ACL transection results in a posterior shift and increased velocity of contact on the medial tibial plateau.

Our objective was to quantify the effect of ACL transection on dynamic knee joint contact force distributions during simulated gait. Given the prevalence of medial compartment osteoarthritis in un-reconstructed ACL ruptured knees, we hypothesized that changes in contact mechanics after ACL transection would be most prevalent in the medial compartment. Twelve human cadaveric knees were tested using a dynamic knee gait simulator which was programmed to mimic a clinical Lachman exam and gait. An electronic pressure sensor was placed on the medial and lateral tibial plateaus under the menisci to quantify dynamic contact forces before and after ACL transection. Tibial translations and rotations, medial and lateral plateau peak contact stress, and position and velocity of the Weighted Center of Contact (WCoC) were computed. After ACL transection, the tibia translated more anteriorly in the Lachman examination and at heel strike during gait. Changes in contact mechanics across the medial tibial plateau during simulated gait were: an increase in the velocity of WCoC and a posterior shift in the WCoC, both of which occurred at heel strike; increased peak contact forces in the posterior-peripheral quadrant of the tibial plateau at 45% of the gait cycle; and an additional posterior shift in WCoC from 25 to 55% of the gait cycle. The only change in contact mechanics in the lateral plateau was a decrease in WCoC velocity in late stance. This data is suggested to further the study of biomechanical pathways (biomechanical biomarkers) in the relationship between altered knee contact mechanics and chondrocyte metabolic responses after ACL transection.

Effect of Articular Surface Compression on Cartilage Extracellular Matrix Deformation.

Early stage osteoarthritis is characterized by disruption of the superficial zone (SZ) of articular cartilage, including collagen damage and proteoglycan loss, resulting in "mechanical softening" of the extracellular matrix (ECM). The role of the SZ in controlling fluid exudation and imbibition during loading and unloading, respectively, was studied using confined creep compression tests. Bovine osteochondral (OC) plugs were subjected to either a static (88 kPa) or cyclic (0-125 kPa at 1 Hz) compressive stress for five minutes, and the cartilage deformation and recovery were measured during tissue loading and unloading, respectively. During unloading, the articular surface of the cartilage was either loaded with a small 1% tare load (∼1 kPa) applied through a porous load platen (covered), or completely unloaded (uncovered). Then the SZ (∼10%) of the cartilage was removed and the creep tests were repeated. Randomized tests were performed on each OC specimen to assess variability within and between plugs. Static creep strain was always greater than cyclic creep strain except at the beginning of loading (10-20 cycles). Uncovering the articular surface after creep deformation resulted in faster thickness recovery compared to the covered recovery. Removal of the SZ resulted in increased static and cyclic creep strains, as well as an increase in the cyclic peak-to-peak strain envelope. Our results indicate that an intact SZ is essential for normal cartilage mechanical function during joint motion by controlling fluid exudation and imbibition, and concomitantly ECM deformation and recovery, when loaded and unloaded, respectively.

Squeeze-film properties of synovial fluid and hyaluronate-based viscosupplements.

The rheological properties of synovial fluid and hyaluronate (HA) solutions have been studied using a variety of viscometers and rheometers. These devices measure the viscosity of the fluid's resistance to shearing forces, which is useful when studying the lubrication and frictional properties of movable joints. Less commonly used is a squeeze-film fluid test, mechanistically similar to when two joint surfaces squeeze interposed fluid. In our study, we used squeeze-film tests to determine the rheological response of normal bovine synovial fluid and 10 mg/ml HA-based solutions, Hyalgan/Hyalovet, commercially available 500-700 kDa HA viscosupplements, and a 1000 kDa sodium hyaluronate (NaHy) solution. We found similar rheological responses (fluid thickness, viscosity, viscosity-pressure relationship) for all three fluids, though synovial fluid's minimum squeeze-film thickness was slightly thicker. Squeeze-film loading speed did not affect these results. Different HA concentrations and molecular weights also did not have a significant or consistent effect on the squeeze-film responses. An unexpected result for the HA-solutions was a linear increase in minimum fluid-film thickness with increasing initial fluid-film thickness. This result was attributed to faster gelling of thicker HA-solutions, which formed at a lower squeeze-film strain and higher squeeze-film strain rate compared to thinner layers. Also included is a review of the literature on viscosity measurements of synovial fluid and HA solutions.

