The structure and function of the pericellular matrix of articular cartilage.

Chondrocytes in articular cartilage are surrounded by a narrow pericellular matrix (PCM) that is both biochemically and biomechanically distinct from the extracellular matrix (ECM) of the tissue. While the PCM was first observed nearly a century ago, its role is still under investigation. In support of early hypotheses regarding its function, increasing evidence indicates that the PCM serves as a transducer of biochemical and biomechanical signals to the chondrocyte. Work over the past two decades has established that the PCM in adult tissue is defined biochemically by several molecular components, including type VI collagen and perlecan. On the other hand, the biomechanical properties of this structure have only recently been measured. Techniques such as micropipette aspiration, in situ imaging, computational modeling, and atomic force microscopy have determined that the PCM exhibits distinct mechanical properties as compared to the ECM, and that these properties are influenced by specific PCM components as well as disease state. Importantly, the unique relationships among the mechanical properties of the chondrocyte, PCM, and ECM in different zones of cartilage suggest that this region significantly influences the stress-strain environment of the chondrocyte. In this review, we discuss recent advances in the measurement of PCM mechanical properties and structure that further increase our understanding of PCM function. Taken together, these studies suggest that the PCM plays a critical role in controlling the mechanical environment and mechanobiology of cells in cartilage and other cartilaginous tissues, such as the meniscus or intervertebral disc.

High resistance of the mechanical properties of the chondrocyte pericellular matrix to proteoglycan digestion by chondroitinase, aggrecanase, or hyaluronidase.

In articular cartilage, the extracellular matrix (ECM) and chondrocyte-associated pericellular matrix (PCM) are characterized by a high concentration of proteoglycans (PGs) and their associated glycosaminoglycans (GAGs). These molecules serve important biochemical, structural, and biomechanical roles in the tissue and differences in their regional distributions suggest that different GAG/PG species contribute to the specific biomechanical properties of the ECM and PCM. The objective of this study was to investigate region-specific contributions of aggrecan, chondroitin and dermatan sulfate, and hyaluronan to the micromechanical properties of articular cartilage PCM and ECM in situ. Cryosections of porcine cartilage underwent digestion with ADAMTS-4, chondroitinase ABC, bacterial hyaluronidase or human leukocyte elastase. Guided by immunofluorescence for type VI collagen, AFM stiffness mapping was used to evaluate the elastic properties of matched PCM and ECM regions in paired control and digested cartilage sections. These methods were used to test the hypotheses that specific enzymatic digestion of GAGs or PGs would reduce both PCM and ECM elastic moduli. Elastase, which digests a number of PGs, some types of collagen, and non-collagenous proteins, was used as a positive control. ECM elastic moduli were significantly reduced by all enzyme treatments. However, PCM micromechanical properties were unaffected by enzymatic digestion of aggrecan, chondroitin/dermatan sulfate, and hyaluronan but were significantly reduced by 24% following elastase digestion. Our results provide new evidence for high resistance of PCM micromechanical properties to PG digestion and suggest a potential role for elastase in the degradation of the ECM and PCM.

Atomic force microscopy reveals regional variations in the micromechanical properties of the pericellular and extracellular matrices of the meniscus.

Regional variations in the composition and architecture of the extracellular matrix (ECM) and pericellular matrix (PCM) of the knee meniscus play important roles in determining the local mechanical environment of meniscus cells. In this study, atomic force microscopy was used to spatially map the mechanical properties of matched ECM and perlecan-labeled PCM sites within the outer, middle, and inner porcine medial meniscus, and to evaluate the properties of the proximal surface of each region. The elastic modulus of the PCM was significantly higher in the outer region (151.4 ± 38.2 kPa) than the inner region (27.5 ± 8.8 kPa), and ECM moduli were consistently higher than region-matched PCM sites in both the outer (320.8 ± 92.5 kPa) and inner (66.1 ± 31.4 kPa) regions. These differences were associated with a higher proportion of aligned collagen fibers and lower glycosaminoglycan content in the outer region. Regional variations in the elastic moduli and some viscoelastic properties were observed on the proximal surface of the meniscus, with the inner region exhibiting the highest moduli overall. These results indicate that matrix architecture and composition play an important role in the regional micromechanical properties of the meniscus, suggesting that the local stress-strain environment of meniscal cells may vary significantly among the different regions.

