195-208

  Abstract

  The lack of quantitative and objective metrics to assess cartilage and meniscus cell phenotypes contributes to the challenges in fibrocartilage tissue engineering. Although functional assessment of the final resulting tissue is essential, initial characterization of cell sources and quantitative description of their progression towards the natural, desired cell phenotype would provide an effective tool in optimizing cell-based tissue engineering strategies. The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus). The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions. We additionally demonstrate a possible application of these quantifiable metrics in evaluating the use of serially passaged chondrocytes as a possible cell source in fibrocartilage engineering. Comparing the ratios of the passaged chondrocytes and the native meniscal cells may provide direction to optimize towards the desired cell phenotype. We have thus shown that measurable markers defining the characteristics of the native meniscus can establish a standard by which different tissue engineering strategies can be objectively assessed. Such metrics could additionally be useful in exploring the different stages of meniscal degradation in osteoarthritis and provide some insight in the disease progression.

  View details for Web of Science ID 000307554200015

  View details for PubMedID 22442006

• Comparison of osmotic swelling influences on meniscal fibrocartilage and articular cartilage tissue mechanics in compression and shear JOURNAL OF ORTHOPAEDIC RESEARCH Nguyen, A. M., Levenston, M. E. 2012; 30 (1): 95-102

  Abstract

  Although the contribution of the circumferential collagen bundles to the anisotropic tensile stiffness of meniscal tissue has been well described, the implications of interactions between tissue components for other mechanical properties have not been as widely examined. This study compared the effects of the proteoglycan-associated osmotic swelling stress on meniscal fibrocartilage and articular cartilage (AC) mechanics by manipulating the osmotic environment and tissue compressive offset. Cylindrical samples were obtained from the menisci and AC of bovine stifles, equilibrated in phosphate-buffered saline solutions ranging from 0.1× to 10×, and tested in oscillatory torsional shear and unconfined compression. Biochemical analysis indicated that treatments and testing did not substantially alter tissue composition. Mechanical testing revealed tissue-specific responses to both increasing compressive offset and decreasing bath salinity. Most notably, reduced salinity dramatically increased the shear modulus of both axially and circumferentially oriented meniscal tissue explants to a much greater extent than for cartilage samples. Combined with previous studies, these findings suggest that meniscal proteoglycans have a distinct structural role, stabilizing, and stiffening the matrix surrounding the primary circumferential collagen bundles.

  View details for DOI 10.1002/jor.21493

  View details for Web of Science ID 000297913600013

  View details for PubMedID 21735474

• FIBRONECTIN- AND COLLAGEN-MIMETIC LIGANDS REGULATE BONE MARROW STROMAL CELL CHONDROGENESIS IN THREE-DIMENSIONAL HYDROGELS EUROPEAN CELLS & MATERIALS Connelly, J. T., Petrie, T. A., Garcia, A. J., Levenston, M. E. 2011; 22: 168-177

  Abstract

  Modification of tissue engineering scaffolds with bioactive molecules is a potential strategy for modulating cell behavior and guiding tissue regeneration. While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM)-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D) hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10) and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs) were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli.

  View details for Web of Science ID 000295874600013

  View details for PubMedID 21932193

• Cyclic Tensile Culture Promotes Fibroblastic Differentiation of Marrow Stromal Cells Encapsulated in Poly(Ethylene Glycol)-Based Hydrogels TISSUE ENGINEERING PART A Doroski, D. M., Levenston, M. E., Temenoff, J. S. 2010; 16 (11): 3457-3466

  Abstract

  To inform future efforts in tendon/ligament tissue engineering, our laboratory has developed a well-controlled model system with the ability to alter both external tensile loading parameters and local biochemical cues to better understand marrow stromal cell differentiation in response to both stimuli concurrently. In particular, the synthetic, poly(ethylene glycol)-based hydrogel material oligo(poly(ethylene glycol) fumarate) (OPF) has been explored as a cell carrier for this system. This biomaterial can be tailored to present covalently incorporated bioactive moieties and can be loaded in our custom cyclic tensile bioreactor for up to 28 days with no loss of material integrity. Human marrow stromal cells encapsulated in these OPF hydrogels were cultured (21 days) under cyclic tensile strain (10%, 1  Hz, 3  h of strain followed by 3  h without) or at 0% strain. No difference was observed in cell number due to mechanical stimulation or across time (n = 4), with cells remaining viable (n = 4) through 21 days. Cyclic strain significantly upregulated all tendon/ligament fibroblastic genes examined (collagen I, collagen III, and tenascin-C) by day 21 (n ≥ 6), whereas genes for other pathways (osteogenic, chondrogenic, and adipogenic) did not increase. After 21 days, the presence of collagen I and tenascin-C was observed via immunostaining (n = 2). This study demonstrates the utility of this hydrogel/bioreactor system as a versatile, yet well-controlled, model environment to study marrow stromal cell differentiation toward the tendon/ligament phenotype under a variety of conditions.

  View details for DOI 10.1089/ten.tea.2010.0233

  View details for Web of Science ID 000283899600017

  View details for PubMedID 20666585

• Meniscus and cartilage exhibit distinct intra-tissue strain distributions under unconfined compression OSTEOARTHRITIS AND CARTILAGE Lai, J. H., Levenston, M. E. 2010; 18 (10): 1291-1299

  Abstract

  To examine the functional behavior of the surface layer of the meniscus by investigating depth-varying compressive strains during unconfined compression.Pairs of meniscus and articular cartilage explants (n=12) site-matched at the tibial surfaces were subjected to equilibrium unconfined compression at 5, 10, 15, and 20% compression under fluorescence imaging. Two-dimensional (2D) deformations were tracked using digital image correlation (DIC). For each specimen, local compressive engineering strains were determined in 200 μm layers through the depth of the tissue. In samples with sharp strain transitions, bilinear regressions were used to characterize the surface and interior tissue compressive responses.Meniscus and cartilage exhibited distinct depth-dependent strain profiles during unconfined compression. All cartilage explants had elevated compressive engineering strains near the surface, consistent with previous reports. In contrast, half of the meniscus explants tested had substantially stiffer surface layers, as indicated by surface engineering strains that were ∼20% of the applied compression. In the remaining samples, surface and interior engineering strains were comparable. 2D Green's strain maps revealed highly heterogeneous compressive and shear strains throughout the meniscus explants. In cartilage, the maximum shear strain appeared to be localized at 100-250 μm beneath the articular surface.Meniscus was characterized by highly heterogeneous strains during compression. In contrast to cartilage, which consistently had a compliant surface region, meniscal explants were either substantially stiffer near the surface or had comparable compressive stiffness through the depth. The relatively compliant interior may allow the meniscus to maintain a consistent surface contour while deforming during physiologic loading.

  View details for DOI 10.1016/j.joca.2010.05.020

  View details for Web of Science ID 000283452600009

  View details for PubMedID 20633686

• Tensile Loading Modulates Bone Marrow Stromal Cell Differentiation and the Development of Engineered Fibrocartilage Constructs TISSUE ENGINEERING PART A Connelly, J. T., Vanderploeg, E. J., Mouw, J. K., Wilson, C. G., Levenston, M. E. 2010; 16 (6): 1913-1923

  Abstract

  Mesenchymal progenitors such as bone marrow stromal cells (BMSCs) are an attractive cell source for fibrocartilage tissue engineering, but the types or combinations of signals required to promote fibrochondrocyte-specific differentiation remain unclear. The present study investigated the influences of cyclic tensile loading on the chondrogenesis of BMSCs and the development of engineered fibrocartilage. Cyclic tensile displacements (10%, 1 Hz) were applied to BMSC-seeded fibrin constructs for short (24 h) or extended (1-2 weeks) periods using a custom loading system. At early stages of chondrogenesis, 24 h of cyclic tension stimulated both protein and proteoglycan synthesis, but at later stages, tension increased protein synthesis only. One week of intermittent cyclic tension significantly increased the total sulfated glycosaminoglycan and collagen contents in the constructs, but these differences were lost after 2 weeks of loading. Constraining the gels during the extended culture periods prevented contraction of the fibrin matrix, induced collagen fiber alignment, and increased sulfated glycosaminoglycan release to the media. Cyclic tension specifically stimulated collagen I mRNA expression and protein synthesis, but had no effect on collagen II, aggrecan, or osteocalcin mRNA levels. Overall, these studies suggest that the combination of chondrogenic stimuli and tensile loading promotes fibrochondrocyte-like differentiation of BMSCs and has the potential to direct fibrocartilage development in vitro.

  View details for DOI 10.1089/ten.tea.2009.0561

  View details for Web of Science ID 000278164800011

  View details for PubMedID 20088686

• DOSE-DEPENDENT EFFECTS OF INTERLEUKIN-1ALPHA ON FUNCTIONAL DEGRADATION OF LATERAL AND MEDIAL MENISCI 12th ASME Summer Bioengineering Conference Ling, C. H., Lai, J. H., Nishimuta, J. F., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2010: 45–46

  View details for Web of Science ID 000290705300023

• OUTSIDE-IN VS. INSIDE-OUT: CONTRASTING PATTERNS OF COMPRESSIVE DEFORMATION IN CARTILAGE AND MENISCUS 12th ASME Summer Bioengineering Conference Lai, J. H., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2010: 327–328

  View details for Web of Science ID 000290705300164

• ARE PASSAGED CHONDROCYTES PHENOTYPICALLY SIMILAR TO MENISCAL FIBROCHONDROCYTES? 12th ASME Summer Bioengineering Conference Son, M., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2010: 943–944

  View details for Web of Science ID 000290705300472

• Nondestructive assessment of sGAG content and distribution in normal and degraded rat articular cartilage via EPIC-mu CT OSTEOARTHRITIS AND CARTILAGE Xie, L., Lin, A. S., Guldberg, R. E., Levenston, M. E. 2010; 18 (1): 65-72

  Abstract

  The objective of this study was to evaluate the feasibility of quantifying the Equilibrium Partitioning of an Ionic Contrast agent via Microcomputed Tomography (EPIC-microCT) to nondestructively assess sulfated glycosaminoglycan (sGAG) content and distribution in rat articular cartilage ex vivo, and in doing so to establish a paradigm for extension of this technique to other small animal models.After determination of an appropriate incubation time for the anionic contrast agent, EPIC-microCT was used to examine age-related differences in cartilage sGAG content between 4-, 8-, and 16-week old (n=5 each) male Wistar rats and to evaluate sGAG depletion in the right femora of each age group after 60 min of digestion with chondroitinase ABC. The EPIC-microCT measurements were validated by histological safranin-O staining, and reproducibility was evaluated by triplicate scans of six femora.Cartilage attenuation gradually increased with cumulative digestion time and reached a plateau at approximately 60 min with a 16.0% temporal increase (P<0.01). Average femoral articular cartilage attenuation increased by 14.2% from 4- to 8-weeks of age (P<0.01) and further increased by 2.5% from 8 to 16 weeks (P<0.05). After 60 min of digestion, femoral articular cartilage attenuations increased by 15-17% in each age group (P<0.01). Correspondingly, sGAG optical density decreased with age and digestion, and showed a linear correlation (r=-0.88, slope=-1.26, P<0.01, n=30) with EPIC-microCT cartilage attenuation. High reproducibility was indicated by a low coefficient of variation (1.5%) in cartilage attenuation.EPIC-microCT imaging provides high spatial resolution and sensitivity to assess sGAG content and three-dimensional distribution in rat femoral articular cartilage.