Collagen peptide simulated bending after applied axial deformation.

Structural proteins in the extracellular matrix are subjected to a range of mechanical loading conditions, including varied directions of force application. Molecular modeling suggests that these mechanical forces directly affect collagen's conformation and the subsequent mechanical response at the molecular level is complex. For example, tensile forces in the axial direction result in collagen triple helix elongation and unwinding, while perpendicular forces can cause local triple helix disruption. However, the effects of more complicated mechanical loading, such as the effect of axial pretension on collagen bending and triple helix microunfolding are unknown. In this study we used steered molecular dynamics to first model a collagen peptide under axial tension and then apply a perpendicular bending force. Axial tension causes molecular elongation and increased the subsequent perpendicular bending stiffness, but surprisingly did not increase the predicted collagen triple helix microunfolding threshold. We believe these results elucidate a key potential mechanism by which microscale mechanical loads translate from cellular and micro scales down to the nano and atomistic. Further, these data predict that cryptic force-induced collagen triple helix unwinding is axial-deformation independent, supporting the possibility that cell traction forces could be a key molecular mechanism to alter the cellular matrix microenvironment to facilitate collagen enzymatic degradation and subsequent cellular migration, such as in tumor extravasation.

Ultraviolet light (365 nm) transmission properties of articular cartilage as a function of depth, extracellular matrix, and swelling.

Current tissue engineering approaches for treatment of injured or diseased articular cartilage use ultraviolet light (UV) for in situ photopolymerization of biomaterials to fill chondral and osteochondral defects as well as resurfacing, stiffening and bonding the extracellular matrix and tissue interfaces. The most commonly used UV light wavelength is UVA 365 nm, the least cytotoxic and deepest penetrating. However, little information is available on the transmission of UVA 365 nm light through the cartilage matrix. In the present study, 365 nm UV light transmission was measured as a function of depth through 100 µm thick slices of healthy articular cartilage removed from mature bovine knees. Transmission properties were measured in normal (Native) cartilage and after swelling equilibration in phosphate-buffered saline (Swollen). Single-factor and multiple linear regression analyses were performed to determine depth-dependencies between the effective attenuation coefficients and proteoglycan, collagen and water contents. For both cartilages, a significant depth-dependency was found for the effective attenuation coefficients, being highest at the articular surface (superficial zone) and decreasing with depth. The effective attenuation coefficients for full-thickness cartilages were approximately a third lower than the total attenuation coefficients calculated from the individual slices. Analysis of absorption and scattering effects due to the ECM and chondrocytes found that UV light scatter coefficients were ∼10 times greater than absorption coefficients. The greater transmittance of UV light through the thicker cartilage was attributed to the collagen within the ECM causing significant backscatter forward reflectance.

Effect of interface mechanical discontinuities on scaffold-cartilage integration.

A consistent lack of lateral integration between scaffolds and adjacent articular cartilage has been exhibited in vitro and in vivo. Given the mismatch in mechanical properties between scaffolds and articular cartilage, the mechanical discontinuity that occurs at the interface has been implicated as a key factor, but remains inadequately studied. Our objective was to investigate how the mechanical environment within a mechanically loaded scaffold-cartilage construct might affect integration. We hypothesized that the magnitude of the mechanical discontinuity at the scaffold-cartilage interface would be related to decreased integration. To test this hypothesis, chondrocyte seeded scaffolds were embedded into cartilage explants, pre-cultured for 14 days, and then mechanically loaded for 28 days at either 1N or 6N of applied load. Constructs were kept either peripherally confined or unconfined throughout the duration of the experiment. Stress, strain, fluid flow, and relative displacements at the cartilage-scaffold interface and within the scaffold were quantified using biphasic, inhomogeneous finite element models (bFEMs). The bFEMs indicated compressive and shear stress discontinuities occurred at the scaffold-cartilage interface for the confined and unconfined groups. The mechanical strength of the scaffold-cartilage interface and scaffold GAG content were higher in the radially confined 1N loaded groups. Multivariate regression analyses identified the strength of the interface prior to the commencement of loading and fluid flow within the scaffold as the main factors associated with scaffold-cartilage integration. Our study suggests a minimum level of scaffold-cartilage integration is needed prior to the commencement of loading, although the exact threshold has yet to be identified. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Light Absorptive Properties of Articular Cartilage, ECM Molecules, Synovial Fluid, and Photoinitiators as Potential Barriers to Light-Initiated Polymer Scaffolding Procedures.

