Experimental Study On Human Umbilical Cord Mesenchymal Stem Cells In Combination With Calcium Alginate For Tissue Engineered Cartilage

Objective:To isolate and culture of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro,identify their biological characteristic,and induce hUCMSCs differentiate into cartilage cells in different conditions.Calcium alginate column was manipulated and observed its biocompatibility,and investigate the potential of tissue engineered cartilage to heal a segmental defect in the rabbits.Methods:The hUCMSCs were isolated from human umbilical cord by digested with collagenase. After serial subcultivation in vitro, the stem cells were passaged. Morphologic appearance of hUCMSC was observed by an optical microscope. The proliferation rate was measured by MTT assay. Cell cycle and surface antigens were measured by Flow cytometry. The hUCMSC were induced to differentiate into chondrocytes by low density, high density, micromass groups culture of the environment.The structure of cartilage differentiation was observed by by an optical microscope.After 3 weeks,different groups were identified by toluidine blue staining,collagen typesâ…¡immunohistochemical assay.Preparation calcium alginate hydrogel scaffold materials.The calcium alginate column was co-cultured with hUCMSCs,then this composite material scaffolds were being cultured in cartilage medium for three weeks,and the growth and adhesion of cells on the scaffold were observed with scanning electron microscope and the cell proliferation was detected by MTT assay so as to evaluate the tissue response.New Zealand white rabbits femoral condyle osteochondral defect were created and subsequently filled with the scaffolds, healing of the defect was evaluated with histological examination.Results:The isolated hUCMSCs by digested with collagenase is efficient. The isolated hUCMSCs were exhibiting a large expansive potential.Flow cytometry analysis revealed that CD29,CD44 and CD 105 were highly expressed on the surface of passages 3 cells, but there was negative for CD34,CD45 and HLA-DR. After culture in inducing medium,the low density and control groups were low positive for toluidine blue staining,collagen typesâ…¡immunohistochemical assay,the high density were positive for toluidine blue staining,collagen typesâ…¡immunohistochemical assay,the micromass groups were high positive for those dying. The cells grow well on the surface of the the calcium alginate column through scanning electron microscope. The MTT test assay showed that the calcium alginate column scaffolds couldn't inhibit the proliferation of hUCMSCs. The composite of hUCMSCs and calcium alginate can be used to construct tissue engineered cartilage,in order to heal cartilage defects.Conclusions:An in vitro method for the isolation and purification of hUCMSCs from human umbilical cord has been established. The cultured cells were only composed of undifferentiated cells and biological properties was stabilization. Culture expanded hUCMSCs maintained their ability to cartilage direction differentiation in different environment.The calcium alginate has good biocompatibility. The composite by combining hUCMSCs with calcium alginate is able to repair of cartilage defects.