Experimental Study On Artificial Biological Heart Valve Materials Crosslinked By Natural Occurring Reagents

PartⅠ:Crosslinking effects of quercetin(QCT) on porcine heart valve matrixBioprosthetic heart valve,prepared by glutaraldehyde crosslinking,has some limitations due to poor durability,calcification and immunogenic reactions.The aim of this study was to evaluate the crosslinking effect of a natural product quercetin on decellularized porcine heart valve matrix(ECM).After crosslinking,the mechanical properties,stability,anticalfication and cytocompatibility were examined.The results showed that the tensile strength of quercetincrosslinked ECM was higher than that of GA-crosslinked.Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any decrease of ultimate tensile strength and elasticity.After soaking in D-Hanks solution for 36 days,there was only 11.55%noncrosslinked excess quercetin released and no further release thereafter.Cell culture study shows that no inhibition on proliferation of vascular endothelial cells when quercetin concentration was lower than 1μg/ml.This non-cytotoxicity concentration was 100 times higher than that of glutaraldehyde.The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of glutaraldehyde crosslinked ECM.In vitro anti-calcification experiment showed that quercetin-crosslinking could protect ECM from deposition of minerals in simulated body fluid.The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively,which suggests that quercetin might be a new crosslinking reagent for preparation bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.PartⅡ:Crosslinking effect of Nordihydroguaiaretic acid(NDGA) on decellularized heart valve scaffold for tissue engineeringThe quest to design an optimized tissue engineering heart valve(TEHV) scaffold incorporates a similar micro-architecture to that of the native valve,adequate mechanical properties,ECM regeneration capabilities and a cytocompatible surface. The aim of this study was to explore crosslinking effect of Nordihydroguaiaretic acid (NDGA) on decellularized heart valve extracellular matrix as a TEHV scaffold.After crosslinking the tensile strength,the in vitro enzymatic degradation resistance,NDGA release rate and the cytocompability of NDGA to valvular related cells were examined. The results showed that the tensile strength of NDGA crosslinked scaffold was superior to that of GA crosslinked valve scaffold.Scaffold crosslinked by 2.5 mg/ml NDGA could be stored in PBS solution for a long period over 120 days without any decline in ultimate tensile strength and maintained.Furthermore,NDGA was found to release gentlely when the crosslinked heart valve stored in D-Hanks solution.After 8 days,there was no detactable free NDGA released from scaffolds.Under the concentration of 10μg/ml,NDGA has no toxic effect to valvular cells.In vitro enzymatic degradation revealed that crosslinked scaffolds could resist the enzymatic hydrolysis,and the resistant capacity was approximately the same as GA crosslinked valve scaffolds.The SEM images show that endothelial cells can attach and spread on the surface of NDGA-crosslinked valvular scaffolds after 24 hours.This study shows that NDGA can crosslink decellularized porcine heart valve matrix effectively as scaffolds for tissue engineering without toxicity.Our results indicated that this method might be a feasible approach for preparation of TEHV scaffolds.