Primary Experimental Study Of Tissue Engineered Heart Valve Based On Acellular Porcine Aortic Valve

Despite distinct advantages over mechanical cardiac valve prostheses, theuse of bioprosthetic valves remains limited, due to poor long term durability, primarily as a result of tissues calcification. Tissue-engineered valves hold the promise of generating completely biological, autologous "living" valves with functional features of their native counterparts, could possibly overcome these problems. The principal idea of tissue engineering is the use of an biodegradable matrix, such as acellularized porcine valve tissue(APHV), followed seeding seeded cells. 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 cell source from which a living tissue is grown; and (c) the in vitro culture conditions of the living construct before implantation.This study compared the effects of different cell extraction procedurs and different chemicals (TritonX-100, DOA, and SDS) on porcine aortic valve cellularity, structure, and mechanical properties. In the study ,we evaluated theeffects of acellularity procedure on porcine endogenous retrovirus. Interstitial cells(ICs) and endothelial cells (ECs)of aortic valve, aorta and saphenous vein were isloted and campared. We seeded ICs and ECs on leaflets and roots of acellular porcine aortic in different conditions . The repopulation results of APAV were compared. We implanted APAV as a valve patch in the abdominal aorta in a canine model. The changes were observed 1 month upto 12 months after operation.The study were divided into four parts. Parti:APAV were obtained through different cellular exrtaction processes and detergents(TritonX-100,DOA,SDS). We make our option on the effects of different processes and detergents on structure characteristics, mechanical properties of porcine aortic valve. Biocompatibility and degradation rate of porcine acellular aortic valve were stuied in a growing rabbit model. Results: Treatment of porcine aortic valve with DOA could not remove all the cells. Treatmnet with SDS disrupted collagen fiber structure .resulting in poor mechnical properties. Treatment with TritonX-100 result in an acellular porcine aortic valve matrix with retention of near normal structure and mechanical properties. Using hypotonicand (hypertonic) solutions could benefit the remove of the cells. Temperature and shaking rhythm have some effects on efficacy of acellularised process. The results showed that aortic valve had no significant inflammatory reaction. The degrees in inflammatory reaction for the APAV were signicantly less than NPAV and native rabbits' aorta, and similar to acellular rabbits' aorta. The difference in degradation rate between the native and acellular tissues were signicant.The subcutaneous degradation duration of the porcine cellular aortic valve, and their acellualr countparts are 12 weeks, and 16-18 weeks, in respectively.Part 2:This study examined potential transpeciese infection of PERV by in vivo canine model to assess the safety of tissue engineered heart valve based on APAV. We screened for PERV infection among Chinese pigs of different species. APAV were implanted in the abdominal aorta in 8 dogs. Blood samples of dogs were collected 1 months to 12 months after operation and tested for PERV by means of polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). Results:PERV was easily detected in native porcine aortic valves and porcine blood samples; No PERV sequences were detected in APAV and canine blood samples. Our acelluarisation process can prevent cross-species transmission of PERV. Acellularised porcine matrix scaffold can be used for cardiovascular tissue engineered grafts without risk of PERV transmission. Part3:The ICs and ECs from canine aortic wall, aortic valve and saphenous veins wall were isolated, cultured and compared..The expression of smooth musclea-actin, microstructure characteristics and cellular growth curves of these cells were studied. Optimal precondition of the scaffolds and intervals for seeding cells were investigated using the MTT assay. Results: There are little differece in cell phenotypes from different tissues. Transparent electron microscopy shows there are muscle actin and rough endogenous reticulum (RER) in the cytoplasm of some of the interstitial cells from different tissues. Immunohistochemistry also showed that part of the canine interstitial cells from different tissues express smooth muscle a -actin.However,there are more a -actins in the interstitial cells from the root of the valve than cells from other tissues. There are no significant differnce in growth curve between cellsfrom valves and saphenous vein.. The VIII factor expression of ECs from different tisuues were similar. Regarding clinical feasibility,we chose cells from saphenous veins as seeded cells. Adherence of seeded cells to acellular matrix precoated with autologous serum was greater than adherence to acellular matrix precoated with fetal bovine serum and fibronectin The results also showed that repeated seeding of cells can improve the efficacy of cell seeding. Part 4:The porcine acellular aortic valve were implanted into the canine abdominal aorta as a valve patch to observe their morphologic and histologic changes. Doppler echocardiography examinations were performed to evaluate valves station at 1, 3, 6, 12 months postoperation. The animals were sacrificed ,and the valves were examined histologically and biochemically.Result:Leaflet motion could observed on two-dimensional echocardiography at 1 month, 3 months and 6 months of follow-up. The aorta with acellular valve patch were patent with no narrowing,without thrombus organization; The leaflet montion disappeared at 12 months. However, the aorta with acellular velve patch were still patent with no narrowing,without thrombus organization at 12 months. The histological examination revealed the following features:(1)no inflammatory response to the valve leaflets or adjacent aortic wall;(2)ingrowth of fibroblasts-like cells through the body to the tips of the leaflets;(3)partial endothelialization across surface of leaflets(covered by flattered endothelial-like cells);(4).partial repopulation of the root Conclusion:It could be concluded that acellular porcine aortic valve matrix can offer the prospect of a graft "platform" which, through healing by endothelial cells...