Repair Mechanisms in Articular Cartilage—A Porcine in Vitro Study

Explants are excellent systems for studying homeostasis in cartilage. The systems are very useful in pharmacological studies involving OA-treatment and in studies of repair mechanisms during injury to hyaline cartilage. The purpose of this study was to evaluate the reparative processes occurring in a young age porcine cartilage explant model examining tissue by Light (LM) and Transmission Electron Microscopy (TEM). Explants of articular cartilage were dissected from the femoral condyles of immature one-year-old pigs and cultured in DMEM/F12 medium with FCS (stimulated explant) or in medium without FCS (control explant) for up to 4 weeks. After 1 - 4 weeks of culture with FCS, LM showed migration and proliferation of chondrocytes in cartilage close to the injured surface differentiating two areas: proliferative zone and necrotic zone. The chondrocytes present in the necrotic zone showed a polarization towards the injured surface. After budding through the injured surface, the chondrocytes formed repair tissue in an interface repair zone and in outer repair tissue. TEM showed chondrocytes in expanded lacunae involving the proliferative zone. The pericellular matrix of the expanded lacunae was partly dissolved, indicating release of matrix-degrading enzymes during proliferation and remodeling. Migratory chondrocytes were identified in oval lacunae close to the injured surface. The pericellular matrix of these oval lacunae was significantly dissolved and immunohistochemistry demonstrated strong staining with a polyclonale collagenase antibody around these units, suggesting release of matrix-degrad- ing collagenase contributing to chondrocyte mobility. We describe an explant model comprising two different repair systems in immature articular cartilage. This model provides us with new reference points that are important in understanding the repair mechanisms.

Intra-Articular Cartilage Calcification Associated with Type II Pro-Collagen Accumulation in Chondrocytes and Abnormal Fibrils in the Extra Cellular Matrix (ECM): Case Report

Objective: The purpose of this case-study was to perform morphological and molecular analysis of articular cartilage biopsies from the femoral condyle of a 33 year old woman with intra-articular calcification in the right knee joint and compare the findings with those of normal cartilage. Methods: Femural condyle cartilage biopsies were used for Light Microscopy (LM), Transmission Electron Microscopy (TEM), explant culturing and DNA mutation analysis of the COL2A1 gene. Results: X-ray of the affected knee joint showed intra-articular calcifications on the femur condyle, tibia and meniscus. Pathological LM and TEM examination of cartilage biopsies revealed calcified islands morphologically identical to calcium pyro-phosphate dehydrate (CPPD) and hydroxyapatite (HA)-like crystals. In addition, chondrocytes showed accumulation of pro-collagen molecules. With explant culturing and immunochemistry, it was confirmed that matrix calcification correlated with high intracellular matrix accumulation and expression of type X collagen. The induction of hypertrophy in chondrocytes was further associated with matrix vesicle (MV) release and a prominent calcification zone. Surprisingly, TEM showed crystal development on thick abnormal type II collagen fibrils suggesting that these ECM components might nucleate and contribute to calcification. Conclusions: We suggest that intra-articular calcification may be associated with type II pro-collagen accumulation in chondrocytes. In particular, we hypothesize that matrix accumulation may induce hypertrophy and type X collagen expression in cartilage cells and release of MV’s into the ECM, which together with thick abnormal type II collagen hetero-fibrils, are responsible for crystal deposition in the ECM.

Patella Alta and Trochlea Dysplasia Is Associated with Abnormal Type Ⅱ Collagen and Matrix Accumulation in Chondrocytes

The purpose of this study was to perform morphological and molecular analyses of articular cartilage from a 14-year-old boy with unusual cartilage lesions, patella alta and trochlea dysplasia in both knee joints and clinically examine two family members (sister, mother), also affected in their knee joints. Biopsies from the boy’s patella were used for: histological examination, Transmission Electron Microscopy (TEM) and DNA sequencing of the COL2A1 gene including Multiplex Ligation-dependent Probe Amplification (MLPA), for detection of DNA deletions and duplications. Clinical and radiological examination showed patella alta and trochlea dysplasia for the brother (type D), sister (type A) and mother (type A) with Insall-Salvati ratios of 1.50, 1.46 and 1.3. Light Microscopy (LM) of biopsies from the patient showed rhomboid chondrocytes in lacuna with deposition of protein aggregates in the ECM. TEM revealed abnormal type II collagen fibrils in aggregates and chondrocytes with abnormal matrix accumulation in rough Endoplasmic Reticulum (rER). Immunostaining showed that type II collagen was deposited intracellularly and in protein aggregates, together with type I collagen, indicating alterations in chondrocyte function and turnover of these molecules. DNA sequencing of 54 exons including extended DNA analysis with MLPA was non-conclusive. Conclusions: We suggest that patella alta and trochlea dysplasia for this patient is associated with collagen accumulation in chondrocytes, abnormal type II collagen heterofibrils in the ECM, cell death and cartilage with subnormal strength and increased risk of premature patellofemoral arthritis. A family with these disorders suggests that phenotype might be transmitted as an autosomal dominant trait.