Engineering of Hypertrophic Cartilage – preliminary results

Objectives:Using a tissue-engineered template that can mineralize in vitro or in vivo (e.g. hypertrophic cartilage) could be an alternative approach to bone tissue engineering. Cartilage has the advantage that it can survive in a relatively hypoxic environment, which may allow more time for vascularisation and mineralization of the engineered graft to develop post-implantation. The aim of this research was to investigate different cell sources and conditions that promote generation of a cartilage construct with characteristics of hypertrophic tissue. Methods: Chondrocytes isolated from articular and nasal cartilages were expanded in monolayer culture. When sufficient cell numbers were obtained, chondrocytes were cultured as pellets for 28 days under conditions which promote chondrogenic or osteogenic differentiation (DMEM with 10% FCS, 1ug/ml insulin, 50 µg/ml L-ascorbic acid or DMEM with 10% FCS, 50 µg/ml L-ascorbic acid with 10mM -glycerophosphate and 10-8M dexamethasone, respectively). In this 3D environment, chondrocyte differentiation is induced. The stage of chondrocyte differentiation was evaluated by analysis of the extracellular matrix by immunolocalization of collagens (types I and II) and histochemical detection of proteoglycans, calcium deposition and alkaline phosphatase. Results: Preliminary data showed successful process of chondrogenic re-differentiation in pellet culture. Alkaline phosphatase activity was detected in a limited proportion of the samples and was suggestive that chondrocytes were entering the hypertrophic stage. Studies in monolayer cultures showed that addition of dexamethasone to the culture medium results in lower expression of alkaline phosphatase in both cell types. Conclusion: Chondrocytes successfully re-differentiated in our pellet culture system. The cells expressed some characteristics of hypertrophic tissue and therefore show some potential for use in bone tissue repair strategies. However, the use of dexamethasone in the culture medium appeared to inhibit the terminal differentiation of chondrocytes. Ongoing work is directed at determining culture conditions to accelerate process of hypertropic differentiation in nasal chondrocytes.