Rapid volumetric gagCEST imaging of knee articular cartilage at 3 T: evaluation of improved dynamic range and an osteoarthritic population.

Chemical exchange saturation transfer of glycosaminoglycans, gagCEST, is a quantitative MR technique that has potential for assessing cartilage proteoglycan content at field strengths of 7 T and higher. However, its utility at 3 T remains unclear. The objective of this work was to implement a rapid volumetric gagCEST sequence with higher gagCEST asymmetry at 3 T to evaluate its sensitivity to osteoarthritic changes in knee articular cartilage and in comparison with T2 and T1ρ measures. We hypothesize that gagCEST asymmetry at 3 T decreases with increasing severity of osteoarthritis (OA). Forty-two human volunteers, including 10 healthy subjects and 32 subjects with medial OA, were included in the study. Knee Injury and Osteoarthritis Outcome Scores (KOOS) were assessed for all subjects, and Kellgren-Lawrence grading was performed for OA volunteers. Healthy subjects were scanned consecutively at 3 T to assess the repeatability of the volumetric gagCEST sequence at 3 T. For healthy and OA subjects, gagCEST asymmetry and T2 and T1ρ relaxation times were calculated for the femoral articular cartilage to assess sensitivity to OA severity. Volumetric gagCEST imaging had higher gagCEST asymmetry than single-slice acquisitions (p = 0.015). The average scan-rescan coefficient of variation was 6.8%. There were no significant differences in average gagCEST asymmetry between younger and older healthy controls (p = 0.655) or between healthy controls and OA subjects (p = 0.310). T2 and T1ρ relaxation times were elevated in OA subjects (p < 0.001 for both) compared with healthy controls and both were moderately correlated with total KOOS scores (rho = -0.181 and rho = -0.332 respectively). The gagCEST technique developed here, with volumetric scan times under 10 min and high gagCEST asymmetry at 3 T, did not vary significantly between healthy subjects and those with mild-moderate OA. This further supports a limited utility for gagCEST imaging at 3 T for assessment of early changes in cartilage composition in OA.

Mechanical confinement regulates cartilage matrix formation by chondrocytes.

Cartilage tissue equivalents formed from hydrogels containing chondrocytes could provide a solution for replacing damaged cartilage. Previous approaches have often utilized elastic hydrogels. However, elastic stresses may restrict cartilage matrix formation and alter the chondrocyte phenotype. Here we investigated the use of viscoelastic hydrogels, in which stresses are relaxed over time and which exhibit creep, for three-dimensional (3D) culture of chondrocytes. We found that faster relaxation promoted a striking increase in the volume of interconnected cartilage matrix formed by chondrocytes. In slower relaxing gels, restriction of cell volume expansion by elastic stresses led to increased secretion of IL-1ß, which in turn drove strong up-regulation of genes associated with cartilage degradation and cell death. As no cell-adhesion ligands are presented by the hydrogels, these results reveal cell sensing of cell volume confinement as an adhesion-independent mechanism of mechanotransduction in 3D culture, and highlight stress relaxation as a key design parameter for cartilage tissue engineering.

Adipokines induce catabolism of newly synthesized matrix in cartilage and meniscus tissues.

