Chondrogenic differentiation of canine myoblasts induced by cartilage-derived morphogenetic protein-2 and transforming growth factor-β1 in vitro

Abstract

The meniscus has limited ability to repair itself following injury. However, tissue engineering provides new means of meniscus repair. Myoblasts, which possess the potential of multi-directional differentiation, may be ideal seed cells in meniscus tissue engineering. Myoblasts from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated myoblasts from canines and induced chondrogenesis in order to establish a new experimental model of seed cells. Myoblasts were isolated and harvested from Beagle canines. To induce chondrogenesis, cartilage-derived morphogenetic protein-2 (CDMP-2) at different concentrations (10, 20, 50 and 100 ng/ml), transforming growth factor-β1 (TGF-β1; 10, 20, 30 and 50 ng/ml), and different concentrations of CDMP-2 (10, 20, 50 and 100 ng/ml) together with TGF-β1 (20 ng/ml) were added to the cultured pellets. After 21 days of in vitro culture, chondrogenic differentiation was evaluated by histological and immunohistochemical techniques. The degree of gene expression was measured by quantitative RT-PCR. Based on the histological staining of glycosaminoglycan, using the toluidine blue dye-binding method, we found that CDMP-2 initiated chondrogenic differentiation of myoblasts, as did TGF-β1. Furthermore, CDMP-2 conferred a stronger stimulatory effect than TGF-β1. The combination of CDMP-2 and TGF-β1 synergistically induced chondrogenesis of myoblasts. This synergistic chondrogenic effect of CDMP-2 together with TGF-β1 was further confirmed by quantification of glycosaminoglycan using dimethylmethylene blue dye-binding assay and immunohistochemical analysis of the expression of cartilage-specific proteins collagen I and II. Canine myoblasts can be induced into chondrocytes by CDMP-2 and TGF-β1 in vitro, suggesting that myoblasts are suitable as seed cells for meniscus tissue engineering.