Enhanced chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells in zirconia microwell substrata

In human cartilage tissue engineering, three-dimensional zirconia substrata have the potential advantage of producing many uniform cell clusters of controlled size without xenobiotic material, allowing easy clinical application. The objective of this study was to evaluate the possibility of using zirconia porous three-dimensional microwell substrata for chondrogenic differentiation of equine bone marrow-derived mesenchymal stem cells (BMMSCs) in vitro. In regular medium, 8 x 10(5), 2 x 10(6), and 5 x 10(6) equine BMMSCs from five thoroughbred horses were cultured on zirconia microwell substrata for 4 days to allow formation of clusters. The medium was replaced by chondrogenic culture medium. After chondrogenic culture for 7, 14 and 21 days, analysis of collagen type II alpha 1 gene (COL2A1) gene expression and observation of chondrogenic aggregates by scanning electron microscopy (SEM) were performed. SEM showed size-controlled cell clusters and increasing extracellular matrix over time when using 5 x 10(6) cells. The expression of COL2A1 on day 7 and 14 with 5 x 10(6) cells was significantly higher than that of conventional pellet culture with 2 x 10(6) cells. Histological evaluation by immunohistochemical staining for type II collagen (ColII) was performed after chondrogenic culture for 7 days. The clusters showed wide distribution of Corn. The results suggest that the zirconia substrata have the potential to enhance the chondrogenic differentiation of equine BMMSCs, allowing effective equine cartilage tissue engineering without xenobiotic materials.