Biochemical and biomechanical properties of lesion and adjacent articular cartilage after chondral defect repair in an equine model.

BACKGROUND: Chondral defects may lead to degradative changes in the surrounding cartilage, predisposing patients to developing osteoarthritis. PURPOSE: To quantify changes in the biomechanical and biochemical properties of the articular cartilage adjacent to chondral defects after experimental defect repair. STUDY DESIGN: Controlled laboratory study. METHODS: Specimens were harvested from tissue within (lesion), immediately adjacent to, and at a distance from (remote area) a full-thickness cartilage defect 8 months after cartilage repair with genetically modified chondrocytes expressing insulin-like growth factor-I or unmodified, control chondrocytes. Biomechanical properties, including instantaneous Young's and equilibrium aggregate moduli, were determined by confined compression testing. Biochemical properties, such as water and proteoglycan content, were also measured. RESULTS: The instantaneous Young's modulus, equilibrium modulus, and proteoglycan content increased, whereas water content decreased with increasing distance from the repaired lesion. The instantaneous Young's and equilibrium moduli of the adjacent articular cartilage were 80% and 50% that of remote area samples, respectively, whereas water content increased 0.9% and proteoglycan content was decreased by 35%. No significant changes in biomechanical and biochemical properties were found either in the lesion tissue or in adjacent cartilage with genetic modification of the chondrocytes. CONCLUSION: Articular cartilage adjacent to repaired chondral defects showed significant remodeling 8 months after chondral defect repair, regardless of whether genetically modified or unmodified cells were implanted. CLINICAL RELEVANCE: Changes in the biochemical and biomechanical properties of articular cartilage adjacent to repaired chondral defects may represent remodeling as part of an adaptive process or degeneration secondary to an altered distribution of joint forces. Quantification of these changes could provide important parameters for assessing progress after operative chondral defect repair.

Effect of tissue maturity on cell viability in load-injured articular cartilage explants.

OBJECTIVE: During joint maturation, articular cartilage undergoes compositional, structural, and biomechanical changes, which could affect how the chondrocytes within the cartilage matrix respond to load-induced injury. The objective of this study was to determine the effects of tissue maturity on chondrocyte viability when explanted cartilage was subjected to load-induced injury. DESIGN: Cartilage explants from immature (4-8-week-old) and mature (1.5-2-year-old) bovine humeral heads were cyclically loaded at 0.5 hertz in confined compression with a stress of 1 or 5 megapascals for 0.5, 1, 3, 6 and 16 h. Cell death was assessed at 0, 24 and 48 h after load removal using cell viability dyes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The organization of pericellular matrix (PCM), biochemical composition and biomechanical properties of the cartilage were also determined. RESULTS: For the immature and mature cartilage, cell death began at the articular surface and increased in depth with loading time up to 6h. No increase of cell death was found after load removal for up to 48 h. In both groups, cell death increased at a faster rate with the increase of stress level. The depth of cell death in the immature cartilage was greater than the mature cartilage, despite the immature cartilage having a higher bulk aggregate modulus. A less organized PCM in immature cartilage was found as indicated by the weak staining of type VI collagen. CONCLUSION: Cells in the mature cartilage are less vulnerable to load-induced injury than those in immature cartilage.

Increased stromelysin-1 (MMP-3), proteoglycan degradation (3B3- and 7D4) and collagen damage in cyclically load-injured articular cartilage.

OBJECTIVE: To determine whether load-induced injury causes alterations in proteoglycan (PG), stromelysin-1 (MMP-3) and collagen in articular cartilage. METHODS: Mature bovine cartilage was cyclically loaded at 0.5 Hz with 1 and 5 MPa for 1, 6 and 24h. Immediately after loading explants were evaluated for cell viability. Alterations in matrix integrity were determined by measuring PG content, PG degradation using 7D4 and 3B3(-) antibodies, broken collagen using COL2-3/4m antibody, and stromelysin-1 content using a MMP-3 antibody. RESULTS: Mechanical load caused cell death and PG loss starting from the articular surface and increasing in depth with loading time. There was a decrease in the 7D4 epitope (native chondroitin sulfate) in the superficial zone of cartilage loaded for longer than 1h, but an increase around chondrocytes in the deep zone. The 3B3(-) staining for degraded/abnormal chondroitin-4-sulfate neoepitope appeared only in cartilage loaded under the most severe condition (5 MPa, 24 h). The elevation of stromelysin-1 was co-localized with broken collagen (COL2-3/4m) at the articular surface in explants loaded with 1 and 5 MPa for 24 h. CONCLUSIONS: Cell death and PG loss occurred within 6h of cyclic loading. The elevation of MMP-3 following cell death was consistently found in the superficial zone of loaded cartilage. Since MMP-3 can degrade PG and super-activate procollagenase, the increase of MMP-3 can therefore induce matrix degradation and PG depletion in mechanically injured articular cartilage, both of which are important to the development of osteoarthritis.

Chondrocyte survival and material properties of hypothermically stored cartilage: an evaluation of tissue used for osteochondral allograft transplantation.