Depth-dependent anisotropy of the micromechanical properties of the extracellular and pericellular matrices of articular cartilage evaluated via atomic force microscopy.

The extracellular matrix (ECM) of articular cartilage is structurally and mechanically inhomogeneous and anisotropic, exhibiting variations in composition, collagen fiber architecture, and pericellular matrix (PCM) morphology among the different zones (superficial, middle, and deep). Joint loading exposes chondrocytes to a complex biomechanical environment, as the microscale mechanical environment of the chondrocyte depends on the relative properties of its PCM and local ECM. ECM anisotropy and chondrocyte deformation are influenced by the split-line direction, the preferred collagen fiber orientation parallel to the articular surface. While previous studies have demonstrated that cartilage macroscale properties vary with depth and the direction of loading relative to the split-line direction, the potential anisotropic behavior of the ECM and PCM at the microscale has yet to be examined. The goal of this study was to characterize the depth and directional dependence of the microscale biomechanical properties of porcine cartilage ECM and PCM in situ. Cartilage was cryosectioned to generate samples oriented parallel and perpendicular to the split-line direction and normal to the articular surface. Atomic force microscopy (AFM)-based stiffness mapping was utilized to measure ECM and PCM microscale elastic properties in all three directions within each zone. Distinct anisotropy in ECM elastic moduli was observed in the superficial and deep zones, while the middle zone exhibited subtle anisotropy. PCM elastic moduli exhibited zonal uniformity with depth and directional dependence when pooled across the zones. These findings provide new evidence for mechanical inhomogeneity and anisotropy at the microscale in articular cartilage.

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.

Immunofluorescence-guided atomic force microscopy to measure the micromechanical properties of the pericellular matrix of porcine articular cartilage.

The pericellular matrix (PCM) is a narrow region that is rich in type VI collagen that surrounds each chondrocyte within the extracellular matrix (ECM) of articular cartilage. Previous studies have demonstrated that the chondrocyte micromechanical environment depends on the relative properties of the chondrocyte, its PCM and the ECM. The objective of this study was to measure the influence of type VI collagen on site-specific micromechanical properties of cartilage in situ by combining atomic force microscopy stiffness mapping with immunofluorescence imaging of PCM and ECM regions in cryo-sectioned tissue samples. This method was used to test the hypotheses that PCM biomechanical properties correlate with the presence of type VI collagen and are uniform with depth from the articular surface. Control experiments verified that immunolabelling did not affect the properties of the ECM or PCM. PCM biomechanical properties correlated with the presence of type VI collagen, and matrix regions lacking type VI collagen immediately adjacent to the PCM exhibited higher elastic moduli than regions positive for type VI collagen. PCM elastic moduli were similar in all three zones. Our findings provide further support for type VI collagen in defining the chondrocyte PCM and contributing to its biological and biomechanical properties.

Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration.

