| Valve replacement is the very important device for heart valve disease. Nowadays, there are mechannical prosthetic valve and biological valve which were used in clinical application. And tissue valves are utilized more and more extensively. In developped countries, the amount of tissue valves which were used in clinical application is more than the one of mechannical valve, because of the life long anticoagulant therapy and complication with anticoagulation of mechannical valve leading to bad long term effect. On the contrary, only short term anticoagulant therapy after operation were needed with tissue valves, and tissue valves have better hemodynamics. However, the calcification and low durability shuould be pay attention. According to the recent findings, mineralization process in the cusps of bioprosthetic heart valves is initiated predominantly within nonviable connective tissue cells that have been devitalized but not removed by glutaraldehyde pretreatment procedures. This dystrophic calcification mechanism involves reaction of calcium-containing extracellular fluid with membrane-associated phosphorus, causing calcification of the cells. Some investigators have aimed to improve tissue fixatives other than glutaraldehyde. However, all these tissue fixatives could not meet the requirements for the preparation of the heart valve bioprosthesis.According to the study of TEHV, decellularization approaches may reduce host immune response to bioprosthetics and make the bioprosthetic matrix less prone to calcification. However, such an approach has been used on nonfixed homografts which were clinically implanted, yielding unfavorable results . The potential reason may be their inferior enzymatic degradation resistance and mechanical performance because of no crosslinking pretreatment. In this study, procyanidins which are new type biological crosslinking reagent were used to treat the decellularized valve matrix. The morphologic, physiochemical,tissue compatibility and anti-calcification properties were analyzed by in vivo and in vitro.PART I. Morphologic and physiochemical properties of decellularized porcine aortic valves treated with procyanidins. Fresh porcine aortic heart valves procured from a local slaughterhouse were used as raw materials. After Decellularization process, the decellularized valves were crosslinked by soaking in procyanidins solution with a concentration of 10 mg/ml in D-Hanks solution. Thickness, appearance, histology, water content, shrinkage temperature, breaking strength, soluble protein level, durability and crosslinking index of acellular porcine heart valve(DA) fixed with procyanidins(PA), glutaraldehyde(GA), and fresh ones(F) was tested respectively. According to the results, the cellular components of the porcine aortic heart valve leaflets were completely removed and extracelluar matrix were maintained completely after decellularized and crosslinking pretreatment. And the thickness(0.15±0.04)mm, the water content(89.1±3.4)%, shrinking temperature(71±4.1)℃in Group PA were similar to (0.16±0.07)mm,(88.7±4.7)%,(74±2.9)℃in Group F and (0.18±0.11)mm,(90.3±4.3)%,(73.3±5.1)℃in Group DA(p>0.05), while there were significant difference in Group PA and in Group GA(0.27±0.08)mm,(75.1±3.9)%,(87.4±4.3)℃(p<0.05). On the other hand, there were significant difference in soluble protein level(0.71±0.22)% in Group PA and the ones in Group F(2.16±0.32)%, in Group DA(1.47±0.27)%(p<0.05), while there were no difference in Group PA and in Group GA(0.76±0.16)%(p>0.05). The tensile strength of Group PA(11.8±0.91)mPa were more than others[ F(8.71±1.12)mPa,DA(8.25±1.07)mPa,GA(9.15±0.97)mPa ] (p<0.05). The durability experiment revealed that there were no change after conservation in PBS solution. And the 24h antienzyme experiment revealed that valve matrix in Group DA and in Group F were almost totally degradated after 2h, while the rate of weight loss in Group PA and in Group GA are only (10.21±1.8)%, (7.17±1.1)% respectively after 24h. From these results, the cellular components of the porcine aortic heart valve leaflets were completely removed and extracelluar matrix were maintained completely after treatments, and decellular porcine heart valve treated with procyanidins have nice biological and biomechanical characteristics.PART II. Biocompatibility of decellularized porcine aortic valves treated with procyanidins. The release of PA valves in PBS solution were analyzed. And L929 cells were cultured in the extraction botainted from decellular porcine heart valve treated with procyanidins to measure the relative growth rate by methyl—thiazolyl—tetrazolium (MTT)assay; L929 cells were cultured directly with decellular porcine heart valve treated with procyanidins. The cell morphology was observed,and the cell number was counted;And the morphology of L929 cells cultured on the surface of decellular porcine heart valve treated with procyanidins was studied by scanning electron microscopy(SEM).Results:The concentration of procyanidins in solution was 798.32±52.31 ug/ml after 1day, and 7.31±1.12 ug/ml after 4day in relaese analysis. Then there no significant release of procyanidins. The property of decellular porcine heart valve treated with procyanidins was stable and the toxic levels ranged from 0 to 1 grade.There were no signs of cytotoxicity induced by decellular porcine heart valve treated with procyanidins, and the cell morphology was norma1. Conclusion:Decellular porcine heart valve treated with procyanidins has good cytocompatibility in vitro.PART III. Calcification resistance of decellularized porcine aortic valves treated with procyanidins in vivo. The samples of procyanidins crosslinking decellularized valves, glutaraldehyde crosslinking decellularized valves and decellularized valves were implanted in rat subcutaneously. The retrieved grafts were stained with hematoxylin–eosin (H&E) and von Kossa and were analyzed with scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (SEM-EDS) after 21 and 63 days. Results: Histopathologic examination showed that procyanidins crosslinked valve matrix had no significant calcification in 21-day and 63-day rat study. And there were no calcium peaks in the EDS profile of procyanidins crosslinked samples in 21-day rat and 63-day rat study. Conclusion: The procyanidins crosslinked decellularized heart valves can resist calcification to some extent. And they have great tissue compatibility.PART IV. Seeding of human BMSCs on decellularized porcine aortic valves treated with procyanidins in vitro. After isolation, purification, culture and assessment, hBMSCs were seeding on decellularized porcine aortic valves treated with procyanidins with 2×106cm-2. Seeding PA valves were analyzed. Results: According to FCM, CD44 expressed strongly on the surface of the hBMSCs, while CD34 expressed lowly. At the same time, CD44 expressed strongly on the surface of the hBMSCs, while CD34 expressed lowly with immunohistochemistry. After seeding for 3 days, there were a single layer of cells on the the surface of PA valves according to HE stain. And after seeding for 7 days and 14 days, there were a thicker layer of cells on the the surface of PA valves, and cells could excrete ECM according to HE stain and SEM. Conclusion: PA valves can supply to the seeding cells microenvironment for living. And seeding cells can live normally and excrete ECM.In this study, we can prove that decellular porcine heart valve treated with procyanidins have nice biological and biomechanical characteristics, and good cytocompatibility in vitro. The procyanidins crosslinked decellularized heart valves can resist calcification to some extent. And they have great tissue compatibility. PA valves can supply to the seeding cells microenvironment for living. And seeding cells can live normally on the decellularized porcine aortic valves treated with procyanidins.
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