Constructed Primary Vascular Tissue: Seeding Mouse Embryonic Stem Cell-derived Vasculal Cells On PLGA

Objective:Since we have the vessel seed cells that Cultured Esc as embryoid body, then use the inductive factors to induce mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells, RT-PCR and immunohistochemistry were used to detect. The experiment use the cells that induced mouse embryonic stem cells to differentiate into smooth muscle cells and endothelial cells implanted on poly(lactide-co-glycolide)(PLGA)electrospinning,treated by gelatin infiltration, to construct a small-diameter vascular in vitro.Methods:①Detected the contact angle of the scaffold treated with/without 1%gelatin.②VSMc was seeding onto PLGA treated by gelatin, Culture scaffold 60 min ,the compatibility detected at initial stage by Toluidine Bluestaining (TB),proliferation were observed for the 1d and 3d scaffolds by SEM and MTT.③Implanted smooth muscle cells on one side of the PLGA other day seeding again ,then seeding endothelial cells on other side after 3 days. Culture the PLGA with the sheet of smooth muscle cells and endothelial cells on the surface 2 days,examined with the H&E and. Immunohistochemistry.④4-weeks-old Nude mice were implanted subcutaneously with vascular scaffolds and sacrificed 3 days,1 and 3weeks after implantation. The scaffolds were anayslzd using H&E staining and immunohistochemistry.⑤Endothelin were examined by means of radioimmunology in the culture solution, group A simple seeding vascular smooth muscle cell,groupB merely seeding endothelial cell, and group C seeding combined with vascular smooth muscle cell and endothelial cell, each group cultured 2 days after sending cells.Results:①Untreated group contact angle were 88.5°and treated group contact angle were 61.5°.②The VSMc distributed even dense on the treated PLGA ,SME observed cells extended 1h ,1d cell have grown into the pore ,and much layer cell distribution disorder; MTT find that cells proliferation on the treated PLGA is as well as the culture plate in the primary 3days.③H&E and immunohistochemistry prospected the VSMc and Ec distribution the scaffold each side.④The PLGA still conserved after implanted the Nude mice 3W, did not been completely absorbation or degradation ,the graft closed by the connect tissue, examined much layer VSMc at the lateral and one layer Ec on the tubule inside.⑤The levels of endothelin in the culture solution were 0.82±0.37 pg/mml, 5.59±1.23 pg/mml, 4.51±0.59 pg/mml respectively in the group A ,B and C.Conclusion:①The scaffold treated by gelatin improved the vascular smooth muscle cells adsorbability .②The tissue engineering vessel film have two-layer cells of vascular smooth muscle cells and endothelial cells by union seeding the VSMc and EC.③It harvest the engineering vessel with three- lay cells by implanted the Nude mice.