Experimental Study On Tissue Engineering Small-diameter Vascular Prosthestic Model

[ Objection ] To harvest and isolate bone marrow mesenchymal stem cells (BMMSCs), and to induce and differentiate them into vascular smooth muscle and endothelium-like cells.[Methods]1. Isolation, culture and proliferation of BMMSCsBone marrow mononuclear cells, which obtained sterily by Ficoll-Paque density gradient centrifugation, were cultured and expanded. The cells were frozen at ⁸0℃, and then were thawed and replated in expansion medium2. BMMSCs differentiated into vascular smooth muscle-like cellsBMMSCs were cultured in DMEM—LG with 10^-8M all-trans retinoic acid(AT-RA),0.5×10^-3M Dibutyryl c-AMP (db-cAMP) for 3 days . Afterwards, they were maintained in DMEM-LG with 15%FBS for 21 days.3. BMMSCs differentiated into endothelium-like cells:BMMSCs were treated in medium with 10ng/ml VEGF for 21 days. [Results]1. Characterization of BMMSCs derived from bone marrowA total of 5ml bone marrow was obtained from the rib of each adult (n=5 donors). After 5 passages of culture in vitro, approximately 4×10⁸BMMSCs were obtained from each sample, and after 10 passages, about 8×10¹⁰ cells. BMMSCs expressed higher level of CD105 and lower level of KDR.2. Differentiation of BMMSCs into vascular smooth muscle-like cells Expression of smooth mucle actin (SMA) protein was maximal after 12 daysand persisted afterwards. A confluent layer of cells appeared a Peak-valley-like morphology. The level of type I collagen was 18.093±8.968ug...