Experimental Study Of Chondrogenic Differentation Of Human Bone Marrow-derived Mesenchymal Stem Cells Combined With Alginate Scaffold In Vitro

Objective To investigate the feasibility and experimental condition of chondrogenic differentation of human bone marrow-derived mesenchymal stem cells cultured in alginate beads in vitro. To provide experimental evidence for cartilage tissue engineer and clinic apply. Methods bone marrow aspirates were taken from the iliac crest of normal adult donors about 5¹0ml, Nucleated cells were isolated with a density gradient centrifugation, MSCs from nucleated cells were expanded. Observe the morphological changes and growth character of mesenchymal stem cells in monolayer culture. mesenchymal stem cells(5 ×10⁶/ml)were suspended within alginate beads(20μL/each) and cultured in high glucose(25mM) Dulbecco's Modified Eagle's Medium (DMEM) with transferrin, dexamethasone, insulin, and pyruvate, and treated with different doses of TGF-β₁(0,1,10,50,100ng/ml).In 3 weeks, immunohistochemistry and in situ hybridization were applied to detect the expression of collagen type II, Some sections were stained with AB-PAS, assaying proteoglycan. proteoglycan synthesis was quantified with alcian blue staining in 1, 3, 5weeks.Result 95% cells were alive after density gradient centrifugation, BMSCs had a similar spindle-like morphology.latent phase in primary culture lasts for 3 to 5 days, cells grow rapidly in passage culture. The peak doubling rate was seen on day 4, the proliferation of passage 1 to 3 has not much change. Immunohistochemistry and in situ hybridization for type II collagen, and AB-PAS staining for proteoglycan showed positive staining in the lOng/ml...