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

Abstract

Objective The availability of cartilage with or without the potential to ossify and suitable for surgical restoration and resurfacing of joints is an important clinical problem in arthritis-related pathology, trauma and reconstructive 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 biochemical and histological characteristics of the resulting tissue.Design Neonatal porcine epiphyseal and growth plate chondrocytes were cultured on poly(2-hydroxyethyl methacrylate) (polyHEMA)-coated dishes to prevent their adherence to plastic. We previously described that this procedure allows the maintenance of the chondrocyte-specific phenotype for ≥8 months. Chondrocytes were isolated by successive enzymatic digestions and cultured at high density (>2.0×107cells/ml) in DMEM with 10% FBS, 50μg/ml ascorbic acid, glutamine, vitamins, and antibiotics for up to 10 weeks on 60mm plastic culture dishes coated with polyHEMA. The tissues produced during culture were studied histologically and biochemically and were examined for cellular proliferation employing3H-thymidine incorporation and for their collagen production employing biosynthetic labeling with14C-proline and Western blot with specific antibodies. The expression of relevant collagen genes was examined employing RT-PCR.Results Within 24h 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 tissue formed in vitro increased six-fold during the 10-week period of study. Cell proliferation measured by the incorporation of3H-thymidine increased during the first 3 weeks and reached a plateau in subsequent weeks. Histological 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 examined by staining with Masson's Trichrome and Alcian blue, respectively. Deposition of calcium in the deeper layers of the tissue was demonstrated with the von Kossa stain. Western analyses with specific antibodies showed that type II collagen was present from the first week and progressively increased 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 not 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.ConclusionThese studies demonstrate the production in vitro of cartilage-like tissue with similar morphological, histochemical and biochemical characteristics to those of natural growth plate cartilage. The cartilage generated in vitro has the potential to be used in reconstructive surgery and in joint resurfacing and restoration of skeletal defects.