  View details for DOI 10.1016/j.joca.2009.07.014

  View details for Web of Science ID 000273677500011

  View details for PubMedID 19744590

• Photochemical approaches for bonding of cartilage tissues OSTEOARTHRITIS AND CARTILAGE Sitterle, V. B., NISHIMUTA, J. F., Levenston, M. E. 2009; 17 (12): 1649-1656

  Abstract

  The objective of this study was to evaluate photochemical bonding as an approach for adhering live cartilage tissues across a repair interface in a manner that may lead to enhanced integration.Photochemical bonding of both meniscal fibrocartilage and articular cartilage was explored using an anionic, hydrophilic phthalocyanine photosensitizer. Variations on surface preparations and irradiation parameters were explored using overlapped tissue strips and tested using a modified single-lap shear test. Durability of the photochemically induced bonds and cellular viability were examined in an in vitro cartilage defect model for up to 1 week in culture, with bond strength assessed via push-out test.Meniscal tissue strips bonded with no surface treatment, but cartilage strips required enzymatic treatment with chondroitinase-ABC to effectively bond. More aggressive removal of glycosaminoglycans at the interface led to increased bond strengths. Bond strength achieved with a 10min irradiation of treated tissue was on the order of that previously achieved through several weeks of culture. In the defect model, photochemical bonds between a tissue annulus and a press-fit tissue core were maintained for 1 week in culture without substantial increases in cell death near the bonded interface.With appropriate treatment parameters, photochemical bonding rapidly produced a stable structural interface between cartilage tissue samples and may be a promising strategy for enhancing initial attachment in cartilage repair strategies.

  View details for DOI 10.1016/j.joca.2009.06.009

  View details for Web of Science ID 000272966700018

  View details for PubMedID 19591798

• Central and peripheral region tibial plateau chondrocytes respond differently to in vitro dynamic compression OSTEOARTHRITIS AND CARTILAGE Bevill, S. L., Briant, P. L., Levenston, M. E., Andriacchi, T. P. 2009; 17 (8): 980-987

  Abstract

  The objective of this study was to test the hypotheses that chondrocytes from distinct regions of the porcine tibial plateau: (1) display region-specific baseline gene expression, and (2) respond differently to in vitro mechanical loading.Articular cartilage explants were obtained from central (not covered by meniscus) and peripheral (covered by meniscus) regions of porcine tibial plateaus. For baseline gene expression analysis, samples were snap frozen. To determine the effect of mechanical loading, central and peripheral region explants were exposed to equivalent dynamic compression (0-100 kPa) and compared to site-matched free-swelling controls (FSCs). mRNA levels for type II collagen (CII), aggrecan (AGGR), matrix metalloproteinase 1 (MMP-1), MMP-3, MMP-13, A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAM-TS4), ADAM-TS5, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and tumor necrosis factor alpha (TNFalpha) were quantified using real time polymerase chain reaction (RT-PCR).At baseline, mRNA levels for the structural proteins CII and AGGR were approximately twofold greater in the central region compared with peripheral region explants. In vitro dynamic compression strongly affected expression levels for CII, AGGR, MMP-3, and TIMP-2 relative to FSCs. Response differed significantly by region, with greater upregulation of CII, AGGR, and MMP-3 in central region explants.Chondrocytes from different regions of the porcine tibial plateau express mRNA for structural proteins at different levels and respond to equivalent in vitro mechanical loading with distinctive changes in gene expression. These regional biological variations appear to be related to the local mechanical environment in the normal joint, and thus may indicate a sensitivity of the joint to conditions that alter joint loading such as anterior cruciate ligament (ACL) injury, meniscectomy, or joint instability.

  View details for DOI 10.1016/j.joca.2008.12.005

  View details for Web of Science ID 000268654700003

  View details for PubMedID 19157913

• Composition-function relationships during IL-1-induced cartilage degradation and recovery OSTEOARTHRITIS AND CARTILAGE Palmer, A. W., Wilson, C. G., BAUM, E. J., Levenston, M. E. 2009; 17 (8): 1029-1039

  Abstract

  To examine the relationships between biochemical composition and mechanical properties of articular cartilage explants during interleukin-1 (IL-1)-induced degradation and post-exposure recovery.Bovine articular cartilage explants were cultured for up to 32 days with or without 20 ng/mL IL-1. The dynamic shear modulus |G*(dyn)| and equilibrium and dynamic unconfined compression moduli (E(equil) and |E*(dyn)|) were measured at intervals throughout the culture period. In a subsequent recovery study, explants were cultured for 4 days with or without 20ng/mL IL-1 and for an additional 16 days in control media. The dynamic moduli |E*(dyn)| and |G*(dyn)| were measured at intervals during degeneration and recovery. Conditioned media and explant digests were assayed for sulfated glycosaminoglycans (sGAG) and collagen content.Continuous IL-1 stimulation triggered progressive decreases in E(equil), |E*(dyn)|, and |G*(dyn)| concomitant with the sequential release of sGAG and collagen from the explants. Brief IL-1 exposure resulted in a short release of sGAG but not collagen, followed by a gradual and incomplete repopulation of sGAG. The temporary sGAG depletion was associated with decreases in both |E*(dyn)| and |G*(dyn)| which also recovered after removal of IL-1. During IL-1-induced degradation and post-exposure recovery, explant mechanical properties correlated well with tissue sGAG concentration.As previously shown for developing cartilages and engineered cartilage constructs, cytokine-induced changes in sGAG concentration (i.e., fixed charge density) are coincident with changes in compressive and shear properties of articular cartilage. Further, recovery of cartilage mechanical properties can be achieved by relief from proinflammatory stimuli and subsequent restoration of tissue sGAG concentration.

  View details for DOI 10.1016/j.joca.2009.02.009

  View details for Web of Science ID 000268654700010

  View details for PubMedID 19281879

• Chondrocytes and Meniscal Fibrochondrocytes Differentially Process Aggrecan During De Novo Extracellular Matrix Assembly ASME Summer Bioengineering Conference 2007 Wilson, C. G., Nishimuta, J. F., Levenston, M. E. MARY ANN LIEBERT INC. 2009: 1513–22

  Abstract

  Aggrecan is an extracellular matrix molecule that contributes to the mechanical properties of articular cartilage and meniscal fibrocartilage, but the abundance and processing of aggrecan in these tissues are different. The objective of this study was to compare patterns of aggrecan processing by chondrocytes and meniscal fibrochondrocytes in tissue explants and cell-agarose constructs. The effects of transforming growth factor-beta 1 (TGF-beta1) stimulation on aggrecan deposition and processing were examined, and construct mechanical properties were measured. Fibrochondrocytes synthesized and retained less proteoglycans than did chondrocytes in tissue explants and agarose constructs. In chondrocyte constructs, TGF-beta1 induced the accumulation of a 120-kDa aggrecan species previously detected in mature bovine cartilage. Fibrochondrocyte-seeded constructs contained high-molecular-weight aggrecan but lacked aggrecanase-generated fragments found in native, immature meniscus. In addition, reflecting the lesser matrix accumulation, fibrochondrocyte constructs had significantly lower compression moduli than did chondrocyte constructs. These cell type-specific differences in aggrecan synthesis, retention, and processing may have implications for the development of functional engineered tissue grafts.

  View details for DOI 10.1089/ten.tea.2008.0106

  View details for Web of Science ID 000267843700008

  View details for PubMedID 19260779

• Quantitative assessment of articular cartilage morphology via EPIC-mu CT OSTEOARTHRITIS AND CARTILAGE Xie, L., Lin, A. S., Levenston, M. E., Guldberg, R. E. 2009; 17 (3): 313-320

  Abstract

  The objective of the present study was to validate the ability of Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-microCT) to nondestructively assess cartilage morphology in the rat model.An appropriate contrast agent (Hexabrix) concentration and incubation time for equilibration were determined for reproducible segmentation of femoral articular cartilage from contrast-enhanced microCT scans. Reproducibility was evaluated by triplicate scans of six femora, and the measured articular cartilage thickness was independently compared to thickness determined from needle probe testing and histology. The validated technique was then applied to quantify age-related differences in articular cartilage morphology between 4, 8, and 16-week-old (n=5 each) male Wistar rats.A 40% Hexabrix/60% phosphate buffered saline (PBS) solution with 30 min incubation was optimal for segmenting cartilage from the underlying bone tissue and other soft tissues in the rat model. High reproducibility was indicated by the low coefficient of variation (1.7-2.5%) in cartilage volume, thickness and surface area. EPIC-microCT evaluation of thickness showed a strong linear relationship and good agreement with both needle probing (r(2)=0.95, slope=0.81, P<0.01, mean difference 11+/-22 microm, n=43) and histology (r(2)=0.99, slope=0.97, P<0.01, mean difference 12+/-10 microm, n=30). Cartilage volume and thickness significantly decreased with age while surface area significantly increased.EPIC-microCT imaging has the ability to nondestructively evaluate three-dimensional articular cartilage morphology with high precision and accuracy in a small animal model.