PURPOSE: Altered synovial levels of various adipokines (factors secreted by fat as well as other tissues) have been associated with osteoarthritis (OA) onset and progression. However, the metabolic effects of adipokines on joint tissues, in particular the fibrocartilaginous menisci, are not well understood. This study investigated effects of several adipokines on release of recently synthesized extracellular matrix in bovine cartilage and meniscus tissue explants. MATERIALS AND METHODS: After labeling newly synthesized proteins and sulfated glycosaminoglycans (sGAGs) with 3H-proline and 35S-sulfate, respectively; bovine cartilage and meniscus tissue explants were cultured for 6 days in basal medium (control) or media supplemented with adipokines (1 µg/ml of leptin, visfatin, adiponectin, or resistin) or 20 ng/ml interleukin-1 (IL-1). Release of radiolabel and sGAG to the media during culture and the final explant water, DNA, sGAG, and retained radiolabel were measured. Matrix metalloproteinase (MMP-2) and MMP-3 activities were assessed using gelatin and casein zymography, respectively. RESULTS: Water and DNA contents were not significantly altered by any treatment. Visfatin, adiponectin, resistin, and IL-1 stimulated sGAG release from meniscus, whereas only IL-1 stimulated sGAG release from cartilage. Release of 3H and 35S was stimulated not only by resistin and IL-1 in meniscus but also by IL-1 in cartilage. Retained 3H was unaltered by any treatment, while retained 35S was reduced by visfatin, resistin, and IL-1 in meniscus and by only IL-1 in cartilage. Resistin and IL-1 elevated active MMP-2 and total MMP-3 in meniscus, whereas cartilage MMP-3 activity was elevated by only IL-1. CONCLUSIONS: Resistin stimulated rapid and extensive catabolism of meniscus tissue, similar to IL-1, whereas adipokines minimally affected cartilage. Release of newly synthesized matrix was similar to overall release in both tissues. These observations provide further indications that meniscal tissue is more sensitive to pro-inflammatory factors than cartilage and also suggest further study of resistin's role in OA.

Co-culture with infrapatellar fat pad differentially stimulates proteoglycan synthesis and accumulation in cartilage and meniscus tissues.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Quantitative imaging of cartilage and bone morphology, reactive oxygen species, and vascularization in a rodent model of osteoarthritis.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Regional variations in the distribution and colocalization of extracellular matrix proteins in the juvenile bovine meniscus.

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.

Validation of watershed-based segmentation of the cartilage surface from sequential CT arthrography scans.

BACKGROUND: This study investigated the utility of a 2-dimensional watershed algorithm for identifying the cartilage surface in computed tomography (CT) arthrograms of the knee up to 33 minutes after an intra-articular iohexol injection as boundary blurring increased. METHODS: A 2D watershed algorithm was applied to CT arthrograms of 3 bovine stifle joints taken 3, 8, 18, and 33 minutes after iohexol injection and used to segment tibial cartilage. Thickness measurements were compared to a reference standard thickness measurement and the 3-minute time point scan. RESULTS: 77.2% of cartilage thickness measurements were within 0.2 mm (1 voxel) of the thickness calculated in the reference scan at the 3-minute time point. 42% fewer voxels could be segmented from the 33-minute scan than the 3-minute scan due to diffusion of the contrast agent out of the joint space and into the cartilage, leading to blurring of the cartilage boundary. The traced watershed lines were closer to the location of the cartilage surface in areas where tissues were in direct contact with each other (cartilage-cartilage or cartilage-meniscus contact). CONCLUSIONS: The use of watershed dam lines to guide cartilage segmentation shows promise for identifying cartilage boundaries from CT arthrograms in areas where soft tissues are in direct contact with each other.

Contrast solution properties and scan parameters influence the apparent diffusivity of computed tomography contrast agents in articular cartilage.

The inability to detect early degenerative changes to the articular cartilage surface that commonly precede bulk osteoarthritic degradation is an obstacle to early disease detection for research or clinical diagnosis. Leveraging a known artefact that blurs tissue boundaries in clinical arthrograms, contrast agent (CA) diffusivity can be derived from computed tomography arthrography (CTa) scans. We combined experimental and computational approaches to study protocol variations that may alter the CTa-derived apparent diffusivity. In experimental studies on bovine cartilage explants, we examined how CA dilution and transport direction (absorption versus desorption) influence the apparent diffusivity of untreated and enzymatically digested cartilage. Using multiphysics simulations, we examined mechanisms underlying experimental observations and the effects of image resolution, scan interval and early scan termination. The apparent diffusivity during absorption decreased with increasing CA concentration by an amount similar to the increase induced by tissue digestion. Models indicated that osmotically-induced fluid efflux strongly contributed to the concentration effect. Simulated changes to spatial resolution, scan spacing and total scan time all influenced the apparent diffusivity, indicating the importance of consistent protocols. With careful control of imaging protocols and interpretations guided by transport models, CTa-derived diffusivity offers promise as a biomarker for early degenerative changes.

