Experimental Study On Anti-calcification Of Interstitial Seed Cells And Acellular Scoffold Of Tissue Engineering Heart Valve

Cardiac valvular disease is a significant cause of mortality and morbidity in human being. The most common therapeutic procedure for the cardiac valvular disease is valve replacement. There are two kinds of valvular prosthesis: mechanical valve and bioprosthetic valve. Though it has been proved that all these valves are effective and has been shown to significantly alter the course of valvular disease, they are still far from an ideal one. Mechanical valves are associated with a significant risk of thromboembolism and require life-long anticoagulation. Bioprosthetic valves do not need life-long anticoagulation, but they are far less durable and are subject to progressive tissue deterioration. Homograft valves also have a limited resources and durability. None of available valve replacements has the capacity for growth and repair.The calcification and deterioration of bioprosthetic valve are hope to be solved by the development of tissue engineering heart valve(TEHV).But this is one hand ,in the another hand,calcification may be come into being when seed cells are culured in vitro and scoffold is remodeling because trauma,anoxic and malnutrition during the construction of TEHV,which will lead to the calcification of TEHV. Calcification of valve begins from cell and is associated with matrix,but the key tache is not clear.In this study, we observed the mechanisum of interistitial seed cells calcification in vitro and proved that bFGF could inhibit this calcification.Acellular scoffold was pretreated by epichlorohydrin (EC) and Triton X100 could alleviate the calcification in vivo.The effect on anti-calcification of acellular allograft preceded acellular xenograft significantly.This article is divided into three parts.Part 1:Canine saphenous vein interstitial ceslls(SVICs) were cultured and compared with canine aortic valve interstitial cells(CAVICs).The effect of bFGFon canine SVICs proliferation, free Ca²⁺i,apoptosis and the calcification in calcific medium was observed. Results: 1.Canine SVICs was mainly composed of myofibroblast,smooth muscle cell and fibroblast which was similarly to the compose of canine AVICs.They were the ideal interstitial seed cells sourse of TEHV.2.bFGF could promote the proliferation of canine SVICs,inhibit the formation of cellular nodules and the apoptosis by provoke Ca²⁺, exfflux to maintain the Ca²⁺j balance during long-term cultured in vitro.3.In calcific medium,CSVICs had osteoblast-like phenotype and cailcified,which was represented by the increase of activity of ALP and level of OC.Calicium deposition of cellular was increased significantly and calcific nodules formed by von kossa staining in calcific cultured. bFGF could inhibit the formation calcific nodules and maintain the low level of calcium deposition by inhibit the expreesion of ALP and OC from protein and transcription(mRNA) .This results suggested that bFGF could inhibit the calcification of canine SVICs as interstitial seed cells of TEHV during the long-term culturing in vitro.Part2: Porcine acellular aortic valve were obtained through a cellular exrtaction procedure with trypsogen and Triton X100.Then,the acellular aortic valve (AV)was pretreated by EC and Triton X100(ET).The structure and physichemical characteristics, cellular compatibility of two kinds acellular valve were compared.Fresh valve(FV),acellular valve(AV) and pretreated by EC and Triton XI00 valve (ET) were implanted into subderm of infant rat to observe the calcification with injection of vitamin D₃ to promote calcification. ET valved patch was fabricated based on posterior sinus then implanted into canine abdomen aorta.After 14 months,the patch was explanted to observe the remodeling. Results :1.The maximal tensile strength,shrinkage temperature(ST) and resisitance of collagenase of ET valve were superior to AV.But,cell toxicity was very lower and canine SVICs could growth very well on the surface of ET valve.2.Pretreated with EC+Triton XI00 could block the free carboxyl of AV.In explanted valve from rat subderm,the calcium content of FV increased significantly began 6w,which increased slowly in AV and maintained lower level in ET valve.After 6 weeks,by von kossa staining,a lot of serious calcific spots were found in FV and there were some scatter calcific dots in AV after 8 weeks,but none of calcific dot in ET after 8 weeks.3.The ET patch was explanted and test by HE, SEM, immunohistochemical, Sirius and von kossa staining.Interstitial cells growth into valve by vemitin staining and secreted type I III collagen and eastin. The surface of leaflets were partially covered with endothelial cells(ECs) .There was not calcific nods in valve .This results suggest that ET valve improved the physichemical characteristics,have good cellular compatibility,could alleviate calcification in vivo and remodeling very...