The effective mode of growth and differentiation factor-5 in promoting the chondrogenic differentiation of adipose-derived stromal cells

Our study aimed to find out the most effective mode for chondrogenic differentiation based on time, dose and culture method. ADSCs were cultured and identified by CD44, CD49d, and CD106 immumohistochemical staining method, and their differentiation potential to chondrocyte were detected by Alizarin red staining. ADSCs induced by different concentrations of GDF-5 for chondrogenic differentiation were detected by blue and toluidine blue staining and collagen type II and X immumohistochemical staining. The expression of collagen I, II, X and aggrecan gene in GDF-induced ADSCs cultured in 2- and 3-dimension was identified by real-time PCR. Cell microstructure and proliferation in three-dimensional scaffolds at day 7, 14, 21 and 28 were analyzed by scanning electron microscopy and MTS assay. The ADSCs were successfully identified by CD44 and CD49d, and their differentiation potential was detected by Alizarin red staining. Real-time PCR showed that collagen and aggrecan were expressed at high levels in 100 or 200 ng/mL GDF-5 treated cells. The collagen types (I, II) and aggrecan genes were higher expressed in GDF-5 induced scaffold group than that in monolayer group. MTS showed that the cell counts were not significantly different among different treated time. Both collagen type II and aggrecan gene were highly expressed at day 14, while collagen types I and X gene expressions peaked at day 21 and 28. The 100 ng/mL GDF-5 is effective and cost-effective for chondrogenic differentiation when cultured at day 14 in vitro under three-dimensional culture conditions.