Chondrogenic differentiation of immortalized human mesenchymal stem cells on zirconia microwell substrata

Human mesenchymal stem cells (hMSCs) that can differentiate into chondrocytes are a potential autologous cell source for repair of damaged tissue. Current methods usually induce the formation of all three chondrocyte phenotypes, hyaline, fibrous, and elastic, without the ability to selectively induce only one of them. By controlling the size of hMSC cell clusters, it may be possible to direct differentiation more uniformly toward hyaline chondrocytes. We designed new cell culture platforms containing microwells of different diameters. The platforms and wells were composed of a zirconia ceramics substratum. hMSCs briefly adhered to the substratum before releasing and entering the microwells. The physical restraints imposed by the microwells enabled hMSC clusters to homogenously differentiate into hyaline chondrocyte-like cells. Chondrogenic aggregates in microwells expressed the hyaline chondrocyte-specific genes Col II, aggrecan (ACAN), and cartilage oligomeric protein (COMP). The cultures also produced hyaline chondrocyte-specific matrix proteins Col II and ACAN homogenously throughout the aggregates. In contrast, chondrogenesis in pellet cultures was heterogeneous with the expression of nonhyaline chondrocyte genes CD105, Col X, and Col I. In these pellet cultures, hyaline and nonhyaline chondrocyte-specific matrix proteins were distributed heterogeneously. Thus, this novel ceramic microwell substratum technology efficiently directed the differentiation of hyaline chondrocyte-like cells from hMSCs. These results indicate that there is a close relationship between hMSC cluster size regulation in the microwells and differentiation tendency. This microwell culture differentiation method will provide a valuable experimental system for both experimental and potential clinical studies.