Fibril reinforced poroelastic model predicts specifically mechanical behavior of normal,proteoglycan depleted and collagen degraded articular cartilage

Degradation of collagen network and proteoglycan (PG) macromolecules are signs of articular cartilage degeneration. These changes impair cartilage mechanical function. Effects of collagen degradation and PG depletion on the time-dependent mechanical behavior of cartilage are different. In this study, numerical analyses, which take the compression-tension nonlinearity of the tissue into account, were carried out using a fibril reinforced poroelastic finite element model. The study aimed at improving our understanding of the stress-relaxation behavior of normal and degenerated cartilage in unconfined compression. PG and collagen degradations were simulated by decreasing the Young's modulus of the drained porous (nonfibrillar) matrix and the fibril network, respectively. Numerical analyses were compared to results from experimental tests with chondroitinase ABC (PG depletion) or collagenase (collagen degradation) digested samples. Fibril reinforced poroelastic model predicted the experimental behavior of cartilage after chondroitinase ABC digestion by a major decrease of the drained porous matrix modulus (-64+/-28%) and a minor decrease of the fibril network modulus (-11+/-9%). After collagenase digestion, in contrast, the numerical analyses predicted the experimental behavior of cartilage by a major decrease of the fibril network modulus (-69+/-5%) and a decrease of the drained porous matrix modulus (-44+/-18%). The reduction of the drained porous matrix modulus after collagenase digestion was consistent with the microscopically observed secondary PG loss from the tissue. The present results indicate that the fibril reinforced poroelastic model is able to predict specifically characteristic alterations in the stress-relaxation behavior of cartilage after enzymatic modifications of the tissue. We conclude that the compression-tension nonlinearity of the tissue is needed to capture realistically the mechanical behavior of normal and degenerated articular cartilage.     

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1^+/− mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.     

Ultrasonic characterization of articular cartilage

Osteoarthrosis is the most important joint disease that threatens health of the musculoskeletal system of elderly people. Today, there is a need for sensitive, quantitative diagnostic methods for successful and early diagnosis of the disorder. In the present study, we aimed at evaluating the applicability of ultrasound for quantitative assessment of cartilage structure and properties. Bovine articular cartilage was investigated both in vitro and in situ using high frequency ultrasound. Cartilage samples were also tested mechanically in vitro to reveal relationships between acoustic and mechanical parameters of the tissue. The collagen organization and proteoglycan content of cartilage samples were mapped, using quantitative polarized light microscopy and digital densitometry, respectively, to reveal their effect on the acoustic properties of tissue. The high frequency pulse-echo ultrasound (20-30 MHz) technique proved to be sensitive in detecting the degeneration of the superficial collagen-rich cartilage zone. In addition, ultrasound was found to be a potential tool for measuring cartilage thickness. When the results from biomechanical indentation measurements and ultrasound measurements of normal and enzymatically degraded articular cartilage were combined, collagen or proteoglycan degradation in the tissue could be sensitively and specifically differentiated from each other. To conclude, high frequency ultrasound is a useful tool for evaluation of the quality of superficial articular cartilage as well as for the measurement of cartilage thickness. Therefore, ultrasound appears to be a valuable supplement to the mechanical measurements of articular cartilage stiffness.     

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction

The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.

Methods

SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.

Results

Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.

Conclusion

Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

Articular cartilage in the knee joint of the African elephant, Loxodonta africana, Blumenbach 1797

Knee joints of one adult and three juvenile African elephants were dissected. The specific features of the articular cartilage with particular reference to matrix components were studied by light and electron microscopy and immunohistochemistry. The elephant knee joint cartilage contains an unusually low concentration of proteoglycans resulting in rather eosinophilic staining properties of the matrix. The very thick collagen fibers of the cartilage possibly represent collagen I. Except for the different thickness of cartilage at the weight-bearing surfaces of femur (approximately 6.7 mm) and tibia (approximately 11.2 mm) in juvenile elephants, light and electron microscopy did not reveal distinct topographical differences in cartilage structure, perhaps because of the high congruency of the articulating surfaces and resulting uniform load distribution in the knee. The number of cell profiles per section area of both femoral (approximately 950 cell profiles/mm(2)) and tibial cartilage (approximately 898 cell profiles/mm(2)) was low, indicating excessive matrix production by the chondrocytes during cartilage development. These unique properties could be a result of the enormous compressive load resting on the elephant knee. Maintenance of the equilibrium between biological function and resistance to compression seems to be crucial in the elephant knee joint cartilage. Any disturbance that interferes with this equilibrium appears to lead to arthrotic alterations, as particularly seen in captive elephants.     