OBJECTIVE: Many in vivo procedures to repair chondral defects use ultraviolet (UV)-photoinitiated in situ polymerization within the cartilage matrix. Chemical species that absorb UV light might reduce the effectiveness of these procedures by acting as light absorption barriers. This study evaluated whether any of the individual native biochemical components in cartilage and synovial fluid interfered with the absorption of light by common scaffolding photosensitizers. MATERIALS: UV-visible spectroscopy was performed on each major component of cartilage in solution, on bovine synovial fluid, and on four photosensitizers, riboflavin, Irgacure 2959, quinine, and riboflavin-5'-phosphate. Molar extinction and absorption coefficients were calculated at wavelengths of maximum absorbance and 365 nm. Intact articular cartilage was also examined. RESULTS: The individual major biochemical components of cartilage, Irgacure 2959, and quinine did not exhibit a significant absorption at 365 nm. Riboflavin and riboflavin-5'-phosphate were more effectual light absorbers at 365 nm, compared with the individual native species. Intact cartilage absorbed a significantly greater amount of UV light in comparison with the native species. CONCLUSION: Our results indicate that none of the individual native species in cartilage will interfere with the absorption of UV light at 365 nm by these commonly used photoinitiators. Intact cartilage slices exhibited significant light absorption at 365 nm, while also having distinct absorbance peaks at wavelengths less than 300 nm. Determining the UV absorptive properties of the biomolecules native to articular cartilage and synovial fluid will aid in optimizing scaffolding procedures to ensure sufficient scaffold polymerization at a minimum UV intensity.

Articular cartilage response to a sliding load using two different-sized spherical indenters1.

BACKGROUND: Cartilage surface contact geometry influences the deformational behavior and stress distribution throughout the extracellular matrix (ECM) under load. OBJECTIVE: To test the correlation between the mechanical and cellular response of articular cartilage when loaded with two different-sized spherical indenters under dynamic reciprocating sliding motion. METHODS: Articular cartilage explants were subjected to a reciprocating sliding load using a 17.6 mm or 30.2 mm spherical ball for 2000 cycles at 10 mm/s and 4 kg axial load. Deformation of the cartilage was recorded and contact parameters were calculated according to Hertzian theory. After mechanical loading cartilage samples were collected and analyzed for ECM collagen damage, gene regulation and proteoglycan (PG) loss. RESULTS: Significantly higher ECM deformation and strain and lower dynamic effective modulus were found for explants loaded with the smaller diameter indenter whereas contact radius and stress remained unaffected. Also, the 17.6 mm indenter increased PG loss and significantly upregulated genes for ECM proteins and enzymes as compared to the 30.2 mm indenter. CONCLUSION: Sliding loads that increase ECM deformation/strain were found to induce enzyme-mediated catabolic processes in articular cartilage explants. These observations provide further understanding of how changes in cartilage contact mechanics under dynamic conditions can affect the cellular response.

Influence of the pericellular and extracellular matrix structural properties on chondrocyte mechanics.

