Characterization of a biomaterial with cartilage-like properties expressing type X collagen generated in vitro using neonatal porcine articular and growth plate chondrocytes

Objective: The availability of cartilage with or without the potential to o ssify and suitable for surgical restoration and resurfacing of joints is an important clinical problem in arthritis-related pathology, trauma and reco nstructive surgery. Here, we designed experiments to generate a biomaterial with cartilage-like properties by culturing neonatal porcine articular and growth plate chondrocytes on a hydrogel substrate and to examine the bioch emical and histological characteristics of the resulting tissue. Design: Neonatal porcine epiphyseal and growth plate chondrocytes were cult ured on poly(2-hydroxyethyl methacrylate) (polyHEMA)coated dishes to preven t their adherence to plastic. We previously described that this procedure a llows the maintenance of the chondrocyte-specific phenotype for greater tha n or equal to8 months. Chondrocytes were isolated by successive enzymatic d igestions and cultured at high density (>2.0 x 10(7) cells/ml) in DMEM with 10% FBS, 50 mug/ml ascorbic acid, glutamine, vitamins, and antibiotics for up to 10 weeks on 60 mm plastic culture dishes coated with polyHEMA. The t issues produced during culture were studied histologically and biochemicall y and were examined for cellular proliferation employing H-3-thymidine inco rporation and for their collagen production employing biosynthetic labeling with C-14-proline and Western blot with specific antibodies. The expressio n of relevant collagen genes was examined employing RT-PCR. Results: Within 24 h of culture, isolated chondrocytes organized into well- formed clusters and in 2 weeks formed structures with gross appearance and consistence similar to those of natural cartilage. The wet weight of the ti ssue formed in vitro increased six-fold during the 10-week period of study. Cell proliferation measured by the incorporation of H-3-thymidine increase d during the first 3 weeks and reached a plateau in subsequent weeks. Histo logical examination showed that the cultures contained rounded chondrocytes embedded in an abundant cartilaginous extracellular matrix. The cartilage formed contained large amounts of collagen and sulfated proteoglycans as ex amined by staining with Masson's Trichrome and Alcian blue, respectively. D eposition of calcium in the deeper layers of the tissue was demonstrated wi th the von Kossa stain. Western analyses with specific antibodies showed th at type II collagen was present from the first week and progressively incre ased in the cultures, whereas type X collagen was first detected at 4 weeks and increased with length of culture. When chondrocytes isolated from the growth plate were included, small amounts of type I collagen were detected in the medium of cultured biomaterial as expected. Type III collagen was no t detected by Western blot over the 10-week period, High levels of type II and type X collagen gene expression were demonstrated by RT-PCR. Conclusion: These studies demonstrate the production in vitro of cartilage- like tissue with similar morphological, histochemical and biochemical chara cteristics to those of natural growth plate cartilage. The cartilage genera ted in vitro has the potential to be used in reconstructive surgery and in joint resurfacing and restoration of skeletal defects. (C) 2001 OsteoArthri tis Research Society International.