Characterizing the transient response of knee cartilage to running: Decreases in cartilage T2 of female recreational runners.

Cartilage transmits and redistributes biomechanical loads in the knee joint during exercise. Exercise-induced loading alters cartilage hydration and is detectable using quantitative magnetic resonance imaging (MRI), where T2 relaxation time (T2 ) is influenced by cartilage collagen composition, fiber orientation, and changes in the extracellular matrix. This study characterized short-term transient responses of healthy knee cartilage to running-induced loading using bilateral scans and image registration. Eleven healthy female recreational runners (33.73 ± 4.22 years) and four healthy female controls (27.25 ± 1.38 years) were scanned on a 3T GE MRI scanner with quantitative 3D double-echo in steady-state before running over-ground (runner group) or resting (control group) for 40 min. Subjects were scanned immediately post-activity at 5-min intervals for 60 min. T2 times were calculated for femoral, tibial, and patellar cartilage at each time point and analyzed using a mixed-effects model and Bonferroni post hoc. There were immediate decreases in T2 (mean ± SEM) post-run in superficial femoral cartilage of at least 3.3% ± 0.3% (p = .002) between baseline and Time 0 that remained for 25 min, a decrease in superficial tibial cartilage T2 of 2.9% ± 0.4% (p = .041) between baseline and Time 0, and a decrease in superficial patellar cartilage T2 of 3.6% ± 0.3% (p = .020) 15 min post-run. There were decreases in the medial posterior region of superficial femoral cartilage T2 of at least 5.3 ± 0.2% (p = .022) within 5 min post-run that remained at 60 min post-run. These results increase understanding of transient responses of healthy cartilage to repetitive, exercise-induced loading and establish preliminary recommendations for future definitive studies of cartilage response to running.

Osmotic Swelling Responses Are Conserved Across Cartilaginous Tissues With Varied Sulfated-Glycosaminoglycan Contents.

Determining the influence of tissue composition on the osmotic swelling stress of articular cartilage and meniscus fibrocartilage is important to enhance our understanding of physiology and disease. This osmotic swelling stress is critical for the load-bearing capability of both tissues and results in part due to the interactions between the negatively charged sulfated glycosaminoglycan (sGAG) chains and the ionic interstitial fluid. Changes in sGAG content, as those occurring during the progression of degenerative joint disease, alter such interactions. Here, we compare the time-varying effects of altered osmotic environments on the confined compression swelling behavior of bovine tissues spanning a range of sGAG concentrations: juvenile articular cartilage, juvenile and adult meniscus, and juvenile cartilage enzymatically degraded to reduce its sGAG content. The transient response to changes in bath conditions was evaluated for explants assigned to one of three compressive offsets (5%, 10%, or 15% strain) and one of three bath conditions (0.1X, 1X, or 10X phosphate-buffered saline). Our results show that relative responses to alterations to the osmotic environment are consistent across native tissues but differ for degraded juvenile cartilage, demonstrating that changes in sGAG do not completely recapitulate the native swelling behaviors. Further, we found a strong correlation between aggregate modulus and sGAG/collagen, as well as between sGAG and collagen contents across native tissue types, suggesting some conservation of composition-function relationships across a range of tissue types with varying sGAG concentrations. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:785-792, 2020.

Interactions between integrin ligand density and cytoskeletal integrity regulate BMSC chondrogenesis.

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.

3D imaging of tissue integration with porous biomaterials.

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.

A modified lap test to more accurately estimate interfacial shear strength for bonded tissues.

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.

