Abstract
The repair of cartilage defects remains a significant clinical challenge. The use of mesenchymal stem cells for cell-based tissue-engineering strategies represents a promising alternative for the repair of the defects. In this study, we investigated the TGF-bate1 dose and cellular density-dependent effect on chondrogenic differentation of human bone marrow-derived mesenchymal stem cells (MSCs) cultured in alginate beads in vitro.
Methods A volume of 6 ml bone marrow was collected from six volunteer donors respectively. MSCs were cultured in different cellular density (1×104, 1×105, 1×106 and 5×106/ml) and treated with different doses of TGF-beta1 (0, 1, 10, 50 and 100 ng/ml). Immunohistochem-istry and in situ hybridization were applied to detect the expression of collagen type II and assay proteoglycan in different time internal.
Results 95% cellss were alive after density gradient centrifugation. BMSCs had a similar spindle-like morphology. Type II collagen and proteoglycan were showed positive staining in the 10 ng/ml TGF-beta1 group, weakly positive in the 50 ng/ml and 100 ng/ml group, negative in the 0 ng/ml and 1 ng/ml group. With time, the proteoglycan quantity increased. All cell density groups except 1×104/ml showed positive expression of collagen type II and proteoglycan synthesis, and better staining with increase of cellular density. Proteoglycan synthesis did not increased until the fifth weeks.
Conclusions The chondrogenesis differentiation of human MSCs is dose-dependent. 10ng/ml TGF-beta1 is a suitable concentration for such inducing. The cellular density is also important for the differentiation of MSCs. Too small density is ineffective. The more cells, the better differentiation. And the time of in vitro culture should not be longer than 4 weeks
Correspondence should be addressed to Mr Carlos Wigderowitz, Senior Lecturer, University Department of Orthopaedic and Trauma Surgery, Ninewells Hospital and Medical School, Dundee DD1 9SY.
