Experimental Study Of Bio-compatibility Of Tissue Engineering Heart Valve Matrix Materials

Valvular heart disease is an important source of morbidity and mortality world wide. Currently, heart valve replacement with either a nonliving mechanical prosthetic valve, homograft or a xenograft valve is the most common and effective surgical therapy of valvular heart disease. Unfortunately, mechanical or biological valves have limitations not be conquered which include the inability to grow, repair, and remodel. Furthermore, complications of mechanical valves such as infection, thromboembolic phenomenon, and life-long anticoagulation-related hemorrhage may occur. Although biological valves (homograft, xenograft) are considered to be hemodynamically excellent and relatively resistant to infections and they do not require long-term anticoagulation treatment, these grafts have a limited durability resulting from their immunogenic potential and foreign body reaction and deterioration over time. In order to overcome the limitations of today's heart valve (HV) replacements, researchers have developed tissue engineering heart valve (TEHV) using the principles and technology of tissue engineering. Living heart valves can be engineered in vitro and the first TEHV leaflets were implanted successfully in pulmonary position in sheep in 1995. It open a new page with milestone significance inthe filed of cardiac valve.The concept of TEHV is autologous cells, expanded and isolated in the laboratory, are seeded onto appropriate carrier structures, the scaffolds, then is implanted into recipients after a certain time culturing in vitro. The cells start to develop a neo-matrix, while the scaffold material is bio-degrading, ultimately rendering a completely autologous, functional and living heart valve, providing the ability to grow, repair, remodel, and avoiding anticoagulation. Therefore, it is an optimal HV including adequate mechanical function, durability, excellent haemodynamic performance, as well as the absence of immunogenic and/or inflammatory reactions. The success of tissue engineered heart valves (TEHV) is dependent on three main issues: (a) the matrix (scaffold) which determines the three-dimensional shape and serves as an initial guiding structure for cell attachment and tissue development; (b) the seeded cells source from which a living tissue is grown; and (c) the in vitro culture conditions of the living construct before implantation.In this study, Endothelial cells (ECs) and interstitial cells (ICs) were served as seeded cells isolated and cultivated from explanted canine saphenous vein. To investgate microstructure, bio-mechanical properties, degradation time and histocompatibility / hemocompatibility / cell-compatibility of collagen-sodium hyaluronate acid composite membrane (C-SHA) and collagen-chitosan composite membrane (C-C) by comparison, and contrast with porcine acellular aortic valve and collagen membrane which have filtrated as TEHV matrix before this study in our lab. To filtrate a kind of promising matrix materials of TEHV that have good bio-compatibility, adequate mechanical function, appropriate degradation time and no toxic degradation and / or inflammatory reactions.Part 1: Microstructures were oberserved by scanning electron microscopy (SEM); Tensile test of bio-mechanical property; C-SHA , C-C compositemembrane and collagen membrane were implanted subcutaneously in rabbit in sterile conditions and extirpated at 2,4,6,8,12,14,16 weeks respectively to study its biodegradability and histocompatibility by general and HE; Biomaterials hemocompatibility were evaluated using the hemolysis test, the dynamic cruor time test, the adhesion of blood patelet test in viro. Result: The pore' s shape of porcine acellular aortic valve showed small round crevice in scanning electron microscopic views. Other three biomaterials showed round or ellipse three-dimensional interconnected pore like a honeycomb sponge, and all of their porosity demonstrated > 90% , and their pore sizes respectively were 112 ±9um,122±5um,131 ± 11um. Bio-mechanical test demonstrated the maximal tensile of matrixs are porcine acellular aortic valve > C-SHA composite membrane = C-C composite membrane > collagen membrane.The homolysis rate of all biomaterials were less than 5%. The curve of OD-cruor time showed three kinds of biomaterials declined slowly and OD value were high. Platelet adhesive test shows that the surface of C-SHA composite membrane and porcine acellular aortic valve and collagen membrane had less platelet than C-C composite membrane, moreover without obvious malformations and pseudbpod. Histocompatibility test showed all matrix materials' inflammatory cells disappeared at the 12th week, so its meet the demands of bio-materials implanted in vivo. Degradation duration C-SHA composite membrane, collagen membrane and C-C composite membrane were 12~ 14weeks, 14~16weeks, more than 16 weeks respectively. Its meet the demands of scaffold of TEHV.Part 2: Endothelial cells(ECs) and interstitial cells (ICs) were isolated and cultivated from explanted canine saphenous vein and characterized by immunohistochemistry. Results: 90% ICs expressed smooth muscle a -actin, it resembled with aortic valve interstitial cells (VICs) and can be served as seeded cells. ECs were characterized with anti-factor VIII antibody, it expressing rate reached 100% and can be served as seeded cells too.