Characterization of articular cartilage by combining microscopic analysis with a fibril-reinforced finite-element model

Load-bearing characteristics of articular cartilage are impaired during tissue degeneration. Quantitative microscopy enables in vitro investigation of cartilage structure but determination of tissue functional properties necessitates experimental mechanical testing. The fibril-reinforced poroviscoelastic (FRPVE) model has been used successfully for estimation of cartilage mechanical properties. The model includes realistic collagen network architecture, as shown by microscopic imaging techniques. The aim of the present study was to investigate the relationships between the cartilage proteoglycan (PG) and collagen content as assessed by quantitative microscopic findings, and model-based mechanical parameters of the tissue. Site-specific variation of the collagen network moduli, PG matrix modulus and permeability was analyzed. Cylindrical cartilage samples (n=22) were harvested from various sites of the bovine knee and shoulder joints. Collagen orientation, as quantitated by polarized light microscopy, was incorporated into the finite-element model. Stepwise stress-relaxation experiments in unconfined compression were conducted for the samples, and sample-specific models were fitted to the experimental data in order to determine values of the model parameters. For comparison, Fourier transform infrared imaging and digital densitometry were used for the determination of collagen and PG content in the same samples, respectively. The initial and strain-dependent fibril network moduli as well as the initial permeability correlated significantly with the tissue collagen content. The equilibrium Young's modulus of the nonfibrillar matrix and the strain dependency of permeability were significantly associated with the tissue PG content. The present study demonstrates that modern quantitative microscopic methods in combination with the FRPVE model are feasible methods to characterize the structure-function relationships of articular cartilage.     

A novel model of a biomechanically induced osteoarthritis-like cartilage for pharmacological in vitro studies

Excessive pressure or overload induces and aggravates osteoarthritic changes in articular cartilage, but the underlying biomechanical forces are largely ignored in existing pharmacological in vitro models that are used to investigate drugs against osteoarthritis (OA). Here, we introduce a novel in vitro model to perform pathophysiological and pharmacological investigations, in which cartilage explants are subjected to intermittent cyclic pressure, and characterize its ability to mimic OA-like tissue reactivity. Mechanical loading time-dependently increased the biosynthesis, content and retention of fibronectin (Fn), whereas collagen metabolism remained unchanged. This protocol upregulated the production and release of proteoglycans (PGs). The release of PGs from explants was significantly inhibited by a matrix metalloproteinase (MMP) inhibitor, suggesting the involvement of such proteinases in the destruction of the model tissue, similar to what is observed in human OA cartilage. In conclusion, the metabolic alterations in our new biomechanical in vitro model are similar to those of early human OA cartilage, and our pharmacological prevalidation with an MMP-inhibitor supports its value for further in vitro drug studies.     

Alterations in structure and properties of collagen network of osteoarthritic and repaired cartilage modify knee joint stresses

Organization of the collagen network is known to be different in healthy, osteoarthritic and repaired cartilage. The aim of the study was to investigate how the structure and properties of collagen network of cartilage modulate stresses in a knee joint with osteoarthritis or cartilage repair. Magnetic resonance imaging (MRI) at 1.5 T was conducted for a knee joint of a male subject. Articular cartilage and menisci in the knee joint were segmented, and a finite element mesh was constructed based on the two-dimensional section in sagittal projection. Then, the knee joint stresses were simulated under impact loads by implementing the structure and properties of healthy, osteoarthritic and repaired cartilage in the models. During the progression of osteoarthritis, characterized especially by the progressive increase in the collagen fibrillation from the superficial to the deeper layers, the stresses were reduced in the superficial zone of cartilage, while they were increased in and under menisci. Increased fibril network stiffness of repair tissue with randomly organized collagen fibril network reduced the peak stresses in the adjacent tissue and strains at the repair–adjacent cartilage interface. High collagen fibril strains were indicative of stress concentration areas in osteoarthritic and repaired cartilage. The collagen network orientation and stiffness controlled the stress distributions in healthy, osteoarthritic and repaired cartilage. The evaluation of articular cartilage function using clinical MRI and biomechanical modeling could enable noninvasive estimation of osteoarthritis progression and monitoring of cartilage repair. This study presents a step toward those goals.     

Electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage

This study presents direct experimental evidence for assessing the electrostatic and non-electrostatic contributions of proteoglycans to the compressive equilibrium modulus of bovine articular cartilage. Immature and mature bovine cartilage samples were tested in unconfined compression and their depth-dependent equilibrium compressive modulus was determined using strain measurements with digital image correlation analysis. The electrostatic contribution was assessed by testing samples in isotonic and hypertonic saline; the combined contribution was assessed by testing untreated and proteoglycan-depleted samples.Though it is well recognized that proteoglycans contribute significantly to the compressive stiffness of cartilage, results demonstrate that the combined electrostatic and non-electrostatic contributions may add up to more than 98% of the modulus, a magnitude not previously appreciated. Of this contribution, about two thirds arises from electrostatic effects. The compressive modulus of the proteoglycan-depleted cartilage matrix may be as low as 3 kPa, representing less than 2% of the normal tissue modulus; experimental evidence also confirms that the collagen matrix in digested cartilage may buckle under compressive strains, resulting in crimping patterns. Thus, it is reasonable to model the collagen as a fibrillar matrix that can sustain only tension. This study also demonstrates that residual stresses in cartilage do not arise exclusively from proteoglycans, since cartilage remains curled relative to its in situ geometry even after proteoglycan depletion. These increased insights on the structure–function relationships of cartilage can lead to improved constitutive models and a better understanding of the response of cartilage to physiological loading conditions.     

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis

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.     

A new method for investigating osteoarthritis using Fast Field-Cycling nuclear magnetic resonance

Osteoarthritis in synovial joints remains a major cause of long-term disability worldwide, with symptoms produced by the progressive deterioration of the articular cartilage. The earliest cartilage changes are thought to be alteration in its main protein components, namely proteoglycan and collagen. Loss of proteoglycans bound in the collagen matrix which maintain hydration and stiffness of the structure is followed by collagen degradation and loss. The development of new treatments for early osteoarthritis is limited by the lack of accurate biomarkers to assess the loss of proteoglycan. One potential biomarker is magnetic resonance imaging (MRI). We present the results of a novel MRI methodology, Fast Field-Cycling (FFC), to assess changes in critical proteins by demonstrating clear quantifiable differences in signal from normal and osteoarthritic human cartilage for in vitro measurements. We further tested proteoglycan extracted cartilage and the key components individually. Three clear signals were identified, two of which are related predominantly to the collagen component of cartilage and the third, a unique very short-lived signal, is directly related to proteoglycan content; we have not seen this in any other tissue type. In addition, we present the first volunteer human scan from our whole-body FFC scanner where articular cartilage measurements are in keeping with those we have shown in tissue samples. This new clinical imaging modality offers the prospect of non-invasive monitoring of human cartilage in vivo and hence the assessment of potential treatments for osteoarthritis. Keywords: Fast Field-Cycling NMR; human hyaline cartilage; Osteoarthritis; T1 dispersion; quadrupolar peaks; protein interactions     

In Vivo Evaluation of a Novel Oriented Scaffold-BMSC Construct for Enhancing Full-Thickness Articular Cartilage Repair in a Rabbit Model

Tissue engineering (TE) has been proven usefulness in cartilage defect repair. For effective cartilage repair, the structural orientation of the cartilage scaffold should mimic that of native articular cartilage, as this orientation is closely linked to cartilage mechanical functions. Using thermal-induced phase separation (TIPS) technology, we have fabricated an oriented cartilage extracellular matrix (ECM)-derived scaffold with a Young''s modulus value 3 times higher than that of a random scaffold. In this study, we test the effectiveness of bone mesenchymal stem cell (BMSC)-scaffold constructs (cell-oriented and random) in repairing full-thickness articular cartilage defects in rabbits. While histological and immunohistochemical analyses revealed efficient cartilage regeneration and cartilaginous matrix secretion at 6 and 12 weeks after transplantation in both groups, the biochemical properties (levels of DNA, GAG, and collagen) and biomechanical values in the oriented scaffold group were higher than that in random group at early time points after implantation. While these differences were not evident at 24 weeks, the biochemical and biomechanical properties of the regenerated cartilage in the oriented scaffold-BMSC construct group were similar to that of native cartilage. These results demonstrate that an oriented scaffold, in combination with differentiated BMSCs can successfully repair full-thickness articular cartilage defects in rabbits, and produce cartilage enhanced biomechanical properties.     

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.     

The effects of selective matrix degradation on the short-term compressive properties of adult human articular cartilage.

The effects of proteoglycan and collagen digestion on the transient response of human articular cartilage when tested in unconfined compression were determined. Small cylindrical specimens of cartilage, isolated from the femoral head of the hip joint and from the femoral condyles of the knee joint, were subjected to a suddenly applied compressive load using a test apparatus designed to yield a transient oscillatory response. From this response values of the elastic stiffness and the viscous damping coefficient were determined. Cathepsin D and cathepsin B1 were used to digest the proteoglycan in some specimens, while in other specimens leukocyte elastase was used to attack the non-helical terminal regions of the Type II tropocollagen molecules and possibly the Type IX collagen molecule and thereby disturb the integrity of the collagen mesh. The results showed that proteoglycan digestion alone reduced the viscous damping coefficient but it did not significantly alter the elastic stiffness as determined from the oscillatory response. In contrast, the action of elastase reduced both the damping coefficient and the elastic stiffness of the cartilage. The results demonstrated the role of proteoglycans in regulating fluid transport in cartilage and hence controlling the time-dependent viscous properties. The elastic stiffness was shown to be dependent on the integrity of the collagen fibre network and not on the proteoglycans.     

