| ObjectivesThe ideal valve substitutes should have biological structures of native valves, can adapt to growing, have auto-repairing potential, life-long durability and no immunological response. Tissue engineering heart valves(TEHVs) constructed from autologous living cells theoretically have the potential to meet the characteristics of an ideal valve substitute because they have.the ability to self-renew, remodel, and thereby should have the best hemodynamic and biomechanical properties, as well as longer durability without the limitation of lifelong anticogulation. Significant stride toward the development of TEHVs has been made ,in recent years. Nevertheless, clinical application of TEHVs requires additional research and improvements in many aspects such as the cell sources, matrix material, cell seeding procedure and the culturing facilities. Seeding cells most commonly used now in are fibroblasts and endothelial cells derived from autologous vessel wall, so it's inevitably traumatic to the body to sacrifice a middle sized functional blood vessel. Creating TEHVs from vascular cells also involves complicated cell harvesting procedures and a relatively longer period of cultivation. Therefore vascular derived cells are not clinically an ideal cell source for the tissue engineering of heart valves. As for scaffold material, acellular porcine aortic valves turn to be an alternative due to incompetence of synthetic biodegradable polymeric scaffolds for constructing a heart valve leaflets and the shortage of allogenic valves. In vivo studies in animals demonstrated that acellular xenogenic valves have minimal immune responses. However early failure of decellularized porcine valve matrix has been reported in human trials, which indicated that decellularization methods involving detergents and/or enzymatic digestion were incomplete in cell extraction and further investigation, for ideal heart valve decellularizing agents are needed. In this study we investigated the feasibility of BMSCs to be induced to differentiate into endothelial cells(ECs) in primary culture in search of more convenient cell sources for TEHVs. Decellularization protocols composed of low concentration of trypsin, deoxycholic acid (DCA) and nuclease.were used here to develop more reliable matrices for TEHVs, in which native cellular remnants were completely eliminated and extra cellular matrix preserved undamaged. To evaluate the feasibility of fabricating TEHVs with BMSCs, differentiated cells from BMSCs were seeded onto the decelluarized matrices, and their growing characteristics were studied.Methods1. In vitro culture and induced differentiation of ovine BMSCsBone marrow was aspirated from iliac crest. BMSCs were obtained from isolation of the aspirate with Percoll step gradient, cultured and expanded in DMEM medium, characterized by flow-cytometry analysis of the expression of a -SMA and immunohistochemical staining of a -SMA and vimentin; Their synthetic function of collagen type I and type III were evaluated by immunohistochemical staining. PrimaryBMSCs were cultured in Ml99 medium supplemented with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and insulin like growth factor (IFGF), and induced to differentiate into ECs with vascular endothelial growth factor (VEGF). The phenotypes of differentiated cells were identified by flow cytometry analysis of CD34, CD31 antigen expression and immunocytochemical staining of CD34 and Factor Vi related antigen. Their functions were evaluated by ulex europaeus agglutinin (UEA) binding test and NO release analysis.2. Preparation of acelluarized porcine aortic valvesPorcine hearts were obtained under clean conditions within 20 minutes of slaughter and washed with 4 saline. Aortic valve conduits or leaflets were removed aseptically. Valve leaflets were sterilized by 75% alcohol and valve conduits by Y irradiation of 30kGray. The porcine aortic valve leaflets and whole aortic roots were acellularized with three different protocols: group with 0.01% trypsin + 1% Triton...
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