  View details for DOI 10.1016/j.joca.2008.07.015

  View details for Web of Science ID 000264239700006

  View details for PubMedID 18789727

• Improved Estimation of Solute Diffusivity Through Numerical Analysis of FRAP Experiments CELLULAR AND MOLECULAR BIOENGINEERING Irrechukwu, O. N., Levenston, M. E. 2009; 2 (1): 104-117

  View details for DOI 10.1007/s12195-009-0042-1

  View details for Web of Science ID 000269822400011

• Development And Finite Element Implementation Of A Nearly Incompressible Structural Constitutive Model For Artery Substitute Design ASME Summer Bioengineering Conference Sun, W., Chaikof, E. L., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2009: 681–682

  View details for Web of Science ID 000263364700341

• ARTICULAR CARTILAGE AND MENISCAL FIBROCARTILAGE MECHANICS: EVIDENCE FOR DIFFERENCES IN ULTRASTRUCTURE AND FUNCTION OF PROTEOGLYCANS ASME Summer Bioengineering Conference Nguyen, A. M., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2009: 927–928

  View details for Web of Science ID 000280089000464

• OSMOTIC EFFECTS ON THE DYNAMIC SHEAR PROPERTIES OF MENISCAL FIBROCARTILAGE ASME Summer Bioengineering Conference Ho, P. H., Nguyen, A. M., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2009: 415–416

  View details for Web of Science ID 000263364700208

• Aggrecanolysis and in vitro matrix degradation in the immature bovine meniscus: mechanisms and functional implications ARTHRITIS RESEARCH & THERAPY Wilson, C. G., Vanderploeg, E. J., Zuo, F., Sandy, J. D., Levenston, M. E. 2009; 11 (6)

  Abstract

  Little is known about endogenous or cytokine-stimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage.Aggrecanase activity in the native immature bovine meniscus was examined by immunolocalization of the aggrecan NITEGE neoepitope. To investigate mechanisms of cytokine-induced aggrecan catabolism in this tissue, explants were treated with interleukin-1alpha (IL-1) in the absence or presence of selective or broad spectrum metalloproteinase inhibitors. The sulfated glycosaminoglycan (sGAG) and collagen contents of explants and culture media were quantified by biochemical methods, and aggrecan catabolism was examined by Western analysis of aggrecan fragments. The mechanical properties of explants were determined by dynamic compression and shear tests.The aggrecanase-generated NITEGE neoepitope was preferentially localized in the middle and outer regions of freshly isolated immature bovine menisci, where sGAG density was lowest and blood vessels were present. In vitro treatment of explants with IL-1 triggered the accumulation of NITEGE in the inner and middle regions. Middle region explants stimulated with IL-1 exhibited substantial decreases in sGAG content, collagen content, and mechanical properties. A broad spectrum metalloproteinase inhibitor significantly reduced sGAG loss, abrogated collagen degradation, and preserved tissue mechanical properties. In contrast, an inhibitor selective for ADAMTS-4 and ADAMTS-5 was least effective at blocking IL-1-induced matrix catabolism and loss of mechanical properties.Aggrecanase-mediated aggrecanolysis, typical of degenerative articular cartilage, may play a physiologic role in the development of the immature bovine meniscus. IL-1-induced release of sGAG and loss of mechanical properties can be ascribed primarily to the activity of MMPs or aggrecanases other than ADAMTS-4 and ADAMTS-5. These results may have implications for the clinical management of osteoarthritis.

  View details for DOI 10.1186/ar2862

  View details for Web of Science ID 000278282100013

  View details for PubMedID 19919704

• Numerical Approximation of Tangent Moduli for Finite Element Implementations of Nonlinear Hyperelastic Material Models JOURNAL OF BIOMECHANICAL ENGINEERING-TRANSACTIONS OF THE ASME Sun, W., Chaikof, E. L., Levenston, M. E. 2008; 130 (6)

  Abstract

  Finite element (FE) implementations of nearly incompressible material models often employ decoupled numerical treatments of the dilatational and deviatoric parts of the deformation gradient. This treatment allows the dilatational stiffness to be handled separately to alleviate ill conditioning of the tangent stiffness matrix. However, this can lead to complex formulations of the material tangent moduli that can be difficult to implement or may require custom FE codes, thus limiting their general use. Here we present an approach, based on work by Miehe (Miehe, 1996, "Numerical Computation of Algorithmic (Consistent) Tangent Moduli in Large Strain Computational Inelasticity," Comput. Methods Appl. Mech. Eng., 134, pp. 223-240), for an efficient numerical approximation of the tangent moduli that can be easily implemented within commercial FE codes. By perturbing the deformation gradient, the material tangent moduli from the Jaumann rate of the Kirchhoff stress are accurately approximated by a forward difference of the associated Kirchhoff stresses. The merit of this approach is that it produces a concise mathematical formulation that is not dependent on any particular material model. Consequently, once the approximation method is coded in a subroutine, it can be used for other hyperelastic material models with no modification. The implementation and accuracy of this approach is first demonstrated with a simple neo-Hookean material. Subsequently, a fiber-reinforced structural model is applied to analyze the pressure-diameter curve during blood vessel inflation. Implementation of this approach will facilitate the incorporation of novel hyperelastic material models for a soft tissue behavior into commercial FE software.

  View details for DOI 10.1115/1.2979872

  View details for Web of Science ID 000260835100003

  View details for PubMedID 19045532

• Evaluation Criteria for Musculoskeletal and Craniofacial Tissue Engineering Constructs: A Conference Report TISSUE ENGINEERING PART A Butler, D. L., Lewis, J. L., Frank, C. B., Banes, A. J., Caplan, A. I., De Deyne, P. G., Dowling, M., Fleming, B. C., Glowacki, J., Guldberg, R. E., Johnstone, B., Kaplan, D. L., Levenston, M. E., Lotz, J. C., Lu, E. Y., Lumelsky, N., Mao, J. J., Mauck, R. L., McDevitt, C. A., Mejia, L. C., Murray, M., Ratcliffe, A., Spindler, K. P., Tashman, S., Wagner, C. T., Weisberg, E. M., Williams, C. (., Zhang, R. 2008; 14 (12): 2089-2104

  Abstract

  Over the past 20 years, tissue engineering (TE) has evolved into a thriving research and commercial development field. However, applying TE strategies to musculoskeletal (MSK) and craniofacial tissues has been particularly challenging since these tissues must also transmit loads during activities of daily living. To address this need, organizers invited a small group of bioengineers, surgeons, biologists, and material scientists from academia, industry, and government to participate in a two and half-day conference to develop general and tissue-specific criteria for evaluating new concepts and tissue-engineered constructs, including threshold values of success. Participants were assigned to four breakout groups representing commonly injured tissues, including tendon and ligament, articular cartilage, meniscus and temporomandibular joint, and bone and intervertebral disc. Working in multidisciplinary teams, participants first carefully defined one or two important unmet clinical needs for each tissue type, including current standards of care and the potential impact of TE solutions. The groups then sought to identify important parameters for evaluating repair outcomes in preclinical studies and to specify minimally acceptable values for these parameters. The importance of in vitro TE studies was then discussed in the context of these preclinical studies. Where data were not currently available from clinical, preclinical, or culture studies, the groups sought to identify important areas of preclinical research needed to speed the development process. This report summarizes the findings of the conference.

  View details for DOI 10.1089/ten.tea.2007.0383

  View details for Web of Science ID 000261403500016

  View details for PubMedID 19093294

• A modified lap test to more accurately estimate interfacial shear strength for bonded tissues JOURNAL OF BIOMECHANICS Sitterle, V. B., Sun, W., Levenston, M. E. 2008; 41 (15): 3260-3264

  Abstract

  Effective attachment to relevant anatomical surfaces has long been a critical issue for tissue replacement or repair. This is especially true for cartilage repair where adequate, reliable initial fixation to surrounding tissue and joint surfaces has been a dominant factor affecting clinical outcomes. Due to ease of application and ability to replicate dimensions and rates across multiple experiments, the single-lap test in tension has become a common method to assess interfacial strength for cartilage and other tissues in apposition. The standard single-lap configuration does not, however, provide a true measure of shear strength. The presence of a bending moment and resulting bond rotation create an uneven stress environment; specimens typically fail due to peel stresses at the edges of the interface. This report describes finite element analysis of variations to the single-lap method in which supports were added to either side of the bond interface. These results were then experimentally validated using photochemically bonded articular cartilage. Both the finite element and experimental results show that the addition of supports helps mitigate edge stresses and produces a more uniform stress distribution across the bond interface. Adding supports to prevent bond rotation, even for specimens not fixed to the supports, still produces a better estimate of shear strength than the traditional, non-supported configuration. These findings allow selection of a single-lap approach to more closely approximate shear strength even in those situations where it is not feasible or otherwise desirable to fix the tissue specimens to supports.

  View details for DOI 10.1016/j.jbiomech.2008.09.006

  View details for Web of Science ID 000261657000024

  View details for PubMedID 18986654

• 3D imaging of tissue integration with porous biomaterials BIOMATERIALS Guldberg, R. E., Duvall, C. L., Peister, A., Oest, M. E., Lin, A. S., Palmer, A. W., Levenston, M. E. 2008; 29 (28): 3757-3761

  Abstract

  Porous biomaterials designed to support cellular infiltration and tissue formation play a critical role in implant fixation and engineered tissue repair. The purpose of this Leading Opinion Paper is to advocate the use of high resolution 3D imaging techniques as a tool to quantify extracellular matrix formation and vascular ingrowth within porous biomaterials and objectively compare different strategies for functional tissue regeneration. An initial over-reliance on qualitative evaluation methods may have contributed to the false perception that developing effective tissue engineering technologies would be relatively straightforward. Moreover, the lack of comparative studies with quantitative metrics in challenging pre-clinical models has made it difficult to determine which of the many available strategies to invest in or use clinically for companies and clinicians, respectively. This paper will specifically illustrate the use of microcomputed tomography (micro-CT) imaging with and without contrast agents to nondestructively quantify the formation of bone, cartilage, and vasculature within porous biomaterials.

  View details for DOI 10.1016/j.biomaterials.2008.06.018

  View details for Web of Science ID 000259017000001

  View details for PubMedID 18635260

• Articular chondrocytes derived from distinct tissue zones differentially respond to in vitro oscillatory tensile loading OSTEOARTHRITIS AND CARTILAGE Vanderploeg, E. J., Wilson, C. G., Levenston, M. E. 2008; 16 (10): 1228-1236

  Abstract

  The cell morphology, gene expression, and matrix synthesis of articular chondrocytes are known to vary with depth from the tissue surface. The objective of this study was to investigate if chondrocytes from different zones respond to in vitro oscillatory tensile loading in distinct ways and whether tensile strain, which is most prevalent near the articular surface, would preferentially stimulate superficial zone chondrocytes.Chondrocytes were separately isolated from the superficial, middle, and deep zones of articular cartilage and seeded into three-dimensional fibrin hydrogel constructs. An intermittent protocol of oscillatory tensile loading was applied for 3 days, and the effects on extracellular matrix (ECM) synthesis were assessed by measuring the incorporation of radiolabed precursors, size exclusion gel chromatography, and western blotting.Tensile loading was found to be a potent stimulus for proteoglycan synthesis only in superficial zone chondrocytes. Although overall biosynthesis rates by deep zone chondrocytes were unaffected by tensile loading, the molecular characteristics of proteins and proteoglycans released to the culture medium were significantly altered so as to resemble those of superficial zone chondrocytes.Oscillatory tensile loading differentially affected subpopulations of articular chondrocytes in three-dimensional fibrin hydrogel constructs. Cells isolated from deeper regions of the tissue developed some characteristics of superficial zone chondrocytes after exposure to tensile loading, which may indicate an adaptive response to the new mechanical environment. Understanding how exogenous mechanical stimuli can differentially influence chondrocytes from distinct tissue zones will yield important insights into mechanobiological processes involved in cartilage tissue development, maintenance, disease, and repair.