INTRODUCTION: Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-ß1 (TGF-ß1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-ß1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair. METHODS: A micro-wound assay was used to assess meniscal cell migration and proliferation in response to the following treatments for 0, 24, or 48 hours: 0 to 10 ng/mL IL-1, TNF-α, or TGF-ß1, in the presence or absence of 10% serum. Proliferated and total cells were fluorescently labeled and imaged using confocal laser scanning microscopy and the number of proliferated, migrated, and total cells was determined in the micro-wound and edges of each image. Meniscal cell proliferation was also assessed throughout meniscal repair model explants treated with 0 or 10 ng/mL IL-1, TNF-α, or TGF-ß1 for 14 days. At the end of the culture period, biomechanical testing and histological analyses were also performed. Statistical differences were assessed using an ANOVA and Newman-Keuls post hoc test. RESULTS: IL-1 and TNF-α decreased cell proliferation in both cell and tissue models of meniscal repair. In the presence of serum, TGF-ß1 increased outer zone cell proliferation in the micro-wound and in the cross section of meniscal repair model explants. Both IL-1 and TNF-α decreased the integrative shear strength of repair and extracellular matrix deposition in the meniscal repair model system, while TGF-ß1 had no effect on either measure. CONCLUSIONS: Meniscal cell proliferation in vivo may be diminished following joint injury due to the up-regulation of inflammatory cytokines, thereby limiting native cellular repair of meniscal lesions. Therefore, therapies that can promote meniscal cell proliferation have promise to enhance meniscal repair and improve tissue engineering strategies.

Spatial mapping of the biomechanical properties of the pericellular matrix of articular cartilage measured in situ via atomic force microscopy.

In articular cartilage, chondrocytes are surrounded by a narrow region called the pericellular matrix (PCM), which is biochemically, structurally, and mechanically distinct from the bulk extracellular matrix (ECM). Although multiple techniques have been used to measure the mechanical properties of the PCM using isolated chondrons (the PCM with enclosed cells), few studies have measured the biomechanical properties of the PCM in situ. The objective of this study was to quantify the in situ mechanical properties of the PCM and ECM of human, porcine, and murine articular cartilage using atomic force microscopy (AFM). Microscale elastic moduli were quantitatively measured for a region of interest using stiffness mapping, or force-volume mapping, via AFM. This technique was first validated by means of elastomeric models (polyacrylamide or polydimethylsiloxane) of a soft inclusion surrounded by a stiff medium. The elastic properties of the PCM were evaluated for regions surrounding cell voids in the middle/deep zone of sectioned articular cartilage samples. ECM elastic properties were evaluated in regions visually devoid of PCM. Stiffness mapping successfully depicted the spatial arrangement of moduli in both model and cartilage surfaces. The modulus of the PCM was significantly lower than that of the ECM in human, porcine, and murine articular cartilage, with a ratio of PCM to ECM properties of approximately 0.35 for all species. These findings are consistent with previous studies of mechanically isolated chondrons, and suggest that stiffness mapping via AFM can provide a means of determining microscale inhomogeneities in the mechanical properties of articular cartilage in situ.

Inhibition of integrative repair of the meniscus following acute exposure to interleukin-1 in vitro.

Damage or loss of the meniscus is associated with progressive osteoarthritic degeneration of the knee joint. Injured and degenerative joints are characterized by elevated levels of the pro-inflammatory cytokine interleukin-1 (IL-1), which with prolonged exposure can induce catabolic and anti-anabolic activities that inhibit tissue repair. We used an in vitro model system to examine the hypotheses that acute exposure to IL-1 inhibits meniscal repair, and that an IL-1-mediated increase in matrix metalloproteinase (MMP) activity is associated with the inhibition of repair. Integrative tissue repair was studied between concentric explants of porcine medial menisci that were treated with IL-1alpha acutely (100 pg/mL for 1 or 3 days) or chronically (100 pg/mL for entire culture duration). After 14 and 28 days in culture, biomechanical testing, cell viability, and histology were performed to assess meniscal repair. Total specific MMP activity in the culture media was measured using a quenched fluorescent substrate. As little as 1 day of IL-1 exposure significantly reduced shear strength, cell accumulation, and tissue repair compared to controls. IL-1 exposure for 1 or 3 days significantly increased MMP activity that subsided by day 9. With chronic IL-1 exposure, MMP activity remained elevated for the duration of culture and was negatively correlated with repair strength. Our study shows that short-term exposure to physiologically relevant concentrations of IL-1 significantly reduces meniscal repair in vitro, and thus may potentially inhibit the intrinsic repair response in vivo. The suppression of IL-1 or MMP expression and/or activity warrant investigation as potential strategies for promoting meniscal repair.