The Fabrication Of Bone Stromal Stem Cell Sheet And Culture Of Endothelial Progentior Cells

The fabrication of bone stromal stem cell sheet and culture of endothelial progentior cellsObjective:To purify and amplify canine bone marrow stem cells (BMSCs) and induce the BMSCs to differentiate into osteoblast cells and detect the biological characteristics of the BMSCs, such as bone formation and mineralization in vitro. To purify and amplify canine endothelial progenitor cells (EPCs) and induce EPCs to differentiate into endothelial cells. Application of Temperature-responsive culture dishes are applicated to harvest bone marrow stromal cells and prepare the BMSCs cell sheet. Demineralized bone matrix (DBM) are parpred.The seed cell, scaffold and growth factors are combined to build vascularized tissue engineering bone.Methods:Bone marrow stem cells were isolated with the method of density gradient centrifugation from canine and cultured. BMSCs were induced to differentiate to osteoblasts. The BMSCs were obseved under inverted microscope and scanning electron microscope. The BMSCs induced were transferred to the temperature-responsive culture dishes and cultured in incubator at 37℃,5% CO2 and saturated humidity. The temperature of the incubator was changed from to 37℃to 20℃fabricate BMSCs cell sheet. Endothelial progenitor were isolated with the method of density gradient centrifugation from canine and cultured. EPCs were induced to differentiate into endothelial cells with growth factors. The demineralized bone matrix was prepared. BMSCs cell sheet/BMSCs/EPCs complex was prepared to wrap thoracodorsal artery and vein of the dog.Results:1. The BMSCs cultured were fusiform and grew in whirl-like arrangement.2. The BMSCs induced were oval or polygonal and possess the ability of osteogenesis.3. when the temperature dropped at 20℃, BMSCs sheet detached automatically from the temperature-responsive culture dishes.4. Primary culture of endothelial progenitor cells were small round while the EPCs induced were fusiform.Conclusion:1. Endothelial progenitor cells can be isolated with method of density gradient centrifugation and induced to differentiate into endothelial cells;2. BMSCs cell sheet layer can be fabricated with temperature responsive culture dishes for functional osteogenesis.3. The preparation of BMSCs cell sheet/BMSCs/EPCs/DBM complex is useful for the contruction of vascularized tissue engineered bone.