gms | German Medical Science

Objectives: Cartilage defect treatments often result in fibrous tissue formation. New cartilage therapies are needed to achieve defect regeneration with functional repair tissue. Therefore, pre-clinical evaluation of novel cartilage regeneration therapies and material testing in a 3D osteochondral environment are needed. We established an ex vivo culture platform for long term culture of osteochondral explants (OCE). Our cartilage defect model opens new opportunities to evaluate bio-inspired materials for material-tissue- cell-interaction, addressing biocompatibility and cartilage defect regeneration.

Methods: Reproducible OCE (8mm x 5mm) were isolated from porcine condyles. Full thickness cartilage defects (4mm) were filled with cell free or chondrocyte loaded hydrogels (poly(ethylene glycol)/heparin (PEGh), PEG / methacrylated hyaluronic acid (HA) (MHH), HA thiolated and acrylated PEG (HA-SH-PEG)). Fibrin glue and collagen type I (3mg/ml) served as reference materials. OCE were cultured in custom transwell system with separated media compartments for cartilage and bone without supplementation of growth factors. Cell vitality was analyzed (MTT staining). Matrix deposition and defect regeneration were analyzed (immune-) histologically (Safranin-O, collagen II, aggrecan). Cartilage matrix composition was evaluated in terms of glycosaminoglycan (GAG) and DNA content. mRNA of chondrocyte loaded hydrogels was isolated to perform realtime polymerase chain reaction for chondrocyte markers.

Results and Conclusion: MTT staining demonstrated metabolic active chondrocytes in hydrogels and OCE after 28 days ex vivo culture. Cell migration from OCE was observed in cartilage defect area and into cell free hydrogels revealing elongated, fibroblastic like cell phenotype. Despite GAG release into cartilage media supernatant, GAG content in OCE did not change significantly shown by Safranin-O staining and GAG quantification. Safranin-O staining demonstrated cartilage like tissue formation in cartilage defects treated with cell loaded hydrogels. Cartilaginous matrix production was confirmed by GAG content in hydrogels. GAG content in collagen I, PEGh and MHH hydrogel, significantly increased at day 28 to 2-3 fold of initial GAG amount on day 0. GAG content in fibrin glue and HA-SH-PEG did not change significantly.

Separated media compartments with tissue specific nutrient supply to bone and cartilage is critical in preserving matrix composition and vitality during ex vivo culture of OCE. Proteoglycan release into media supernatant and constant GAG amount in OCE indicates active cartilaginous matrix production. Our results overcome limitation of cartilage degeneration in existing osteohondral models. Presence of subchondral bone stimulates cell migration and matrix production for cartilage defect regeneration. Our osteochondral model represents a pre-clinical platform in cartilage defect regeneration to develop new treatment strategies and studying processes in cartilage regeneration and degeneration pathways.