The Studies Of Epoxy Chloropropane Treatment On Tissue Engineering Heart Valve

BackgroundAt present, tissue engineering heart valve is one of the focuses on valve surgery studies. But among them, biotic scaffolds still have problems such as calcification and progressive tissue failure, due to their physicochemical characters. Our study statistics suggest that EC is a nice anti-calcification agent to biotic valve. Compared to GA treated biological valves, EC treated biological valves exist much less calcification (down to 1/317), and better mechanical performance and tissue stability. And the number of porcine valve of our department (Xijing biotic valve) used clinically accounts to over 300. Previous studies demonstrated less toxic and better cell-living in EC treated biotic scaffold, however, the effect on anti-calcification and the physico-chemical characters are not clear yet. The ideal valve substitutes should have biological structures of native valves, adapt to growing, auto-repairing potential, life-long durability and no or few immunological response. To obtain ideal seed cells is the key point of study on TEHV. So to find a good way to obtain efficient seed cells is the core problem in TEHV study. In this study, we separated EPCs and MSCs from bone marrow. And the cells were cultured, multiplied, differentiated and investigated to discuss the condition and possibility of them as proper seed cells for TEHV.ObjectiveIn this study, we investigated the effect of epoxy chloropropane (EC) treatment on the physico-chemical characters and anti-calcification of decellularised scaffolds of tissue engineering heart valves.Method1. We produced osmotic pressure gradient and trypsinase digestion to decellularise porcine aortic valves. And the accelular proportions of these valves were tested by HE staining. We tested the EC treated and(or) GA treated decellularised scaffolds of porcine aortal valves by measuring plasma protein adsorption, platelet adhesion, ninhydrin reaction, and digestion resistance to collagenase enzyme, to investigate the effect of EC treatment on their blood compatibilities and collagen cross linkages. We HE stained the decellularised scaffolds which had been embedded in rat for 60 d, to investigate the calcification.2. Immunity magnetism bean was used to separate EPCs and MSCs. Cells were cultured, multiplicated and differentiated. Then they were subjected to identify the biology characteristic.①Culture medium for endothelial cells were used to culture cells in fibronectin conglutination covered plate. EGF, VEGF were used to induce EPCs differentiation. Immunohistochemical staining methods to detect CD31,Ⅷfactors, and DiI-Ac-LDL test were used to evaluate the function of endothelial cells.②MSCs were cultured with DMEM and were induced with 5-Aza. The differentiation of cells were analyzed byα-SMA and Vimentin tests, and collagen typeⅠandⅢstaining as well.Results1. We treated the fresh porcine aortal valves with trypsinase, detergents. And we produced osmotic pressure gradient to make the decellularised scaffolds. The HE staining demonstrated that there were few cell components in their valve leaflet, wall and smooth muscle. And there were generous cells in those places of untreated valves. We HE stained the DS which had been embedded in rat for 60 d. The calcification of EC treated DS reduced compared to of GA treated. Resistance to collagenase digestion revealed that modified collagen had greater resistance to enzyme digestion than untreated and GA treated groups did (p <0.01). Ninhydrin assay showed that the cross-linkage of EC treated collagen greatly increased as compared with untreated and GA treated groups(p <0.01). Both the plasma protein adsorption and platelet adhesion of DS reduced greatly after EC treatment comparing to GA treated groups (p <0.01). And the data of EC treated groups after GA pretreated above were even lower than untreated groups'(p <0.01).2. MSCs were separated from bone marrow. The primary culture of MSCs was polygonal morphology or spindle shape. Colonies were formed after 4-7 d.α-SMA, Vimentin, and collagen typeⅠandⅢpositive expressing were observed in these cells by immunohistochemical stain. EPCs showed a typical spindle-shaped morphology in the condition of endothelial culture medium, which were also separated from bone marrow. And cells looked like cobblestone after 12 d culture. Immunohistochemistry staining results showed CD31,Ⅷfactors expressed positively, and DiI-Ac-LDL absorption test was positive as well. ConclusionsIn this study, we investigated the effect of epoxy chloropropane (EC) treatment on the physico-chemical characters and anti-calcification of decellularised scaffolds of tissue engineering heart valves. We produced osmotic pressure gradient and trypsinase digestion to decellularise porcine aortic valves. We HE stained the decellularised scaffolds which had been embedded in rat and found that EC treatment could reduce or delay the process of calcification in DS of porcine aortal valves. And the increase of collagen cross linkage was demonstrated in EC treated DS. Both the plasma protein adsorption and platelet adhesion statistics proved that EC treatment could enhance the blood compatibility of DS. All of above demonstrate that EC treatment improves the physico-chemical character, biological compatibility and anti-calcification of DS of porcine valves. And these support a proper treatment of EC to construct the TEHV and need further studies.In this study, immunomagnetic sorting can efficiently separate and purify EPCs and MSCs from bone marrow. Morphorlogy shows that CD34+/- are similar to EPCs/MSCs as reported. Immunohistochemistry staining results show CD31,Ⅷfactors express positively in EPCs, and DiI-Ac-LDL absorption test is positive as well. These suggest that EPCs are able to differentiate into endothelial cells. MSCs areα-SMA, Vimentin, collagen typeⅠ,Ⅲpositive by immunohistochemistry stain, which suggests they could differentiate into vascular myofibroblasts. MSCs and EPCs obtained from bone marrow, have the great potential after induced differentiation, and show some advantages and possibilities to be seed cells for TEHV. However, further investigations are still needed on the biological, mechanical properties of TEHV both in vitro and in vivo.