Understanding the mechanical factors that drive the biological responses of chondrocytes is central to our interpretation of the cascade of events that lead to osteoarthritic changes in articular cartilage. Chondrocyte mechanics is complicated by changes in tissue properties that can occur as osteoarthritis (OA) progresses and by the interaction between macro-scale, tissue level, properties, and micro-scale pericellular matrix (PCM) and local extracellular matrix (ECM) properties, both of which cannot be easily studied using in vitro systems. Our objective was to study the influence of macro- and micro-scale OA-associated structural changes on chondrocyte strains. We developed a multi-scale finite element model of articular cartilage subjected to unconfined loading, for the following three conditions: (i) normal articular cartilage, (ii) OA cartilage (where macro and micro-scale changes in collagen content, matrix modulus, and permeability were modeled), and (iii) early-stage OA cartilage (where only micro-scale changes in matrix modulus were modeled). In the macro-scale model, we found that a depth-dependent strain field was induced in both healthy and OA cartilage and that the middle and superficial zones of OA cartilage had increased tensile and compressive strains. At the micro-scale, chondrocyte shear strains were sensitive to PCM and local ECM properties. In the early-OA model, micro-scale spatial softening of PCM and ECM resulted in a substantial increase (30%) of chondrocyte shear strain, even with no structural changes in macro-scale tissue properties. Our study provides evidence that micromechanical changes at the cellular level may affect chondrocyte activities before macro-scale degradations at the tissue level become apparent. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:721-729, 2018.

Photocrosslinked tyramine-substituted hyaluronate hydrogels with tunable mechanical properties improve immediate tissue-hydrogel interfacial strength in articular cartilage.

Articular cartilage lacks the ability to self-repair and a permanent solution for cartilage repair remains elusive. Hydrogel implantation is a promising technique for cartilage repair; however for the technique to be successful hydrogels must interface with the surrounding tissue. The objective of this study was to investigate the tunability of mechanical properties in a hydrogel system using a phenol-substituted polymer, tyramine-substituted hyaluronate (TA-HA), and to determine if the hydrogels could form an interface with cartilage. We hypothesized that tyramine moieties on hyaluronate could crosslink to aromatic amino acids in the cartilage extracellular matrix. Ultraviolet (UV) light and a riboflavin photosensitizer were used to create a hydrogel by tyramine self-crosslinking. The gel mechanical properties were tuned by varying riboflavin concentration, TA-HA concentration, and UV exposure time. Hydrogels formed with a minimum of 2.5 min of UV exposure. The compressive modulus varied from 5 to 16 kPa. Fluorescence spectroscopy analysis found differences in dityramine content. Cyanine-3 labelled tyramide reactivity at the surface of cartilage was dependent on the presence of riboflavin and UV exposure time. Hydrogels fabricated within articular cartilage defects had increasing peak interfacial shear stress at the cartilage-hydrogel interface with increasing UV exposure time, reaching a maximum shear stress 3.5× greater than a press-fit control. Our results found that phenol-substituted polymer/riboflavin systems can be used to fabricate hydrogels with tunable mechanical properties and can interface with the surface tissue, such as articular cartilage.

Use of novel chitosan hydrogels for chemical tissue bonding of autologous chondral transplants.

The objective of this study was to evaluate the effect of chemical tissue bonding (CTB) on adhesion strength, fluid permeability, and cell viability across a cartilaginous graft-host interface in an in vitro autologous chondral transplant (ACT) model. Chitosan-based cross-linkers; Chitosan-Rose Bengal [Chi-RB (Ch-ABC)], Chitosan-Genipin [Chi-GP (Ch-ABC)], and Chitosan-Rose Bengal-Genipin [Chi-RB-GP (Ch-ABC)] were applied to bovine immature cartilage explants after pre-treatment with surface degrading enzyme, Chondroitinase-ABC (Ch-ABC). Adhesion strength, fluid permeability and cell viability were assessed via mechanical push-out shear testing, fluid transport and live/dead cell staining, respectively. All three chitosan-based cross-linkers significantly increased the adhesion strength at the graft-host interface, however, only a statistically significant decrease in fluid permeability was noted in Chi-GP (Ch-ABC) specimen compared to untreated controls. Cell viability was maintained for 7 days of culture across all three treatment groups. These results show the potential clinical relevance of novel chitosan-based hydrogels in enhancing tissue integration and reducing synovial fluid penetration after ACT procedures in diarthoidal joints such as the knee and ankle. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1139-1146, 2016.

