The Differentiation Of Bone Mesenchymal Stem Cells On Artificial Scaffolds Into Esophageal Cells Towards Regenerating The Histological Esophagous

ObjectivesTo investigate the differentiation conditions and meschanism of bone mesenchymal stem cellsinto esophageal smooth muscle cell and epithelial cell in vitro and in vivo, aming at regeneratingthe defected esophagus with histological structure using the technology of MSC combining withthe artificial scaffolds of electrospining polycarbonate/silk fibroin (PCL/SF) sheet, de-cellulariedmucosa/submucosa, micro-channel patterned polyester urethane(PU).MethodsMSC were isolated from rabbit’s bone marrow with the method of density gradient centrifuge.Cells’ morphology and stem specificity were measured by immunefluorescence staining andflow cytometry. In order to induce them to differentiate into smooth muscle cells, MSCs werecocultured with primary smooth muscle cells which digested from rabbit esophageal muscle tissue.For differentiation into epithelial cells, MSCs were cultured in1640medium containing10%FBS,supplemented with20ng/mL epidermal growth factor (EGF),5ng/mL fibroblast growth factor(bFGF),5μM all-trans-retinoic acid (ATRA), and0.1μM dexamethasone.MSCs (3rd) were labeled with4,6-diamidino-2-phenylindole (DAPI) in the nucleus, and seededonto the PCL/SF/PU composite scaffold.The in vitro activity on those scaffolds were verified usingMTT method. These scaffolds were transplanted subcutaneously on rabbit back skin to evaluatetheir in vivo biocompatibilities. After that, these scaffolds with and without cells were transplantedinto esophagus of New Zealand rabbits whose esophagous had been partially defected, to followthe tissue remodeling and regeneration.ResultsFlow cytomety confirmed mesenchymal stem cells which were generated from rabbit bonemarrow using the method of gradient density centrifuge; immunohistological positive for CD29,CD44and CD90, and negative for CD34and CD45. The MSC were induced successfully into esophageal smooth muscle cells and epithelial cells, botn which were verified from proteinsecretion of α-smooth muscle actin (α-actin) for SMC and Cytokeratin14(CK14) for epithelialcell, respectively. For the in vivo implantation, all scaffolds (with or without cell seeding) exhibitedgood biocompatibility; the wound recovered comletely after30d of the operation, epithelializationand smooth muscle regeneration occurred after80d.ConclusionRabbit bone MSC were indcued effectively into smooth muscle cell and epithelial cell usingthe present protocols. The constructed-scaffolds with MSC seeding can modele the defectedesophagus without rejection and infection, via promoting the reepithelialization of mucosamuscular regeneration. Therefore it was considered to be a promising reconstruction for tissueengineered esophagous.