Experimental Study On Human Umbilical Cord Mesenchymal Stem Cells In Combination With Chitosan/phosphonic Chitosan Sponge For Bone Tissue Engineering

Objective:To explore the method of isolate and culture of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro,and identify their biological characteristic. Observe its biocompatibility with chitosan/phosphonic chitosan sponge,and investigate the potential of tissue engineered bone 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 UCMSCs was observed by an optical microscope and atomic force microscopy. The proliferation rate was measured by MTT assay. Cell cycle and surface antigens were measured by Flow cytometry. The osteogenic and adipogenic differentiation were tested and evaluated by specific stain methods. The ultrastructure of osteogenic differentiation was observed by the Atomic Force Microscope. The chitosan/phosphonic chitosan sponge was produced and co-cultured with hUCMSCs,then this composite material scaffolds were being cultured in osteogenic medium for two 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. hUCMSCs-loaded scaffolds were implanted intramuscularly and the samples were analyzed by histological examination. New Zealand white rabbits femur defects were created and subsequently filled with hUCMSCs-loaded scaffolds, healing of the defect was evaluated with radiographs analysis and histological examination.Results:The isolated hUCMSCs by digested with collagenase is efficient. The similar growth curves of passage3 and 7 were exhibiting a large expansive potential.Flow cytometry analysis revealed thatCD29,CD44,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 cells were successfully induced into osteogenic and adipogenic lineages. These cells were high positive for alkaline phosphate staining and also showed mineralization demonstrated with mineralized nodules staining after culture induction of osteogenic differentiation. Futhermore, liquid vacuoles were detected by Oil red O staining after 3 weeks culture induction of adipogenic differentiation. Under atomic force microscopy, the induced cells possessed mature osteoblast property. The cells grow well on the surface of the chitosan/phosphonic chitosan sponge. The MTT test assay showed that the chitosan/ phosphonic chitosan sponge scaffolds couldn't inhibit the proliferation of hUCMSCs. The composite of hUCMSCs and Chitosan/Phosphonic chitosan Sponge can be used to construct tissue-engineered bone.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 undergo osteogenic and adipogenic differentiation. The phenotype changes during the process of MSC differentiation to osteoblast could be distinctly observed under atomic force microscopy. The chitosan/ phosphonic chitosan sponge has good biocompatibility. Tissue engineering of bone by combining hUCMSCs with chitosan/phosphonic chitosan sponge revealed bone formation potential in ectopic implant. The tissue engineering of bone is able to repair of bone defect.