Tissue engineering strategies for cartilage generation--micromass and three dimensional cultures using human chondrocytes and a continuous cell line

Utilizing ATDC5 murine chondrogenic cells and human articular chondrocytes, this study sought to develop facile, reproducible three-dimensional models of cartilage generation with the application of tissue engineering strategies, involving biodegradable poly(glycolic acid) scaffolds and rotating wall bioreactors, and micromass pellet cultures. Chondrogenic differentiation, assessed by histology, immunohistochemistry, and gene expression analysis, in ATDC5 and articular chondrocyte pellets was evident by the presence of distinct chondrocytes, expressing Sox-9, aggrecan, and type II collagen, in lacunae embedded in a cartilaginous matrix of type II collagen and proteoglycans. Tissue engineered explants of ATDC5 cells were reminiscent of cartilaginous structures composed of numerous chondrocytes, staining for typical chondrocytic proteins, in lacunae embedded in a matrix of type II collagen and proteoglycans. In comparison, articular chondrocyte explants exhibited areas of Sox-9, aggrecan, and type II collagen-expressing cells growing on fleece, and discrete islands of chondrocytic cells embedded in a cartilaginous matrix.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.     

A biomechanical role for perlecan in the pericellular matrix of articular cartilage

Chondrocytes are surrounded by a narrow pericellular matrix (PCM) that is biochemically, structurally, and biomechanically distinct from the bulk extracellular matrix (ECM) of articular cartilage. While the PCM is often defined by the presence of type VI collagen, other macromolecules such as perlecan, a heparan sulfate (HS) proteoglycan, are also exclusively localized to the PCM in normal cartilage and likely contribute to PCM structural integrity and biomechanical properties. Though perlecan is essential for normal cartilage development, its exact role in the PCM is unknown. The objective of this study was to determine the biomechanical role of perlecan in the articular cartilage PCM in situ and its potential as a defining factor of the PCM. To this end, atomic force microscopy (AFM) stiffness mapping was combined with dual immunofluorescence labeling of cryosectioned porcine cartilage samples for type VI collagen and perlecan. While there was no difference in overall PCM mechanical properties between type VI collagen- and perlecan-based definitions of the PCM, within the PCM, interior regions containing both type VI collagen and perlecan exhibited lower elastic moduli than more peripheral regions rich in type VI collagen alone. Enzymatic removal of HS chains from perlecan with heparinase III increased PCM elastic moduli both overall and locally in interior regions rich in both perlecan and type VI collagen. Heparinase III digestion had no effect on ECM elastic moduli. Our findings provide new evidence for perlecan as a defining factor in both the biochemical and biomechanical properties of the PCM.     

The deformation behavior and viscoelastic properties of chondrocytes in articular cartilage

Chondrocytes in articular cartilage utilize mechanical signals in conjunction with other environmental factors to regulate their metabolic activity. However, the sequence of biomechanical and biochemical events involved in the process of mechanical signal transduction has not been fully deciphered. A fundamental step in determining the role of various factors in regulating chondrocyte activity is to characterize accurately the biophysical environment within the tissue under physiological conditions of mechanical loading. Microscopic imaging studies have revealed that chondrocytes as well as their nuclei undergo shape and volume changes in a coordinated manner with deformation of the tissue matrix. Through micromechanical experiments, it has been shown that the chondrocyte behaves as a viscoelastic solid material with a mechanical stiffness that is several orders of magnitude lower than that of the cartilage extracellular matrix. These properties seem to be due to the structure of the chondrocyte cytoskeleton, and in part, the viscoelastic properties of the cell nucleus. The mechanical properties of the pericellular matrix that immediately surrounds the chondrocyte significantly differ from those of the chondrocyte and the extracellular matrix, suggesting that the pericellular matrix plays an important role in defining the mechanical environment of the chondrocyte. These experimentally measured values for chondrocyte and cartilage mechanical properties have been used in combination with theoretical constitutive modeling of the chondrocyte within articular cartilage to predict the non-uniform and time-varying stress-strain and fluid flow environment of the cell. The ultimate goal of these studies has been to elucidate the sequence of biomechanical and biochemical events through which mechanical stress influences chondrocyte activity in both health and in disease.