  View details for DOI 10.1016/j.joca.2008.02.016

  View details for Web of Science ID 000259916700015

  View details for PubMedID 18400525

• Interactions between integrin ligand density and cytoskeletal integrity regulate BMSC chondrogenesis JOURNAL OF CELLULAR PHYSIOLOGY Connelly, J. T., Garcia, A. J., Levenston, M. E. 2008; 217 (1): 145-154

  Abstract

  Interactions with the extracellular matrix play important roles in regulating the phenotype and activity of differentiated articular chondrocytes; however, the influences of integrin-mediated adhesion on the chondrogenesis of mesenchymal progenitors remain unclear. In the present study, agarose hydrogels were modified with synthetic peptides containing the arginine-glycine-aspartic acid (RGD) motif to investigate the effects of integrin-mediated adhesion and cytoskeletal organization on the chondrogenesis of bone marrow stromal cells (BMSCs) within a three-dimensional culture environment. Interactions with the RGD-modified hydrogels promoted BMSC spreading in a density-dependent manner and involved alphavbeta3 integrin receptors. When cultured with the chondrogenic supplements, TGF-beta1 and dexamethasone, adhesion to the RGD sequence inhibited the stimulation of sulfated-glycosaminoglycan (sGAG) production in a RGD density-dependent manner, and this inhibition could be blocked by disrupting the F-actin cytoskeleton with cytochalasin D. In addition, interactions with the RGD-modified gels promoted cell migration and aggrecanase-mediated release of sGAG to the media. While adhesion to the RGD sequence inhibited BMSC chondrogenesis in the presence of TGF-beta1 and dexamethasone, osteocalcin and collagen I gene expression and alkaline phosphatase activity were enhanced by RGD interactions in the presence of serum-supplemented medium. Overall, the results of this study demonstrate that integrin-mediated adhesion within a three-dimensional environment inhibits BMSC chondrogenesis through actin cytoskeleton interactions. Furthermore, the effects of RGD-adhesion on mesenchymal differentiation are lineage-specific and depend on the biochemical composition of the cellular microenvironment.

  View details for DOI 10.1002/jcp.21484

  View details for Web of Science ID 000258899900016

  View details for PubMedID 18452154

• Characterization of proteoglycan production and processing by chondrocytes and BMSCs in tissue engineered constructs OSTEOARTHRITIS AND CARTILAGE Connelly, J. T., Wilson, C. G., Levenston, M. E. 2008; 16 (9): 1092-1100

  Abstract

  The goal of this study was to characterize the proteoglycan (PG) production and processing by bone marrow stromal cells (BMSCs) within a tissue engineered construct.Bovine BMSCs and articular chondrocytes (ACs) were isolated from an immature calf, seeded into agarose gels, and cultured up to 32 days in the presence of TGF-beta1. The localization of various PGs was examined by immunofluorescence and histological staining. The role of proteolytic enzymes in construct development was further investigated by examining the effects of aggrecanase and MMP inhibitors on PG accumulation, aggrecan processing, and construct mechanics.BMSCs developed a matrix rich in sulfated-glycosaminoglycans (sGAG) and full-length aggrecan, but had low levels of versican. The BMSC constructs had less collagen II and aggrecan compared to the AC constructs cultured under identical conditions. AC constructs also had high levels of pericellular collagen VI, while BMSCs had a pericellular matrix containing little collagen VI and greater levels of decorin, biglycan, and fibronectin. Treatment with the aggrecanase inhibitor did not affect sGAG accumulation or the dynamic moduli of the BMSC constructs. The MMP inhibitor slightly but significantly inhibited sGAG accumulation and lowered the dynamic moduli of BMSC constructs.The results of this preliminary study indicate that long-term culture of BMSCs with TGF-beta1 promotes the development of an aggrecan-rich matrix characteristic of native articular cartilage; however, BMSCs accumulate significantly lower levels of sGAG and assemble distinct pericellular microenvironments compared to ACs. PG turnover does not appear to play a major role in the development of tissue engineered cartilage constructs by BMSCs.

  View details for DOI 10.1016/j.joca.2008.01.004

  View details for Web of Science ID 000259379400018

  View details for PubMedID 18294870

• Contrast enhanced micro-CT imaging of vascular and cartilaginous tissues Annual Tissue-Engineering-and-Regenerative-Medicine-International-Society-European-Chapter Meeting Guldberg, R. E., Xie, L., Duvall, C. L., Palmer, A. W., Taylor, W. R., Levenston, M. E. MARY ANN LIEBERT INC. 2008: 715–15

  View details for Web of Science ID 000256239800074

• Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties MATRIX BIOLOGY Wilson, C. G., Palmer, A. W., Zuo, F., Eugui, E., Wilson, S., Mackenzie, R., Sandy, J. D., Levenston, M. E. 2007; 26 (4): 259-268

  Abstract

  Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

  View details for DOI 10.1016/j.matbio.2006.11.001

  View details for Web of Science ID 000246755000006

  View details for PubMedID 17174540

• Dynamic compression regulates the expression and synthesis of chondrocyte-specific matrix molecules in bone marrow stromal cells STEM CELLS Mouw, J. K., Connelly, J. T., Wilson, C. G., Michael, K. E., Levenston, M. E. 2007; 25 (3): 655-663

  Abstract

  The overall objective of the present study was to investigate the mechanotransduction of bovine bone marrow stromal cells (BMSCs) through the interactions between transforming growth factor beta1 (TGF-beta1), dexamethasone, and dynamic compressive loading. Overall, the addition of TGF-beta1 increased cell viability, extracellular matrix (ECM) gene expression, matrix synthesis, and sulfated glycosaminoglycan content over basal construct medium. The addition of dexamethasone further enhanced extracellular matrix gene expression and protein synthesis. There was little stimulation of ECM gene expression or matrix synthesis in any medium group by mechanical loading introduced on day 8. In contrast, there was significant stimulation of ECM gene expression and matrix synthesis in chondrogenic media by dynamic loading introduced on day 16. The level of stimulation was also dependent on the medium supplements, with the samples treated with basal medium being the least responsive and the samples treated with TGF-beta1 and dexamethasone being the most responsive at day 16. Both collagen I and collagen II gene expressions were more responsive to dynamic loading than aggrecan gene expression. Dynamic compression upregulated Smad2/3 phosphorylation in samples treated with basal and TGF-beta1 media. These findings suggest that interactions between mechanical stimuli and TGF-beta signaling may be an important mechanotransduction pathway for BMSCs, and they indicate that mechanosensitivity may vary during the process of chondrogenesis.

  View details for DOI 10.1634/stemcells.2006-0435

  View details for Web of Science ID 000244847100013

  View details for PubMedID 17124008

• Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels BIOMATERIALS Connelly, J. T., Garcia, A. J., Levenston, M. E. 2007; 28 (6): 1071-1083

  Abstract

  The goal of this study was to investigate the effects of adhesion to the arginine-glycine-aspartic acid (RGD) sequence on the chondrogenesis of bone marrow stromal cells (BMSCs). Synthetic RGE- and RGD-containing peptides were conjugated to sodium alginate, and bovine BMSCs were seeded onto 2D alginate surfaces or encapsulated in 3D gels. BMSCs spread specifically on RGD-modified surfaces, and spreading was inhibited by a soluble RGD peptide and by anti-beta1 and anti-alpha(v)beta3 integrin blocking antibodies. After 7 days in 3D gel culture, the chondrogenic supplements (TGF-beta1 and dexamethasone) significantly stimulated chondrocytic gene expression (collagen II, aggrecan, and Sox-9) and matrix accumulation (collagen II and sGAG) in RGE-modified gels, but this response was inhibited in the RGD-modified gels. Inhibition of sGAG synthesis increased with increasing RGD density, and synthesis was partially rescued by adding a soluble RGD peptide. Addition of an anti-alpha(v)beta3 integrin blocking antibody had no effect on chondrogenesis, while an anti-alpha5 antibody reduced sGAG accumulation. Overall, this study demonstrates that interaction with the RGD motif significantly inhibits the initial chondrogenesis of BMSCs within 3D alginate gels. These results provide new insights into the role of cell-matrix interactions in regulating chondrogenesis and highlight the importance of choosing appropriate biomaterials for tissue engineering therapies.

  View details for DOI 10.1016/j.biomaterials.2006.10.006

  View details for Web of Science ID 000243219000018

  View details for PubMedID 17123602

• Ion-channel regulation of chondrocyte matrix synthesis in 3D culture under static and dynamic compression BIOMECHANICS AND MODELING IN MECHANOBIOLOGY Mouw, J. K., Imler, S. M., Levenston, M. E. 2007; 6 (1-2): 33-41

  Abstract

  Inhibition of various ion channels alters chondrocyte mechanotransduction in monolayer, but the mechanisms involved in chondrocyte mechanotransduction in three- dimensional culture remain unclear. The objective of this study was to investigate the effects of inhibiting putative ion-channel influenced mechanotransduction mechanisms on the chondrocyte responses to static and dynamic compression in three-dimensional culture. Bovine articular cartilage explants were used to investigate the dose-dependent inhibition and recovery of protein and sulfated glycosaminoglycan (sGAG) syntheses by four ion-channel inhibitors: 4-Aminopyridine (4AP), a K(+) channel blocker; Nifedipine (Nf), a Ca(2+) channel blocker; Gadolinium (Gd), a stretch-activated channel blocker; and Thapsigargin (Tg), which releases intracellular Ca(2+) stores by inhibiting ATP-dependent Ca(2+) pumps. Chondrocyte-seeded agarose gels were used to examine the influence of 20 h of static and dynamic loading in the presence of each of the inhibitors. Overall, treatment with the ion-channel inhibitors had a greater effect on sGAG synthesis, with the exception of Nf, which more substantially affected protein synthesis. Treatment with Tg significantly impaired both overall protein and sGAG synthesis, with a drastic reduction in sGAG synthesis. The inhibitors differentially influenced the responses to mechanical stimuli. Dynamic compression significantly upregulated protein synthesis but did not significantly affect sGAG synthesis with Nf or Tg treatment. Dynamic compression significantly upregulated both protein and sGAG synthesis rates with Gd treatment. There was no significant stimulation of either protein or sGAG synthesis by dynamic compression with 4AP treatment. Interruption of many ion-channel signaling mechanisms affected sGAG synthesis, suggesting a complicated, multi-pathway signaling process. Also, Ca(2+) signaling may be critical for the transduction of mechanical stimulus in regulating sGAG synthesis. This modulation potentially occurs through direct interactions with the extracellular matrix.

