Experimental Study On Human Umbilical Cord Mesenchymal Stem Cells In Combination With Hydroxyapatite/Collagen Scaffold For Bone Defect Repair

Objective: Aim to isolate and culture the human umbilical cord mesenchymal stemcells (hUCMSCs) in vitro and induce it to osteogenic differentiation beforeidentifying their biological characteristics. Meanwhile, explore a method toconstruct hydroxyapatite/collagen scaffold material,and exam itsbiocompatibility with hUCMSCs.Finally, the effect of human umbilical cordmesenchymal stem cells combinate with hydroxyapatite/collagen scaffold forbone defect repairing will be investigated.Methods: The hUCMSCs were isolated from human umbilical cord by digested ofcollagenase. After serial subcultivationin vitro. Morphologic appearance ofUCMSCs was observed by an optical microscope and atomic force microscope. Theproliferation rate was measure by MTT assay. Cell cycle and surface antigenswere measured by Flow cytometry.The osteogenic differentiation was inducedby chemical drugs and evaluated by specific stain methods. After2weeksco-culture of hUCMSCs and hydroxyapatite/collagen scaffold, MTT assay wereperformed to measure cell proliferation rate,while cell adhesion and growthwas observed by opti cal microscope and SEM (scanning electron microscope),and histocompatibility were evaluated.Finally, the compound were implantedinto New Zealand rabblt with femur defect,healing of the defect of was evaluatedwith radiographs and histological examination in comparing with control.Results: The hUCMSCs isolated by digested with collagenase is efficient. TheMTT showed a large expansive potential, Flow cytometry analysis revealed thatCD13、CD29、CD44、 CD54、 CD73、 CD90、CD105were highly expressed on the surfaceof passage3cells, but there was negative for CD14、 CD31、 CD34、 CD40、CD45�'�HLA一DR。 Morphologic appearance of hUCMSCs was comfirmed by Atomic forcemicroscope.After successfully being induced into osteogenic lineages,thesecells show positive for alkaline phosphate staining and calcium mineralizationfor alizarin red stainingl. Both MTT and optical microscope along with SEMcomfirmed that osteoblast showed good adhesion, prolification andbiocompatibility to hydroxyapatite/collagen scaffold material. Morphologicaland histological examination with Radiology tests showed that the tissue-enggneeing bone can repair the femur defect effectively by ectopicosteogenesisConclusions: The method for isolation and purification of hUCMSCs from humanumbilical cord in vitro have been set up. The hUCMSCs were proliferate activelyand its biological characteristics was stabilization when cultured in vitroand have been successfully induced into osteogenic lineages, which showed goodadhesion, prolification and biocompatibility to hydroxyapatite/collagenscaffold material. These tissue-enggneeing bone can repair the femur defecteffectively by ectopic osteogenesis, which suggested its great potential onhealing...