BACKGROUND: There is little information available on the material properties of hypothermically stored allograft specimens used to repair osteochondral defects. PURPOSE: To analyze the effect of hypothermic storage on the material properties of fresh knee specimens over a 60-day interval. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve sheep knee condyles were isolated. The femoral and tibial condyles and the patella were stored in a nutritive medium containing Dulbecco's Modified Eagle's Medium for 1, 8, 15, 29, 45, or 60 days. Total chondrocyte density, chondrocyte viability, matrix proteoglycan content, matrix water content, and matrix dynamic modulus of elasticity were determined. RESULTS: Mean chondrocyte viability decreased significantly over the storage interval: 100% (day 1), 98.2% (day 8), *80.2% (day 15), *80.6% (day 29), *64.6% (day 45), and *51.6% (day 60) (* P < 0.05). Qualitative analysis demonstrated a preponderance of nonviable chondrocytes in the superficial cartilage layer. Significant decreases in matrix proteoglycan were observed in day 15 through day 60 specimens (P < 0.05). The matrix dynamic modulus significantly decreased from day 1 through day 60 (P < 0.05). CONCLUSION: The material properties of hypothermically stored knee condyles progressively decline over 60 days. CLINICAL RELEVANCE: This observed decline may have significant ramifications on long-term graft survival following stored-allograft implantation.

Time, stress, and location dependent chondrocyte death and collagen damage in cyclically loaded articular cartilage.

We investigated the effect of light (0.1 MPa), moderate (1 MPa) or heavy (5 MPa) cyclical stresses applied continuously or intermittently for 0 to 72 h on cell death and collagen damage in adult bovine cartilage explants. No increase in cell death was observed in the cartilage loaded with a continuous cyclic stress at 0.1 MPa for up to 72 h. Cell death occurred in the uppermost superficial tangential zone (STZ) of explants after loading for 1 h at 1 MPa, and reached a maximum depth of 61+/-23 micro m by 6 h (at the rate of 9+/-6 micro m/h). At 5 MPa, cell death occurred in the STZ after as little as 1 min (30 cycles) of loading, and reached a maximum depth of 70+/-2 micro m by 60 min (47+/-8 micro m/h). When an intermittent (with 2 s on, 2 s off) stress of 5 MPa was applied, cell death appeared in the STZ after 2 min (30 cycles) and increased to a depth of 63+/-2 micro m at 60 min (45+/-11 micro m/h). No significant differences were observed between the continuous and intermittent loading conditions. Both collagenase-cleaved and denatured collagen fibers were found in the STZ of explants loaded at 1 and 5 MPa. We concluded that load-induced cell death depends on load duration and magnitude, and that the chondrocytes in the STZ are more vulnerable to load-induced injury than those in the middle and deep zones.

Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7.

BACKGROUND: Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair. METHODS: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control, an adenovirus vector encoding an irrelevant gene (Escherichia coli cytosine deaminase, AdCD) were implanted into extensive (15 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses. Biopsies were performed to evaluate early healing at 4 weeks. At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells. RESULTS: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control. Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted. By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups. CONCLUSIONS: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects. CLINICAL RELEVANCE: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life. This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing.

Matrix fixed-charge density as determined by magnetic resonance microscopy of bioreactor-derived hyaline cartilage correlates with biochemical and biomechanical properties.

OBJECTIVE: To use noninvasive magnetic resonance imaging (MRI), biochemical analyses, and mechanical testing of engineered neocartilage grown in a hollow- fiber bioreactor (HFBR) to establish tissue properties, and to test the hypothesis that MRI can be used to monitor biochemical and biomechanical properties of neocartilage. METHODS: Chondrocytes from day 16 embryonic chick sterna were inoculated into an HFBR and maintained for up to 4 weeks with and without exposure to chondroitinase ABC. The fixed-charge density (FCD) of the cartilage was determined using the MRI gadolinium exclusion method. The sulfated glycosaminoglycan (S-GAG), hydroxyproline, and DNA contents were determined using biochemical procedures, while dynamic and equilibrium moduli were determined from mechanical indentation tests. RESULTS: S-GAG content, tissue cross-sectional area, and equilibrium modulus of the neocartilage increased with development time. There was a gradient of S-GAG content across the length of control neocartilage at the 4-week time point, with higher values being found toward the inflow region. Exposure to chondroitinase ABC resulted in a decrease in tissue area, negative FCD, proteoglycan content, and equilibrium and dynamic moduli. The treated bioreactors displayed a lengthwise variation in S-GAG content, with higher values toward the outflow end. Linear correlations were established among FCD, proteoglycan content, and biomechanical properties. CONCLUSION: HFBR-derived neocartilage showed regional variation in S-GAG content under control conditions, and in the decrease of S-GAG in response to enzyme treatment. In addition, the results support the hypothesis that tissue parameters derived from MRI can be used to noninvasively monitor focal neocartilage formation and biochemical and biomechanical properties.