  View details for DOI 10.1007/s10237-006-0034-1

  View details for Web of Science ID 000243873500005

  View details for PubMedID 16767453

• Chondrocytes and fibrochondrocytes differentially process aggrecan during de novo extracellular matrix assembly ASME Summer Bioengineering Conference Wilson, C. G., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2007: 1041–1042

  View details for Web of Science ID 000252105700521

• Contrast-enhanced micro-CT imaging of soft tissues International Symposium on Advanced Biotechnologies for Assessing Quality of Bone and Scaffold Biomaterials Lin, A. S., Palmer, A. W., Duvall, C. L., Robertson, G. C., Oest, M. E., Rai, B., Levenston, M. E., Guldberg, R. E. SPRINGER-VERLAG BERLIN. 2007: 239–256

  View details for Web of Science ID 000250578800015

• Depth dependent diffusivity profile in bovine articular cartilage: Comparing transverse and axial diffusivities ASME Summer Bioengineering Conference Irrechukwu, O. N., Levenston, M. E. AMER SOC MECHANICAL ENGINEERS. 2007: 123–124

  View details for Web of Science ID 000252105700062

• Analysis of cartilage matrix fixed charge density and three-dimensional morphology via contrast-enhanced microcomputed tomography PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Palmer, A. W., Guldberg, R. E., Levenston, M. E. 2006; 103 (51): 19255-19260

  Abstract

  Small animal models of osteoarthritis are often used for evaluating the efficacy of pharmacologic treatments and cartilage repair strategies, but noninvasive techniques capable of monitoring matrix-level changes are limited by the joint size and the low radiopacity of soft tissues. Here we present a technique for the noninvasive imaging of cartilage at micrometer-level resolution based on detecting the equilibrium partitioning of an ionic contrast agent via microcomputed tomography. The approach exploits electrochemical interactions between the molecular charges present in the cartilage matrix and an ionic contrast agent, resulting in a nonuniform equilibrium partitioning of the ionic contrast agent reflecting the proteoglycan distribution. In an in vitro model of cartilage degeneration we observed changes in x-ray attenuation magnitude and distribution consistent with biochemical and histological analyses of sulfated glycosaminoglycans, and x-ray attenuation was found to be a strong predictor of sulfated glycosaminoglycan density. Equilibration with the contrast agent also permits direct in situ visualization and quantification of cartilage surface morphology. Equilibrium partitioning of an ionic contrast agent via microcomputed tomography thus provides a powerful approach to quantitatively assess 3D cartilage composition and morphology for studies of cartilage degradation and repair.

  View details for DOI 10.1073/pnas.0606406103

  View details for Web of Science ID 000243166600009

  View details for PubMedID 17158799

• A constitutive model for protein-based materials BIOMATERIALS Wu, X., Levenston, M. E., Chaikof, E. L. 2006; 27 (30): 5315-5325

  Abstract

  Protein-based materials are critical to the construction of tissue substitutes that exhibit precisely defined mechanical properties. Under physiologically relevant conditions, materials derived from natural or synthetic structural proteins are characterized by nonlinear elastic responses at medium and large deformations, time-dependent or viscoelastic behavior, and display the effects of strain-induced structural changes. Although a constitutive model that accurately describes mechanical behavior is essential for the rational design of tissue constructs, few models account for all of these characteristics. In this report, we present a new constitutive model for protein based materials, in which nonlinear elasticity is captured by the Arruda-Boyce eight-chain model, time dependant viscoelasticity is described by a generalized Maxwell model, and the effect of strain-induced structural change is incorporated using a network alteration theory originally proposed by Tobolsky. The model was applied to a number of protein-based materials and cell containing constructs, including recombinant elastin-mimetic protein polymers and fibroblast populated collagen gel matrices. Significantly, numerical implementation of this model is straightforward and mechanical behavior accurately described under a variety of loading conditions. Moreover, when calibrated using stress relaxation data alone, the model accurately predicted cyclic loading responses. Although limitations exist, this model provides a convenient tool to correlate viscoelastic data obtained by different testing modes and may assist in reducing the number of experimental tests required to fully capture the range of viscoelastic responses of protein-based materials.

  View details for DOI 10.1016/j.biomaterials.2006.06.003

  View details for Web of Science ID 000240506400019

  View details for PubMedID 16815545

• Indentation testing of human articular cartilage: Effects of probe tip geometry and indentation depth on intra-tissue strain JOURNAL OF BIOMECHANICS Bae, W. C., Lewis, C. W., Levenston, M. E., Sah, R. L. 2006; 39 (6): 1039-1047

  Abstract

  Experimental determination of intra-tissue deformation during clinically applicable rapid indentation testing would be useful for understanding indentation biomechanics and for designing safe indentation probes and protocols. The objectives of this study were to perform two-dimensional (2-D) indentation tests, using indenters and protocols that are analogous to those in clinically oriented probes, of normal adult-human articular cartilage in order to determine: (1) intra-tissue strain maps and regions of high strain magnitude, and (2) the effects on strain of indenter geometry (rectangular prismatic and cylindrical) and indentation depth (40-190 microm). Epifluorescence microscopy of samples undergoing indentation and subsequent video image correlation analysis allowed determination of strain maps. Regions of peak strain were near the "edges" of indenter contact with the cartilage surface, and the strain magnitude in these regions ranged from approximately 0.05 to approximately 0.30 in compression and shear, a range with known biological consequences. With increasing indentation displacement, strain magnitudes generally increased in all regions of the tissue. Compared to indentation using a rectangular prismatic tip, indentation with a cylindrical tip resulted in slightly higher peak strain magnitudes while influencing a smaller region of cartilage. These results may be used to refine clinical indenters and indentation protocols.

  View details for DOI 10.1016/j.jbiomech.2005.02.018

  View details for Web of Science ID 000236834900007

  View details for PubMedID 16549094

• Biomechanical analysis of silicon microelectrode-induced strain in the brain. Journal of neural engineering Lee, H., Bellamkonda, R. V., Sun, W., Levenston, M. E. 2005; 2 (4): 81-89

  Abstract

  The ability to successfully interface the brain to external electrical systems is important both for fundamental understanding of our nervous system and for the development of neuroprosthetics. Silicon microelectrode arrays offer great promise in realizing this potential. However, when they are implanted into the brain, recording sensitivity is lost due to inflammation and astroglial scarring around the electrode. The inflammation and astroglial scar are thought to result from acute injury during electrode insertion as well as chronic injury caused by micromotion around the implanted electrode. To evaluate the validity of this assumption, the finite element method (FEM) was employed to analyze the strain fields around a single Michigan Si microelectrode due to simulated micromotion. Micromotion was mimicked by applying a force to the electrode, fixing the boundaries of the brain region and applying appropriate symmetry conditions to nodes lying on symmetry planes. Characteristics of the deformation fields around the electrode including maximum electrode displacement, strain fields and relative displacement between the electrode and the adjacent tissue were examined for varying degrees of physical coupling between the brain and the electrode. Our analysis demonstrates that when physical coupling between the electrode and the brain increases, the micromotion-induced strain of tissue around the electrode decreases as does the relative slip between the electrode and the brain. These results support the use of neuro-integrative coatings on electrode arrays as a means to reduce the micromotion-induced injury response.

  View details for PubMedID 16317231

• Biomechanical analysis of silicon microelectrode-induced strain in the brain JOURNAL OF NEURAL ENGINEERING Lee, H., Bellamkonda, R. V., Sun, W., Levenston, M. E. 2005; 2 (4): 81-89

  View details for DOI 10.1088/1741-2560/2/4/003

  View details for Web of Science ID 000209672600009

• Alterations in physical cross-linking modulate mechanical properties of two-phase protein polymer networks BIOMACROMOLECULES Wu, X. Y., Sallach, R., Haller, C. A., Caves, J. A., Nagapudi, K., Conticello, V. P., Levenston, M. E., Chaikof, E. L. 2005; 6 (6): 3037-3044

  Abstract

  Physically cross-linked protein-based materials possess a number of advantages over their chemically cross-linked counterparts, including ease of processing and the ability to avoid the addition or removal of chemical reagents or unreacted intermediates. The investigations reported herein sought to examine the nature of physical cross-links within two-phase elastin-mimetic protein triblock copolymer networks through an analysis of macroscopic viscoelastic properties. Given the capacity of solution processing conditions, including solvent type and temperature to modulate the microstructure of two-phase protein polymer networks, viscoelastic properties were examined under conditions in which interphase block mixing had been either accentuated or diminished during network formation. Protein networks exhibited strikingly different properties in terms of elastic modulus, hysteresis, residual deformability, and viscosity in response to interdomain mixing. Thus, two-phase protein polymer networks exhibit tunable responses that extend the range of application of these materials to a variety of tissue engineering applications.

  View details for DOI 10.1021/bm0503468

  View details for Web of Science ID 000233392100021

  View details for PubMedID 16283724

• Variations in matrix composition and GAG fine structure among scaffolds for cartilage tissue engineering OSTEOARTHRITIS AND CARTILAGE Mouw, J. K., Case, N. D., Guldberg, R. E., Plaas, A. H., Levenston, M. E. 2005; 13 (9): 828-836

  Abstract

  To compare matrix composition and glycosaminoglycan (GAG) fine structure among five scaffolds commonly used for in vitro chondrocyte culture and cartilage tissue engineering.Bovine articular chondrocytes were seeded into agarose, alginate, collagen I, fibrin and polyglycolic acid (PGA) constructs and cultured for 20 or 40 days. In addition to construct DNA and sulfated GAG (sGAG) contents, the delta-disaccharide compositions of the chondroitin/dermatan sulfate GAGs were determined for each scaffold group via fluorophore-assisted carbohydrate electrophoresis (FACE).Significant differences were found in cell proliferation and extracellular matrix accumulation among the five scaffold groups. Significant cell proliferation was observed for all scaffold types but occurred later (20-40 days) in PGA constructs compared to the other groups (0-20 days). By 40 days, agarose constructs had the highest sGAG to DNA ratio, while alginate and collagen I had the lowest levels. Quantitative differences in the Delta-disaccharide composition of the GAGs accumulated in the different scaffolds were also found, with the most striking variations in unsulfated and disulfated delta-disaccharides. Agarose constructs had the highest fraction of disulfated residues and the lowest fraction of unsulfated residues, with a 6-sulfated/4-sulfated disaccharide ratio most similar to that of native articular cartilage.The similarities and differences among scaffolds in proteoglycan accumulation and GAG composition suggest that the scaffold material directly or indirectly influences chondrocyte proteoglycan metabolism and may have an influence on the quality of tissue engineered cartilage.