A Model to Study Articular Cartilage Mechanical and Biological Responses to Sliding Loads.

In physiological conditions, joint function involves continuously moving contact areas over the tissue surface. Such moving contacts play an important role for the durability of the tissue. It is known that in pathological joints these motion paths and contact mechanics change. Nevertheless, limited information exists on the impact of such physiological and pathophysiological dynamic loads on cartilage mechanics and its subsequent biological response. We designed and validated a mechanical device capable of applying simultaneous compression and sliding forces onto cartilage explants to simulate moving joint contact. Tests with varying axial loads (1-4 kg) and sliding speeds (1-20 mm/s) were performed on mature viable bovine femoral condyles to investigate cartilage mechanobiological responses. High loads and slow sliding speeds resulted in highest cartilage deformations. Contact stress and effective cartilage moduli increased with increasing load and increasing speed. In a pilot study, changes in gene expression of extracellular matrix proteins were correlated with strain, contact stress and dynamic effective modulus. This study describes a mechanical test system to study the cartilage response to reciprocating sliding motion and will be helpful in identifying mechanical and biological mechanisms leading to the initiation and development of cartilage degeneration.

Shape of chondrocytes within articular cartilage affects the solid but not the fluid microenvironment under unconfined compression.

Metabolic activity of the chondrocytes in articular cartilage is strongly related to their zone-specific shape and the composition and mechanical properties of their surrounding extracellular matrix (ECM). However the mechanisms by which cell shape influences the response of the ECM microenvironment to mechanical loading is yet to be elucidated. This relationship was studied using a biphasic multiscale finite element model of different shaped chondrocytes in the superficial and deep zones of the ECM during unconfined stress relaxation. For chondrocytes in the superficial zone, increasing the cell's initial aspect ratio (length/height) increased the deformation and solid stresses of the chondrocyte and pericellular matrix (PCM) during the loading phase; for chondrocytes in the deep zone the effect of the cell shape on the solid microenvironment was time and variable dependent. However, for superficial and deep zone chondrocytes the cell shape did not affect the fluid pressure and fluid shear stress. These results suggest that mechanotransduction of chondrocytes in articular cartilage may be regulated through the solid phase rather than the fluid phase, and that high stresses and deformations in the solid microenvironment in the superficial zone may be essential for the zone-specific biosynthetic activity of the chondrocyte. The biphasic multiscale computational analysis suggests that maintaining the cell shape is critical for regulating the microenvironment and metabolic activity of the chondrocyte in tissue engineering constructs. STATEMENT OF SIGNIFICANCE: We investigated the effect of chondrocyte shape on the cellular microenvironment using a biphasic multiscale finite element analysis. Our study showed that cell shapes affects the solid but not the fluid microenvironment of the chondrocyte, and that maintaining the cell shape is critical for regulating the microenvironment and metabolic activity of the chondrocyte in native cartilage and tissue engineering constructs. As far as we know, this is the first study on the mechanotransduction mechanisms by which cell shape influences the response of the microenvironment to mechanical loading. This study is important for understanding cell mechanobiology, not only for regulation of cell phenotype in tissue engineered constructs but, as important, for understanding changes in normal chondrocyte function after post-traumatic injury and in the initiation and progression of osteoarthritis.

Mechanical Loading of Cartilage Explants with Compression and Sliding Motion Modulates Gene Expression of Lubricin and Catabolic Enzymes.

OBJECTIVE: Translation of the contact zone in articulating joints is an important component of joint kinematics, yet rarely investigated in a biological context. This study was designed to investigate how sliding contact areas affect cartilage mechanobiology. We hypothesized that higher sliding speeds would lead to increased extracellular matrix mechanical stress and the expression of catabolic genes. DESIGN: A cylindrical Teflon indenter was used to apply 50 or 100 N normal forces at 10, 40, or 70 mm/s sliding speed. Mechanical parameters were correlated with gene expressions using a multiple linear regression model. RESULTS: In both loading groups there was no significant effect of sliding speed on any of the mechanical parameters (strain, stress, modulus, tangential force). However, an increase in vertical force (from 50 to 100 N) led to a significant increase in extracellular matrix strain and stress. For 100 N, significant correlations between gene expression and mechanical parameters were found for TIMP-3 (r(2) = 0.89), ADAMTS-5 (r(2) = 0.73), and lubricin (r(2) = 0.73). CONCLUSIONS: The sliding speeds applied do not have an effect on the mechanical response of the cartilage, this could be explained by a partial attainment of the "elastic limit" at and above a sliding speed of 10 mm/s. Nevertheless, we still found a relationship between sliding speed and gene expression when the tissue was loaded with 100 N normal force. Thus despite the absence of speed-dependent mechanical changes (strain, stress, modulus, tangential force), the sliding speed had an influence on gene expression.

