生物瓣材料新型化学改性和内皮化的实验研究

New chemical modification and endothelialization of bioprosthetic heart valve materials: experimental study

目的通过对生物瓣材料应用新型化学改性剂2,3-丁二醇去除戊二醛的细胞毒性,实现人工内皮化,观察内皮细胞生长情况。方法对戊二醛固定的生物瓣材料牛心包片分两组处理:A组应用2,3-丁二醇改性处理,再用Ⅰ型胶原蛋白预覆;B组为对照组,不作任何处理。用消化法获取猪血管内皮细胞,体外培育至第3代,以高浓度种植至牛心包片表面,计算种植后第1、3、5、8天的活细胞数。体外孵育8 d后行第Ⅷ因子检测和扫描电镜(SEM)观察。结果 A组牛心包片表面细胞生长活跃,在第3、5、8天的细胞数明显多于B组,在第5天以后B组细胞全部死亡。种植后第8天,A组第Ⅷ因子相关抗原检测阳性率达90%,SEM显示A组牛心包片表面形成内皮细胞单层,细胞与基质间有微丝连接形成。而对照组牛心包片表面无细胞生长。结论经过2,3-丁二醇化学改性后可实现生物瓣材料的人工内皮化,并能形成稳定的微丝连接结构,具有良好的临床应用前景。

Objective To study a new chemical modification with the 2,3-butanediol for elimination of cell toxicity of glutaraldehyde（GA） and endothelialization of bioprosthetic heart valve material.Methods Bovine pericardia（BPs） fixed with GA were divided into two groups： Group A,modified with the 2,3-butanediol and precoated with collagen type I;Group B,non-treated as control.Endothelial cells（ECs） isolated from the porcine femoral vein were cultivated to 3rd generation in vitro,and seeded onto the surfaces of BPs with high density.Viable cell count was performed on day 1,3,5 and 8.Factor Ⅷ staining and scanning electron microscopy（SEM） were carried out on day 8.Results The amounts of viable ECs in Group A were significantly more than those in Group B On day 3,5 and 8（1.9±0.24 vs 0.2±0.02,3.8±0.45 vs 0,4.8±0.76 vs 0,respectively,P〈0.01）.ECs proliferated rapidly in Group A,while all died in group B after day 5.Factor Ⅷ positive cells were accounted for 90% in Group A on day 8.SEM showed ECs layer,spider-like junctions between ECs and matrix was formed in group A while no ECs were observed in group B.Conclusion Endothelialization of bioprosthetic materials with the 2,3-butanediol modified treatment in vitro is effective and potentially available for clinical use.