Experimental Study On Human Umbilical Cord Mesenchymal Stem Cells In Gradient Nano-fiber Scaffolds For Tissue Engineered Osteochondral

Objective: To isolate and culture the stem cells from healthy full-term infants umbilical cord invitro and explore the biological characteristics of hUCMSCs.Meanwhile,explore a method toconstruct gradient Nano-fiber scaffolds material,and exam its biocompatibility withhUCMSCs.Finally, the effect of human umbilical cord mesenchymal stem cells combinatewith gradient Nano-fiber scaffolds and the ability of hUCMSCs into osteochondral will beinvestigated.Methods: We get the hUCMSCs from human umbilical cord by digested of collagenase, andculture,Morphologic appearance of hUCMSCs was observed by an optical microscope and atomicforce microscope.The proliferation rate and the experiment of cell proliferation toxicity wasmeasure by MTT assay.Cell cycle and surface antigens were measured by Flow cytometry.Theosteogenic and Cartilage differentiation was induced by chemical drugs and evaluated by specificstain methods;To observe the changes in the structure of the cytoskeleton.The combination ofhUCMSCs and gradient Nano-fiber scaffolds is induced to osteogenic and Cartilage differentiationfor2weeks,the cell proliferation rate was measured by MTT assay,while we observed the celladhesion and growth using optical microscope and SEM (scanning electron microscope),andhistocompatibility were evaluated.To evaluate the effect of the combination of hUCMSCs andgradient Nano-fiber scaffolds is induced to osteogenic and Cartilage differentiation by specific stainmethods.Results: The hUCMSCs that we isolated is efficient and dynamic.The result of MTT showed thecells has a large expansive potential,Flow cytometry analysis revealed that CD13、CD29、CD44、CD54、CD73、CD90、CD105were highly expressed on the surface of passage3cells,but there wasnegative for CD14、 CD31、 CD34、 CD40、 CD45和HLA-DR.Morphologic appearance andcytoskeleton of hUCMSCs was comfirmed by Atomic force microscope.After successfully beinginduced into osteogenic lineages,these cells show positive for alkaline phosphate staining、calciummineralization for alizarin red stainingl and Von Kossa mineralized nodules for Von Kossastainingl;The hUCMSCs have the biological characteristics of osteoblast cells by using theatomic force microscope to observe.After successfully being induced into cartilage lineages,thesecells show positive for toluidine blue staining and Alberto new blue staining;The hUCMSCs havethe biological characteristics of cartilage cells by using the atomic force microscope toobserve.Both MTT and optical microscope along with SEM comfirmed that osteoblast andchondrocytes showed good adhesion,prolification and biocompatibility to gradient Nano-fiberscaffold material.The effect of the combination of hUCMSCs and gradient Nano-fiber scaffolds is induced to osteogenic and cartilage differentiation is fine by specific stain methods.Conclusions: The method that isolated and amplified of hUCMSCs from human umbilical cord invitro have been set up.The hUCMSCs were proliferate actively and its biological characteristicswas stabilization when cultured in vitro and have been successfully induced into osteogenic andcartilage lineages.The gradient Nano-fiber scaffolds have fine biocompatibility and it is usefulmaterials for tissue-enggneeing bone and cartilage with a wide range of application.