Mechanical properties of the airway tree: heterogeneous and anisotropic pseudoelastic and viscoelastic tissue responses.

Airway obstruction and pulmonary mechanics remain understudied despite lung disease being the third cause of death in the United States. Lack of relevant data has led computational pulmonary models to infer mechanical properties from available material data for the trachea. Additionally, the time-dependent, viscoelastic behaviors of airways have been largely overlooked, despite their potential physiological relevance and utility as metrics of tissue remodeling and disease progression. Here, we address the clear need for airway-specific material characterization to inform biophysical studies of the bronchial tree. Specimens from three airway levels (trachea, large bronchi, and small bronchi) and two orientations (axial and circumferential) were prepared from five fresh pig lungs. Uniaxial tensile tests revealed substantial heterogeneity and anisotropy. Overall, the linear pseudoelastic modulus was significantly higher axially than circumferentially (30.5 ± 3.1 vs. 8.4 ± 1.1 kPa) and significantly higher among circumferential samples for small bronchi than for the trachea and large bronchi (12.5 ± 1.9 vs. 6.0 ± 0.6 and 6.6 ± 0.9 kPa). Circumferential samples exhibited greater percent stress relaxation over 300 s than their axial counterparts (38.0 ± 1.4 vs. 23.1 ± 1.5%). Axial and circumferential trachea samples displayed greater percent stress relaxation (26.4 ± 1.6 and 42.5 ± 1.7%) than corresponding large and small bronchi. This ex vivo pseudoelastic and viscoelastic characterization reveals novel anisotropic and heterogeneous behaviors and equips us to construct airway-specific constitutive relations. Our results establish necessary fundamentals for airway mechanics, laying the groundwork for future studies to extend to clinical questions surrounding lung injury, and further directly enables computational tools for lung disease obstruction predictions. NEW & NOTEWORTHY Understanding the mechanics of the lung is necessary for investigating disease progression. Trachea mechanics comprises the vast majority of ex vivo airway tissue characterization despite distal airways being the site of disease manifestation and occlusion. Furthermore, viscoelastic studies are scarce, whereas time-dependent behaviors could be potential physiological metrics of tissue remodeling. In this study, the critical need for airway-specific material properties is addressed, reporting bronchial tree anisotropic and heterogeneous material properties.

Enhancing integration of articular cartilage grafts via photochemical bonding.

The integration of osteochondral grafts to native articular cartilage is critical as the lack of graft integration may lead to continued tissue degradation, poor load transfer and inadequate nutrient transport. Photochemical bonding promotes graft integration by activating a photosensitizer at the interface via a light source and avoids negative effects associated with other bonding techniques. We hypothesized that the bond strength depends on photosensitizer type and concentration in addition to light exposure. Photochemical bonding was evaluated using methylene blue (MB), a cationic phenothiazine photosensitizer, and two phthalocyanine photosensitizers, Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and aluminum phthalocyanine chloride (AlPc). Exposure was altered by varying irradiation time for a fixed irradiance or by varying irradiance with a fixed irradiation time. MB was ineffective at producing bonding at the range of concentrations tested while CASPc produced a peak twofold bond strength increase over controls. AlPc produced substantial bonding at all concentrations with a peak 3.9-fold bond strength increase over controls. Parametric tests revealed that bond strength depended primarily on the total energy delivered to the bonding site rather than the rate of light delivery or light irradiance. Bond strength persisted for 1 week of in-vitro culture, which warrants further exploration for clinical applications. These studies indicate that photochemical bonding is a viable strategy for enhancing articular cartilage graft integration. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2406-2415, 2018.

Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties.

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.

Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels.

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.

Quantitative tracking of passage and 3D culture effects on chondrocyte and fibrochondrocyte gene expression.

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.

A constitutive model for protein-based materials.

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.

Indentation testing of human articular cartilage: effects of probe tip geometry and indentation depth on intra-tissue strain.

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.