Resurfacing damaged articular cartilage to restore compressive properties.

Surface damage to articular cartilage is recognized as the initial underlying process causing the loss of mechanical function in early-stage osteoarthritis. In this study, we developed structure-modifying treatments to potentially prevent, stabilize or reverse the loss in mechanical function. Various polymers (chondroitin sulfate, carboxymethylcellulose, sodium hyaluronate) and photoinitiators (riboflavin, irgacure 2959) were applied to the surface of collagenase-degraded cartilage and crosslinked in situ using UV light irradiation. While matrix permeability and deformation significantly increased following collagenase-induced degradation of the superficial zone, resurfacing using tyramine-substituted sodium hyaluronate and riboflavin decreased both values to a level comparable to that of intact cartilage. Repetitive loading of resurfaced cartilage showed minimal variation in the mechanical response over a 7 day period. Cartilage resurfaced using a low concentration of riboflavin had viable cells in all zones while a higher concentration resulted in a thin layer of cell death in the uppermost superficial zone. Our approach to repair surface damage initiates a new therapeutic advance in the treatment of injured articular cartilage with potential benefits that include enhanced mechanical properties, reduced susceptibility to enzymatic degradation and reduced adhesion of macrophages.

A biphasic finite element study on the role of the articular cartilage superficial zone in confined compression.

The aim of this study was to investigate the role of the superficial zone on the mechanical behavior of articular cartilage. Confined compression of articular cartilage was modeled using a biphasic finite element analysis to calculate the one-dimensional deformation of the extracellular matrix (ECM) and movement of the interstitial fluid through the ECM and articular surface. The articular cartilage was modeled as an inhomogeneous, nonlinear hyperelastic biphasic material with depth and strain-dependent material properties. Two loading conditions were simulated, one where the superficial zone was loaded with a porous platen (normal test) and the other where the deep zone was loaded with the porous platen (upside down test). Compressing the intact articular cartilage with 0.2 MPa stress reduced the surface permeability by 88%. Removing the superficial zone increased the rate of change for all mechanical parameters and decreased the fluid support ratio of the tissue, resulting in increased tissue deformation. Apparent permeability linearly increased after superficial removal in the normal test, yet it did not change in the upside down test. Orientation of the specimen affected the time-dependent biomechanical behavior of the articular cartilage, but not equilibrium behavior. The two tests with different specimen orientations resulted in very different apparent permeabilities, suggesting that in an experimental study which quantifies material properties of an inhomogeneous material, the specimen orientation should be stated along with the permeability result. The current study provides new insights into the role of the superficial zone on mechanical behavior of the articular cartilage.

A biphasic multiscale study of the mechanical microenvironment of chondrocytes within articular cartilage under unconfined compression.

Computational analyses have been used to study the biomechanical microenvironment of the chondrocyte that cannot be assessed by in vitro experimental studies; yet all computational studies thus far have focused on the effect of zonal location (superficial, middle, and deep) on the mechanical microenvironment of chondrocytes. The aim of this paper was to study the effect of both zonal and radial locations on the biomechanical microenvironment of chondrocytes in inhomogeneous cartilage under unconfined stress relaxation. A biphasic multiscale approach was employed and nine chondrocytes in different locations were studied. Hyperelastic biphasic theory and depth-dependent aggregate modulus and permeability of articular cartilage were included in the models. It was found that both zonal and radial locations affected the biomechanical stresses and strains of the chondrocytes. Chondrocytes in the mid-radial location had increased volume during the early stage of the loading process. Maximum principal shear stress at the interface between the chondrocyte and the extracellular matrix (ECM) increased with depth, yet that at the ECM-pericellular matrix (PCM) interface had an inverse trend. Fluid pressure decreased with depth, while the fluid pressure difference between the top and bottom boundaries of the microscale model increased with depth. Regardless of location, fluid was exchanged between the chondrocyte, PCM, and ECM. These findings suggested that even under simple compressive loading conditions, the biomechanical microenvironment of the chondrocytes, PCM and ECM was spatially dependent. The current study provides new insight on chondrocyte biomechanics.