  View details for DOI 10.1016/j.joca.2005.04.020

  View details for Web of Science ID 000232252100010

  View details for PubMedID 16006153

• Oscillatory tension differentially modulates matrix metabolism and cytoskeletal organization in chondrocytes and fibrochondrocytes JOURNAL OF BIOMECHANICS Vanderploeg, E. J., Imler, S. M., Brodkin, K. R., Garcia, A. J., Levenston, M. E. 2004; 37 (12): 1941-1952

  Abstract

  Several modes of mechanical stimulation, including compression, shear, and hydrostatic pressure, have been shown to modulate chondrocyte matrix synthesis, but the effects of mechanical tension have not been widely explored. Since articular cartilage is primarily loaded in compression, tension is not generally viewed as a major contributor to the stress state of healthy tissue. However, injury or attempted repair may cause tension to become more significant. Additionally, fibrocartilaginous tissues experience significant tensile stresses in their normal mechanical environment. In this study we investigated mechanical tension as a means to modulate matrix synthesis and cytoskeletal organization in bovine articular chondrocytes and meniscal fibrochondrocytes (MFCs) in a three-dimensional fibrin construct culture system. Oscillatory tension was applied to constructs at 1.0 Hz and 0-10% displacement variation using a custom device. For nearly all conditions and both cell types, oscillatory tension inhibited matrix synthesis as indicated by 3H-proline and 35S-sulfate incorporation. Additionally, oscillatory tension significantly increased proliferation by chondrocytes but not MFCs. Confocal imaging revealed that all cells initially displayed a rounded morphology, but over time MFCs spontaneously developed a three-dimensional, stellate morphology with numerous projections containing organized cytoskeletal filaments. Interestingly, while unloaded chondrocytes remained rounded, chondrocytes subjected to oscillatory tension developed a similar stellate morphology. Both the biochemical and morphological results of this study have important implications for successfully developing cartilage and fibrocartilage tissue replacements and repair strategies.

  View details for DOI 10.1016/j.jbiomech.2004.02.048

  View details for Web of Science ID 000225192800016

  View details for PubMedID 15519602

• Chondrocyte phenotypes on different extracellular matrix monolayers BIOMATERIALS Brodkin, K. R., Garcia, A. J., Levenston, M. E. 2004; 25 (28): 5929-5938

  Abstract

  Chondrocytes undergo a process of dedifferentiation in monolayer culture that is characterized by a transition to a fibroblast-like phenotype. This behavioral change poses a challenge for tissue-engineered cartilage constructs, as approaches using autologous cells require expansion in vitro. Because chondrocytes express a variety of integrin receptors specific to different adhesive proteins, we hypothesized that chondrocytes expanded on various underlying protein monolayers would have different phenotypic responses. Bovine articular chondrocytes were cultured for up to 2 weeks on tissue culture plastic, fibronectin, collagen type I or collagen type II substrate in the presence or absence of ascorbate. Contrary to our hypothesis, the extracellular matrix protein substrates used in this study did not significantly alter the changes in chondrocyte morphology, gene expression, matrix formation, or cytoskeletal organization. Cells on all substrates assembled equivalent matrices, which may have subsequently regulated cell behavior. In cultures with ascorbate, populations of round and spread cells emerged after 1 week, with round cells expressing collagen type II and the differentiated phenotype and spread cells dedifferentiating. In cultures without ascorbate, chondrocytes rapidly adhered and spread onto organized fibronectin matrices via the alpha5beta1 integrin, which has been associated with survival and proliferation of chondrocytes in vitro. These findings indicate that expanding chondrocytes on protein monolayers may not be an effective solution to preventing dedifferentiation and improving autologous chondrocyte transplantation.

  View details for DOI 10.1016/j.biomaterials.2004.01.044

  View details for Web of Science ID 000223208400003

  View details for PubMedID 15183607

• Combined effects of growth factors and static mechanical compression on meniscus explant biosynthesis OSTEOARTHRITIS AND CARTILAGE Imler, S. M., Doshi, A. N., Levenston, M. E. 2004; 12 (9): 736-744

  Abstract

  To compare the actions of fibroblast growth factor-basic (bFGF), insulin-like growth factor-I (IGF-I), platelet derived growth factor-AB (PDGF-AB), and transforming growth factor-beta 1 (TGF-beta1) on bovine meniscus tissue explants with and without static mechanical compression.Meniscus tissue explants were cultured in a serum-free environment supplemented with an individual growth factor (1) over a range of concentrations for 4 days, (2) at a single concentration for 2-14 days, and (3) at a single concentration for 4 days coupled with graded levels of static compression. Explants were analyzed for accumulation of newly synthesized proteoglycan and total protein as measured by 35S-sulfate and 3H-proline incorporation, respectively.Over the range of chosen concentrations, TGF-beta1 was the most potent stimulator of both protein and proteoglycan production, whereas bFGF was the least effective stimulator. Over a 2-week period for all four growth factors, the stimulation of proteoglycan production was sustained while there was no stimulation of protein production during this period. The superposition of static mechanical compression inhibited matrix production in the presence of each anabolic factor, with comparable inhibition relative to uncompressed controls for all factors.The growth factors chosen exhibited an anabolic effect on the meniscus tissue explants, encouraging matrix production and deposition. The addition of static mechanical compression produced comparable relative inhibition of matrix production for each growth factor, suggesting that static compression and growth factors may modulate meniscal fibrochondrocyte biosynthesis via distinct pathways.

  View details for DOI 10.1016/j.joca.2004.05.007

  View details for Web of Science ID 000224046200008

  View details for PubMedID 15325640

• Maturation and integration of tissue-engineered cartilages within an in vitro defect repair model TISSUE ENGINEERING Hunter, C. J., Levenston, M. E. 2004; 10 (5-6): 736-746

  Abstract

  This study compared the behavior of four different engineered cartilages in a hybrid culture system. First, the growth and maturation of tissue-engineered cartilages in isolation were compared to those grown in an in vitro articular cartilage defect repair model. Tissue-engineered cartilages using fibrin, agarose, or poly(glycolic acid) scaffolds were implanted into annular explants of articular cartilage and cultured for 20 or 40 days. Native tissue had a substantial influence on the DNA, sulfated glycosaminoglycan, and hydroxyproline content of the engineered tissues, suggesting that the presence of living tissue in the culture significantly altered cell proliferation and matrix accumulation. Second, the adhesion strength of various engineered cartilages to native tissue was measured and compared with the biochemical content of the engineered tissues. All scaffold treatments adhered to the native cartilage, but there were statistically significant differences in adhesive strength between the different scaffolds. The adhesive strength of all engineered scaffolds was significantly lower than that of native tissue to itself. In the engineered tissues, neither failure stress nor energy to failure correlated with gross biochemical content, suggesting that adhesion between native and engineered tissues is not purely a function of gross matrix synthesis.

  View details for Web of Science ID 000222534500008

  View details for PubMedID 15265290

• Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness OSTEOARTHRITIS AND CARTILAGE Hunter, C. J., Mouw, J. K., Levenston, M. E. 2004; 12 (2): 117-130

  Abstract

  Various strategies have been tested to direct and control matrix synthesis in tissue engineered cartilage, including mechanical stimulation of the construct both before and after implantation. This study examined the effects of oscillatory compression on chondrocytes in a fibrin-based tissue engineered cartilage.Chondrocyte-seeded fibrin gels were cultured under unconfined mechanical compression for 10 or 20 days (free-swelling, 10% static, or 10+/-4% at 0.1 or 1Hz). During the culture period, accumulation of nitrite, sGAG, and proteolytic enzymes in the culture media were monitored. Following culture, the mechanical stiffness and biochemical content of the gels (DNA, sGAG, and hydroxyproline content and GAG Delta-disaccharide composition) were assessed.Compared to free-swelling conditions, static compression had little effect on the mechanical stiffness or biochemical content of the gels. Compared to static compression, oscillatory compression produced softer gels, inhibited sGAG and hydroxyproline accumulation in the gels, and stimulated accumulation of nitrite and sGAG in the culture media. Minimal differences were observed in DNA content and Delta-disaccharide composition across treatment conditions.In this study, oscillatory compression inhibited formation of cartilage-like tissues by chondrocytes in fibrin gels. These results suggest that the effects of mechanical stimuli on tissue engineered cartilage may vary substantially between different scaffold systems.

  View details for Web of Science ID 000188844000004

  View details for PubMedID 14723871

• The influence of cyclic tension amplitude on chondrocyte matrix synthesis: Experimental and finite element analyses 3rd International Symposium on Mechanobiology of Cartilage and Chondrocyte Connelly, J. T., Vanderploeg, E. J., Levenston, M. E. IOS PRESS. 2004: 377–87

  Abstract

  While not generally viewed as physiologically significant in articular cartilage, substantial tension can develop in fibrocartilage structures and in articular cartilage injuries. This study examined how different amplitudes of cyclic tension influence chondrocyte matrix synthesis. Bovine articular chondrocytes seeded in fibrin gels were loaded continuously for 48 hours at 1.0 Hz with displacements of 5%, 10%, or 20%. Protein and proteoglycan synthesis were measured by (3)H-proline and (35)S-sulfate incorporation, respectively. A poroelastic finite element model of the fibrin gel was developed to determine the strain distributions, hydrostatic pressures, and fluid velocities within the constructs at the various levels of displacement. Compared to unloaded controls, 10% and 20% displacements inhibited proteoglycan synthesis to the same extent, while 5% displacement had no effect. Tensile loading did not significantly affect protein synthesis. The finite element model predicted a wide range of strains and fluid velocities within the region of the gel analyzed for matrix synthesis, and the ranges overlapped for the different levels of displacement. These results indicate that the cyclic tension amplitude influences chondrocyte proteoglycan synthesis and that there may be a threshold in the response.

  View details for Web of Science ID 000225104400024

  View details for PubMedID 15299270

• Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels BIOMATERIALS Hunter, C. J., Imler, S. M., Malaviya, P., Nerem, R. M., Levenston, M. E. 2002; 23 (4): 1249-1259

  Abstract

  Articular cartilage responds to its mechanical environment through altered cell metabolism and matrix synthesis. In this study, isolated articular chondrocytes were cultured in collagen type I gels and exposed to uniaxial static compression of 0%, 25%, or 50% of original thickness for 0.5, 4, and 24 h, and to oscillatory (25 +/- 4%, 1 Hz) compression for 24 h. The cellular response was assessed through competitive and real-time RT-PCR to quantify expression of genes for collagen type I, collagen type II, and aggrecan core protein, and through radiolabelled proline and sulfate incorporation to quantify protein and proteoglycan synthesis rates. Static compression for 24 h inhibited expression of collagen I and II mRNAs and inhibited 3H-proline and 35S-sulfate incorporation. The mRNA expression exhibited transient fluctuations at intermediate time points. Oscillatory compression had no effect upon mRNA expression, and 24 h after release from static compression, there was no difference in collagen II or aggrecan mRNA, while there was an inhibition of collagen I. We conclude that the chondrocytes maintained some aspects of their ability to sense and respond to static compression, despite a biochemical and mechanical environment which is different from that in tissue. This suggests that mechanical stimuli may be useful in modulating chondrocyte metabolism in tissue engineering systems using fibrillar protein scaffolds.

