| Annually more than 175,000 artifical heart valves have been used clinically all over the world. However, neither bioprosthetic heart valves nor mechanical valves could work ideally. Bioprosthetic heart valves have central flow and good hemodynamics. And usually have good thrombo-resistance in comparison to mechanical heart valves, hence, patients having a single bioprosthetic heart valve need not be maintained on anticoagulant treatment. But in the long run, the application of bioprosthetic heart valves is restricted due to calcification-related failure. Therefore, great many of researches on mechanism of calcification and anticalcification of bioprosthetic heart valves have been done and some methods for anticalcification were obtained. The available anticalcification methods can alleviate calcification , but all of them are effective enough. Our lab previously reported a highly efficacious epoxy chloropropan (EC) pretreatment of BPHVs for the prevention of valve calcification. In vivo calcification, calcium in EC pretreated tissue deposition were significantly decreased, when compared with the GA treated tissue. It is one of the most effective method amongcommercialized products, and it also maintain tissue's structure and stability. EC pretreated porcine aortic valves has been used clinically for 15 years. Totally, more than 300 patients have used the bioprosthesis and until now none of them has occurred calcification. Primary research indicated that the anticalcification mechanism of EC pretreatment is probably related to react with the free carboxyl group in protein of the tissue. However, the mechanism has not been clarified precisely. Here we further explored the anticalcification mechanism of EC pretreatment by identifying the reaction between EC and amino acid, EC and protein, as well as the effect of EC pretreatment on the valve structure, surface characteristic, etc. ObjectiveTo explore the anticalcification mechanism of EC pretreatment to bioprosthetic heart valves, and provide theoretical basis to improve EC pretreatment and its clinical application. Method:1. the research of reaction between EC and amino acid: Deduce the possible reaction between EC and amino acids such as glycine, praline, 6-aminohexanoic acid, etc. Detect the chloric ion by silver nitrate titration and chloric electrode measurement; The reaction was also observed by ultraviolet spectroscope and FTIR spectroscope.2. the research of reaction between EC and collagen: Modified collagens(fresh, glutaraldhyde-pretreated and glutaraldhyde plus EC pretreated collagens) were characterized by measuring digestion resistance to collagenase enzyme, and also measured the crosslinking value by ninhydrin reaction .EC-treated collagens after lyophilized were also observed by FTIR spectroscope.3. the research of reaction between EC and valves: Modified valves (fresh, glutaraldhyde-pretreated and glutaraldhyde plus EC pretreated porcine aortic valves) were characterized by measuring shrinkage temperature, digestion resistance to collagenase enzyme, in vitro plasma protein adsorption and platelet adhesion, and also measured the cross-linking value by ninhydrin reaction . EC-treated valves were also observed by FTIR spectroscope.4. The valves were divided into four groups: fresh porcine valve group, GA treated valve group, EC treated valve group, and GA+EC treated group. Two calcification models, calcium deposit experiment in vitro and rat subdermal implantation, were adapted to evaluate the calcification of differently treated valves. The calcium content was measured by atomic absorption spectroscopy. Fresh porcine valve and only porcine valve treated with GA were tested as control.Results:1. Five minutes after EC and amino acid were mixed in solution, a great deal of chloride ion was detected by chloric electrode measurement, and white sediment was observed after silver nitrate was added, which indicated that nucleophilic substitution occurred. At the same time, a strong absorption peak at 295 nm was detected in the solution by ultraviolet spectroscopic analysis, which suggested that a process of polymerization was induced by the reaction of EC with amines. FTIR spectroscopic analysis showed a new absorption peak at 1743.67 cm"1, which suggested carboxyl group may beinvolved in the reaction.2. Resistance to collagenase digestion revealed that modified collagen had greater resistance to enzyme digestion than did fresh collagen and GA treated collagen. Ninhydrin assay showed that the cross-linking of EC treated collagen increased as compared with fresh group and GA treated group. An overlay of ATR-FTIR spectra for collagens (control and EC-pretreated) is obtained by FTIR spectroscope. There were considerable changes in the carbonyl stretching region (amide I) due to the EC pretreatment. After EC pretreatment, the band at 1743 cm"1 was considerably reduced in intensity and the band at 1649 cm"1 was increased in intensity as compared to controls. There were no changes in other regions of the spectra.3. Collagenase assay indicated that the glutaraldhyde plus EC pretreated valves showed better ability to resist collagenase degradation. The thermal denaturation temperature of the valves is higher than control group. Ninhydrin assay showed that the cross-linking of EC treated tissue increased as compared with fresh tissue and GA treated tissue. Lower protein adsorption and platelet adhesion were observed on modified tissue than non-modified tissue.4. In the calcium deposit experiment in vitro, the calcification of each group increased as time prolonged, and at the third day, the calcium content of each group began to show difference between the groups, and at the 21st day, the calcium content of each group is: GA group 584.2±56.9umol/g, EC group 312.8±43.1umol/g, fresh valve group 205.7±31.7umol/g, GA+EC group 175.3±21.6umol/g. In the rat subdermal implantation test, at the 6th week, the...
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