Glycation cross-linking induced mechanical-enzymatic cleavage of microscale tendon fibers.

Recent molecular modeling data using collagen peptides predicted that mechanical force transmitted through intermolecular cross-links resulted in collagen triple helix unwinding. These simulations further predicted that this unwinding, referred to as triple helical microunfolding, occurred at forces well below canonical collagen damage mechanisms. Based in large part on these data, we hypothesized that mechanical loading of glycation cross-linked tendon microfibers would result in accelerated collagenolytic enzyme damage. This hypothesis is in stark contrast to reports in literature that indicated that individually mechanical loading or cross-linking each retards enzymatic degradation of collagen substrates. Using our Collagen Enzyme Mechano-Kinetic Automated Testing (CEMKAT) System we mechanically loaded collagen-rich tendon microfibers that had been chemically cross-linked with sugar and tested for degrading enzyme susceptibility. Our results indicated that cross-linked fibers were >5 times more resistant to enzymatic degradation while unloaded but became highly susceptible to enzyme cleavage when they were stretched by an applied mechanical deformation.

Characterization of a macroporous polyvinyl alcohol scaffold for the repair of focal articular cartilage defects.

Focal cartilage defects reduce the ability of articular cartilage to resist mechanical loading and provide lubrication during joint motion. The limitations in current surgical treatments have motivated the use of biocompatible scaffolds as a future treatment option. Here we describe a second generation macroporous, polyvinyl alcohol (PVA) scaffold with independently tunable morphological and mechanical properties. The compressive moduli of the PVA scaffold increased with increasing polymer concentration and applied compressive strain, with values in the range for human articular cartilage (HA > 1000 kPa, EY > 500 kPa). Scaffolds also possessed strain-dependent permeability and Poisson's ratio. The interconnected macroporous network was found to facilitate chondrocyte seeding and proliferation through the scaffold over one week in culture. Overall, these promising characteristics demonstrate the potential of this macroporous scaffold for future studies in focal cartilage defect repair.

An in vitro model for the pathological degradation of articular cartilage in osteoarthritis.

The objective of this study was to develop an in vitro cartilage degradation model that emulates the damage seen in early-stage osteoarthritis. To this end, cartilage explants were collagenase-treated to induce enzymatic degradation of collagen fibers and proteoglycans at the articular surface. To assess changes in mechanical properties, intact and degraded cartilage explants were subjected to a series of confined compression creep tests. Changes in extracellular matrix structure and composition were determined using biochemical and histological approaches. Our results show that collagenase-induced degradation increased the amount of deformation experienced by the cartilage explants under compression. An increase in apparent permeability as well as a decrease in instantaneous and aggregate moduli was measured following collagenase treatment. Histological analysis of degraded explants revealed the presence of surface fibrillation, proteoglycan depletion in the superficial and intermediate zones and loss of the lamina splendens. Collagen cleavage was confirmed by the Col II-3/4Cshort antibody. Degraded specimens experienced a significant decrease in proteoglycan content but maintained total collagen content. Repetitive testing of degraded samples resulted in the gradual collapse of the articular surface and the compaction of the superficial zone. Taken together, our data demonstrates that enzymatic degradation with collagenase can be used to emulate changes seen in early-stage osteoarthritis. Further, our in vitro model provides information on cartilage mechanics and insights on how matrix changes can affect cartilage's functional properties. More importantly, our model can be applied to develop and test treatment options for tissue repair.