  View details for Web of Science ID 000172738500031

  View details for PubMedID 11791929

• The influence of repair tissue maturation on the response to oscillatory compression in a cartilage defect repair model 2nd International Symposium on Mechanobiology - Cartilage and Chondrocyte Hunter, C. J., Levenston, M. E. IOS PRESS. 2002: 79–88

  Abstract

  This study examined the effects of mechanical compression on engineered cartilage in a novel hybrid culture system. Cylindrical holes were cut in discs of bovine articular cartilage and filled with agarose gels containing chondrocytes. These constructs were compressed in radiolabeled medium under static or oscillatory unconfined compression. Oscillatory compression at 1 Hz significantly stimulated synthesis above static control levels. Control experiments indicate that oscillatory compression does not stimulate freshly cast gels (without annuli), but does so after several weeks. This may be because physiologic fluid flow levels do not occur until sufficient extracellular matrix has accumulated. Finite element models predict minimal fluid flow in the gel core, and minimal differences in flow patterns between free and constrained gels. However, the models predict fluid pressures in constrained gels to be substantially higher than those in free gels. Our results suggest that pressure variations may influence synthesis of engineered cartilage matrices, with implications for construct development and post-implantation survival.

  View details for Web of Science ID 000175769100010

  View details for PubMedID 12082270

• A versatile shear and compression apparatus for mechanical stimulation of tissue culture explants JOURNAL OF BIOMECHANICS Frank, E. H., Jin, M., Loening, A. M., Levenston, M. E., GRODZINSKY, A. J. 2000; 33 (11): 1523-1527

  Abstract

  We have developed an incubator housed, biaxial-tissue-loading device capable of applying axial deformations as small as 1 microm and sinusoidal rotations as small as 0.01 degrees. Axial resolution is 50 nm for applying sinewaves as low as 10 microm (or 1% based on a 1 mm thickness) or as large as 100 microm. Rotational resolution is 0.0005 degrees. The machine is small enough (30 cm high x 25 cm x 20 cm) to be placed in a standard incubator for long-term tissue culture loading studies. In metabolic studies described here, application of sinusoidal macroscopic shear deformation to articular cartilage explants resulted in a significant increase in the synthesis of proteoglycan and proteins (uptake of (35)S-sulfate and (3)H-proline) over controls held at the same static offset compression.

  View details for Web of Science ID 000089948900023

  View details for PubMedID 10940414

• Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Loening, A. M., James, I. E., Levenston, M. E., Badger, A. M., Frank, E. H., Kurz, B., Nuttall, M. E., Hung, H. H., Blake, S. M., Grodzinsky, A. J., Lark, M. W. 2000; 381 (2): 205-212

  Abstract

  A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.

  View details for Web of Science ID 000089557300004

  View details for PubMedID 11032407

• Cartilage tissue remodeling in response to mechanical forces ANNUAL REVIEW OF BIOMEDICAL ENGINEERING Grodzinsky, A. J., Levenston, M. E., Jin, M., Frank, E. H. 2000; 2: 691-?

  Abstract

  Recent studies suggest that there are multiple regulatory pathways by which chondrocytes in articular cartilage sense and respond to mechanical stimuli, including upstream signaling pathways and mechanisms that may lead to direct changes at the level of transcription, translation, post-translational modifications, and cell-mediated extracellular assembly and degradation of the tissue matrix. This review focuses on the effects of mechanical loading on cartilage and the resulting chondrocyte-mediated biosynthesis, remodeling, degradation, and repair of this tissue. The effects of compression and tissue shear deformation are compared, and approaches to the study of mechanical regulation of gene expression are described. Of particular interest regarding dense connective tissues, recent experiments have shown that mechanotransduction is critically important in vivo in the cell-mediated feedback between physical stimuli, the molecular structure of newly synthesized matrix molecules, and the resulting macroscopic biomechanical properties of the tissue.

  View details for Web of Science ID 000089887400025

  View details for PubMedID 11701528

• Electrokinetic and poroelastic coupling during finite deformations of charged porous media JOURNAL OF APPLIED MECHANICS-TRANSACTIONS OF THE ASME Levenston, M. E., Frank, E. H., GRODZINSKY, A. J. 1999; 66 (2): 323-333

  View details for Web of Science ID 000081476100005

• Proximal Femoral Density Patterns are Consistent with Bicentric Joint Loads. Computer methods in biomechanics and biomedical engineering Fischer, K. J., Jacobs, C. R., Levenston, M. E., Cody, D. D., Carter, D. R. 1999; 2 (4): 271-283

  Abstract

  We developed an alternate method for density-based load estimation and applied it to estimate hip joint load distributions for two femora. Two-dimensional finite element models were constructed from single energy quantitative computed tomography (QCT) data. Load estimation was performed using five loading regions on the femoral head. Within each loading region, individual nodal loads, normal to the local surface, were supplied as input to the load estimation. An optimization procedure independently adjusted individual nodal load magnitudes in each region, and the magnitudes of muscle forces on the greater trochanter, such that the applied tissue stimulus approached the reference stimulus throughout the model. Dominant estimated load resultant directions were generally consistent with published experimental data for loads during gait. The estimated loads also suggested that loads near the extremes of the articulating surface may be important (even required) for development and maintenance of normal bone architecture. Estimated load distributions within nearly all regions predicted bicentric loading patterns, which are consistent with observations of hip joint incongruity. Remodeling simulations with the estimated loads predicted density distributions with features qualitatively similar to the QCT data sets. This study illustrates how applications of density-based bone load estimation can improve understanding of dominant loading patterns in other bones and joints. The prediction of bicentric loading suggests a very fine level of local adaptation to details of joint loading.

  View details for PubMedID 11264832

• A variational formulation for coupled physicochemical flows during finite deformations of charged porous media INTERNATIONAL JOURNAL OF SOLIDS AND STRUCTURES Levenston, M. E., Eisenberg, S. R., GRODZINSKY, A. J. 1998; 35 (34-35): 4999-5019

  View details for Web of Science ID 000075969000027

• An energy dissipation-based model for damage stimulated bone adaptation JOURNAL OF BIOMECHANICS Levenston, M. E., Carter, D. 1998; 31 (7): 579-586

  Abstract

  Based on experimental observations, several researchers have proposed a role for damage processes in stimulating an adaptive response in bone. In the current study we propose a model for bone adaptation based on cyclic energy dissipation as a measure of bone damage creation. By reanalyzing the fatigue data of Pattin et al. (1996), we derive a uniaxial form of the damage based formulation applicable to cortical regions experiencing primarily longitudinal stresses. Because of the experimentally observed difference between damage formation under tension and compression (Pattin et al., 1996), this formulation naturally predicts a difference in the adaptive response to tensile and compressive loading. This feature distinguishes the new formulation from existing strain energy based adaptation theories which treat tensile and compressive strains identically. Thus, developmental adaptation in response to unequal generation of damage provides one possible explanation for the experimentally observed difference between peak tensile and compressive bone surface strains.

  View details for Web of Science ID 000076346300001

  View details for PubMedID 9796679

• Variationally derived 3-field finite element formulations for quasistatic poroelastic analysis of hydrated biological tissues COMPUTER METHODS IN APPLIED MECHANICS AND ENGINEERING Levenston, M. E., Frank, E. H., GRODZINSKY, A. J. 1998; 156 (1-4): 231-246

  View details for Web of Science ID 000073491400012

• Bone Load Estimation for the Proximal Femur Using Single Energy Quantitative CT Data. Computer methods in biomechanics and biomedical engineering Fischer, K. J., Jacobs, C. R., Levenston, M. E., Cody, D. D., Carter, D. R. 1998; 1 (3): 233-245

  Abstract

  A density-based load estimation method was applied to determine femoral load patterns. Two-dimensional finite element models were constructed using single energy quantitative computed tomography (QCT) data from two femora. Basic load cases included parabolic pressure joint loads and constant tractions on the greater trochanter. An optimization procedure adjusted magnitudes of the basic load cases, such that the applied mechanical stimulus approached the ideal stimulus throughout each model. Dominant estimated load directions were generally consistent with published experimental data for gait. Other estimated loads suggested that loads at extreme joint orientations may be important to maintenance of bone structure. Remodeling simulations with the estimated loads produced density distributions qualitatively similar to the QCT data sets. Average nodal density errors between QCT data and predictions were 0.24 g/cm(3) and 0.28 g/cm(3). The results indicate that density-based load estimation could improve understanding of loading patterns on bones.

  View details for PubMedID 11264806

• Loading Mode Interactions in Simulations of Long Bone Cross-Sectional Adaptation. Computer methods in biomechanics and biomedical engineering Levenston, M. E., Beaupré, G. S., Carter, D. R. 1998; 1 (4): 303-319

  Abstract

  Although many bone adaptation theories have been formulated to address both trabecular and cortical adaptation, most applications have focused on trabecular adaptation. Thus far, no thorough investigations of the influence of different types of loading on predicted patterns of long bone cross-sectional adaptation have been reported. In the current study, we present a new model for long bone cross-sectional adaptation that incorporates axial, bending and torsional loading components. We found that bending moments have a strong potential to modulate cross-sectional geometry, but can produce unforseen (and unrealistic) geometric instabilities. Torsional moments have the ability to suppress these instabilities, suggesting that torsion may play a more significant role in guiding long bone development than previously recognized. Our results also call into question the concept of strict "remodeling equilibrium," suggesting that long bones do not necessarily approach a state of uniform mechanical stimulation. This modeling approach provides an additional perspective on experimental studies, and may lead to a greater understanding of the interaction between mechanics and biology in long bone adaptation.

