不同方法制备脱细胞猪主动脉瓣膜支架的组织结构

背景：理想的脱细胞方法要求既能完全去除供体细胞，降低免疫原性，又能保留天然瓣膜的胶原纤维、弹力纤维等细胞外基质成分，以保持足够的机械强度。 目的：采用不同洗剂制备脱细胞猪主动脉瓣膜支架，对比其组织结构，探讨最为有效的脱细胞瓣膜支架制备方法。 方法：20个新鲜猪主动脉瓣膜随机分为新鲜对照组、去污剂组、酶消化组和去污剂-酶消化组，后3组分别使用Triton X-100、胰蛋白酶以及二者联合的方法制备脱细胞瓣膜支架，对比支架大体形态、苏木精-伊红染色、Mallory-Heidenhain染色和电镜下超微结构的不同。 结果与结论：经脱细胞处理后，去污剂组瓣叶柔软、光滑，苏木精-伊红染色少量核物质存留、纤维排列规整，Mallory-Heidenhain染色胶原纤维和弹性纤维相交错，电镜下呈波浪状排列、原纤维横纹清楚；酶消化组瓣叶局部塌陷，苏木精伊红染色细胞完全去除、纤维排列较紊乱，Mallory-Heidenhain染色胶原纤维和弹性纤维呈网状排列，电镜下纤维部分断裂、原纤维横纹存在；去污剂-酶消化组瓣叶柔软、光滑，苏木精伊红染色细胞完全去除、纤维完整，Mallory-Heidenhain染色胶原纤维和弹性纤维平行排列，电镜下纤维完好，但排列稀疏，原纤维横纹清晰。说明3种方法均可有效去除供体细胞，保持纤维结构相对完整，在完全清除供体细胞并保持纤维支架完整性方面，Triton X-100联合胰蛋白酶的方法更为有效。

Histological structure of porcine aortic valve scaffolds decellularized by using different methods

BACKGROUND:An ideal decellularization method cannot only remove donorcells completely to decrease immunogenicity, but also reserve the extracellular matrix of natural valve, such as collagen and elastic fibers to keep sufficient mechanical strength. OBJECTIVE:To prepare porcine aortic valve scaffolds decellularized by different methods and to compare their tissue structures, as well as to explore the most effective methods for preparing decellularized valve scaffolds. METHODS:Totally 20 fresh porcine aortic valve scaffolds were randomly divided into four groups: control group, detergent group, enzymatic digestion group and detergent+enzymatic group. Decellularized valve scaffolds in the latter three groups were prepared by Triton X-100, Trypsin and Triton X-100+Trypsin methods, respectively. The valve scaffolds were contrasted by gross observation, hematoxylin-eosin and Mallory-Heidenhain staining, scanning and transmission electron microscope. RESULTS AND CONCLUSION:(1) After decellularized, the valve leaflets in the detergent group were soft and smooth. Besides, Hematoxylin-eosin staining showed that the cells were removed from the valve, but few nucleoli remained and fibers were arranged regularly. Mallory-Heidenhain staining showed that the blue collagen and the red elastin were interlaced. Electron microscope observation showed that the fibers presented with wave-like arrangement and lateral stripes could be observed. (2)The valve leaflets in the enzymatic digestion group were collapsed partly, but their endothelium was smooth. Hematoxylin-eosin staining showed that the cells were removed completely and the connective tissue structures were loose. Mallory-Heidenhain staining showed that collagen and elastin were found reticulated interlacing. Electron microscope showed that some fibers were ruptured, but lateral stripe existed. (3)The valve leaflets in detergent + enzymatic group was collapsed partly, but their endothelium was smooth. Hematoxylin- eosin staining showed that the cells were removed completely from valve. Mallory-Heidenhain staining showed that collagen and elastin showed parallel arrangement. Electron microscope observation showed that fibers were intact but scattered, and lateral stripe can be observed. These findings suggest that Triton X-100, Trypsin and Triton X-100+ Trypsin methods can remove valve cells effectively and keep the fibers intact; moreover, the Triton X-100 + Trypsin method is more effective.