  View details for PubMedID 11264811

• Temporal stability of node-based internal bone adaptation simulations JOURNAL OF BIOMECHANICS Levenston, M. E. 1997; 30 (4): 403-407

  Abstract

  Jacobs et al. (1995, J. Biomechanics 28, 449-459) introduced a new implementation of the remodeling theory developed by Beaupre et al. (1990, J. Orthop. Res. 8, 651-661) that eliminates certain spurious spatial instabilities of previous implementations. Due to the highly nonlinear, coupled nature of multi-dimensional adaptation simulations, direct stability analyses of this method are not practical. In this manuscript, a linearized stability analysis was used to derive an expression for the critical time step for the stability of a simple model problem. In addition to accurately predicting the temporal response of the single degree-of-freedom problem, the analysis provided a conservative estimate of the critical time step for a more realistic, multiple degree-of-freedom adaptation simulation.

  View details for Web of Science ID A1997WN13500014

  View details for PubMedID 9075011

• Observations of convergence and uniqueness of node-based bone remodeling simulations ANNALS OF BIOMEDICAL ENGINEERING Fischer, K. J., Jacobs, C. R., Levenston, M. E., Carter, D. R. 1997; 25 (2): 261-268

  Abstract

  Some investigators have indicated that mathematical theories and computational models of bone adaptation may not converge and that the density solutions from such simulations are dependent on the initial density distribution. In this study, two-dimensional finite element models were used to investigate the effect of initial density distribution on the final density distribution produced using a node-based bone remodeling simulation. The first model was a generic long bone, and the second was a proximal femur, For each model, we conducted time-dependent, node-based, linear rate-law bone remodeling simulations. Five initial density conditions were used with the generic long bone and three with the proximal femur. Remodeling simulations were performed, and the largest average nodal density differences at the end of the simulations were 0.000010 g/cm3 and 0.000006 g/cm3 for the generic long bone and proximal femur models, respectively. Results illustrate that, for a given set of loads and a given finite element model, the node-based bone adaptation algorithm can yield a unique density distribution. In conjunction with previous studies, this finding suggests that uniqueness of the density solution is dependent on both the mathematical theory and the computational implementation.

  View details for Web of Science ID A1997WN69400003

  View details for PubMedID 9084831

• Bite-force estimation for Tyrannosaurus rex from tooth-marked bones NATURE Erickson, G. M., VANKIRK, S. D., Su, J. T., Levenston, M. E., CALER, W. E., Carter, D. R. 1996; 382 (6593): 706-708

  View details for Web of Science ID A1996VD33300046

• Different loads can produce similar bone density distributions BONE Fischer, K. J., Jacobs, C. R., Levenston, M. E., Carter, D. R. 1996; 19 (2): 127-135

  Abstract

  Finite element models of a generic long bone and the proximal femur were used to identify important load characteristics and to determine whether small changes in load affect bone adaptation simulations. We also examined the effect of implants on the sensitivity of bone adaptation simulations to changes in loads. For each model, a primary load set was selected and incorporated in a bone adaptation simulation to generate a primary density distribution. A density-based load estimation method was used to determine a secondary set of loading conditions for each model. Each secondary load set was incorporated in a bone adaptation simulation and the resulting density distribution was compared to the corresponding primary density distribution. Nearly identical density distributions were produced for the natural generic long bone model (average nodal density difference 0.02 g/cm3). For the natural proximal femur model, the density distributions were very similar, but differences were apparent (average nodal density difference 0.07 g/cm3). The same primary and secondary load sets were used for bone adaptation simulations with implant models. For the proximal femur model, density distribution differences with the implant were very slightly less than those of the natural model. For the generic long bone model, the implant amplified differences between density distributions (average nodal density difference 0.14 g/cm3). Thus, variations in loading conditions may partially explain variations in long-term total joint outcome. The total equivalent stimulus load magnitudes for the two load sets for the generic long bone model were within 1%, and the stimulus-weighted average load directions were within 1 degree. The similarity of these parameters and the natural generic long bone density distributions indicate that the overall magnitude and average load direction are key factors affecting bone adaptation.

  View details for Web of Science ID A1996VB74000008

  View details for PubMedID 8853856

• Letter to the editor. Journal of biomechanics Levenston, M. E., Beaupre, G. S. 1996; 29 (1): 133-135

  View details for PubMedID 8839026

• PERIOSTEAL BONE-FORMATION STIMULATED BY EXTERNALLY INDUCED BENDING STRAINS JOURNAL OF BONE AND MINERAL RESEARCH Levenston, M. E. 1995; 10 (4): 671-671

  View details for Web of Science ID A1995QQ47900021

  View details for PubMedID 7610940

• NUMERICAL INSTABILITIES IN BONE REMODELING SIMULATIONS - THE ADVANTAGES OF A NODE-BASED FINITE-ELEMENT APPROACH JOURNAL OF BIOMECHANICS Jacobs, C. R., Levenston, M. E., Beaupre, G. S., SIMO, J. C., Carter, D. R. 1995; 28 (4): 449-?

  Abstract

  Long bone structure occurs in two distinct forms. The bone mass near the joint is primarily found in a distributed, porous trabecular structure, while in the diaphyses a tubular cortical structure is formed. It seems likely that these two observed morphologies come about, at least in part, as a mechanical adaptation to the different mechanical demands in the two regions. Mathematical formulations of this dependency have been proposed, thus facilitating numerical simulations of bone adaptation. Recently two types of discontinuities have been observed in these simulations. The first type (near-field) appears in areas near distributed load application and is characterized by a 'checkerboard' pattern of density wherein adjacent remodeled elements alternate between low and high density. The second type of discontinuity (far-field) appears remote from the load application and is characterized by strut or column-like regions of elements which become fully compact bone while adjacent regions are fully resorbed. In fact, the far-field discontinuity is an accurate representation of bone physiology and morphology since it is consistent with the appearance of cortical bone in the diaphysis. On the other hand, the near-field discontinuity, appears in a region where continuous distributions of intermediate apparent densities (trabecular bone) are expected. This finding may cause some to question whether a single continuum formulation of bone remodeling can predict both discontinuous far-field behavior and continuous near-field behavior. We describe a node-based implementation of current continuum bone remodeling theories which eliminates the spurious near-field discontinuities and preserves the anatomically correct far-field discontinuities, thus indicating that a single biological process may be at work in forming and maintaining both far-field and near-field morphologies.

  View details for Web of Science ID A1995QM53600010

  View details for PubMedID 7738054

• IMPROVED METHOD FOR ANALYSIS OF WHOLE BONE TORSION TESTS JOURNAL OF BONE AND MINERAL RESEARCH Levenston, M. E., Beaupre, G. S., VANDERMEULEN, M. C. 1994; 9 (9): 1459-1465

  Abstract

  Structural tests, such as whole bone torsion tests, have become widely accepted methods for assessing average bone material properties. To simplify interpretation of these tests, the nonuniform bone geometry is often analyzed as a tube with a constant cross section (prismatic) and the areal properties of the smallest bone section. This approach may not adequately represent the true torsional behavior of the cross section and does not account for any lengthwise variations in bone geometry. The errors introduced by these approximations are particularly significant when comparing bones of different sizes and geometries. In this paper, we examine the effects of approximating the cross-sectional torsional behavior and of neglecting lengthwise variations in bone geometry. We then present a simple, standardized procedure utilizing a FORTRAN computer program for accurate determination of material properties. We examine first simple idealized bone geometries and then a complex three-dimensional model of the femur from a 26-day-old male Sprague-Dawley rat. For these models, the conventional methods for interpreting torsion tests introduce errors of up to 42% in the shear modulus and up to 48% in the maximum shear stress; a straightforward extension of these methods reduces the errors to within 3%.

  View details for Web of Science ID A1994PD70600018

  View details for PubMedID 7817831

• THE ROLE OF LOADING MEMORY IN BONE ADAPTATION SIMULATIONS BONE Levenston, M. E., Beaupre, G. S., Jacobs, C. R., Carter, D. R. 1994; 15 (2): 177-186

  Abstract

  The concept that bone responds to a time-averaged value of its current mechanical loading forms the basis for many computational bone adaptation algorithms. Some mathematical formulations have incorporated a quantification of the loading experienced during a single "average" day and thus implicitly assume that bone responds abruptly to changes in its loading history. To better reflect the time delays inherent in bone cell recruitment and activation processes, we included a fading memory of past loading. Implementing an exponentially fading memory with time constants of 5, 20, and 100 days, we simulated bone adaptations to abrupt and gradual changes in mechanical loading. Both an idealized single degree-of-freedom model and a finite element model of the proximal femur were studied. A time constant of 5 days produced time-dependent density changes that were negligibly different from those of the standard approach without memory. Models with higher time constants produced significant transient time lags (up to 8.1% difference) in the predicted short-term (3 months) bone density changes. A time constant of 100 days produced overshoots (by approximately 1%) of the eventual steady-state. All models predicted comparable long-term (after several years) steady-state adaptations. Future experimental analyses will be necessary to better determine appropriate fading memory time constants for bone under various loading conditions.

  View details for Web of Science ID A1994NE28000006

  View details for PubMedID 8086235

• BITE FORCE OF TYRANNOSAURUS-REX VANKIRK, S. D., Su, J., Erickson, G., LEVENSTON, M., STANFORD, D. C. SLACK INC. 1994: A3–A3

  View details for Web of Science ID A1994MR21400017

• Computer simulations of stress-related bone remodeling around noncemented acetabular components. journal of arthroplasty Levenston, M. E., Beaupré, G. S., Schurman, D. J., Carter, D. R. 1993; 8 (6): 595-605

  Abstract

  The authors have used computer modeling techniques to examine stress-related bone changes in the acetabular region. Using a previously developed theory for bone development and adaptation, the authors simulated the distribution of bone density in the natural pelvis as well as changes in bone density following total hip arthroplasty. The geometry of the finite element model was based on a two-dimensional slice through the pelvis. Starting from a solid, homogeneous structure, the computer simulations predicted the distribution of bone density throughout the natural pelvis. The predicted bone density distribution in this first simulation agreed well with the actual bone density distribution only when loads representing multiple activities were incorporated. Using the predicted density distribution as a starting point the authors modified the finite element models to study two designs of noncemented, metal-backed acetabular cups. The simulations with fully fixed bone-implant interfaces predicted extensive loss of bone density medial and inferior to the prosthetic components. The simulations with loose interfaces led to more moderate losses of bone density, indicating a load transfer more similar to that which occurs in the natural joint. The differences in simulated bone remodeling between the two component designs were quite minimal. These results indicate that acetabular components with full bony ingrowth may induce significant stress-related bone remodeling due to a nonphysiologic transfer of load.

  View details for PubMedID 8301277

• A NEW IMPLEMENTATION OF FINITE ELEMENT-BASED BONE REMODELING International Symposium on Computer Methods in Biomechanics and Biomedical Engineering Jacobs, C. R., Levenston, M. E., Beaupre, G. S., SIMO, J. C. BOOKS & JOURNALS INT LTD. 1992: 20–29

  View details for Web of Science ID A1992BZ83S00003