两种细胞洗涤剂制备脱细胞同种生物带瓣管道支架的研究

目的比较去氧胆酸钠(deoxycholic acid, DOA)和十二烷基硫酸钠(sodium dodecylsulphate, SDS)两种细胞洗涤剂制备脱细胞的同种生物带瓣管道(homograft bioprosthetic tube valved, HBTV)支架的效果,为体外构建组织工程瓣(tissue-engineering heart valve, TEHV)提供同种生物瓣支架材料. 方法对同种带瓣主动脉管道和带瓣肺动脉管道,分别用含0.5% DOA或0.03% SDS的Tris低渗缓冲液共同孵育48 h,脱去经液氮保存、具有活性的HBTV表面的内皮细胞,固定剂固定后分别进行HE、胶原纤维和弹力纤维染色的光镜观察,以及扫描电镜观察,摄片. 结果两种洗涤剂均完全地脱去了HBTV表面的内皮细胞,中心部位的成纤维细胞有部分存留;细胞外基质,如胶原纤维和弹力纤维等均较完整地保留下来,其形态结构与脱细胞前无明显改变. 结论 0.03%～0.1% SDS或0.5% DOA的低渗缓冲液对制备HBTV脱内皮细胞支架效果均较佳,有利于HBTV再内皮化时种植细胞的黏附和扩增,可进一步应用于体外构建TEHV的研究.对于脱主动脉壁的细胞,SDS浓度应增至0.1%为佳.

FABRICATION OF DECELLULARIZED SCAFFOLD OF HOMOGRAFT BIOPROSTHETIC TUBE VALVED WITH TWO KINDS OF CELL DETERGENTS

Objective To compare the effect of fabricating decellularized scaffold of homograft bioprosthetic tube valved (HBTV) with two kinds of cell detergents and to provide a homograft bioprosthetic scaffold for fabrication of tissue-engineering heart valve (TEHV). Methods The active cells in the HBTV, which conserved by liquid nitrogen, were decellularized by low osmotic pressure of Tris buffer, in which containing sodium dodecylsulphate (SDS) and deoxycholic acid (DOA) respectively. The leaflets or aortic wall was fixed with fixative and stained with hematoxylin and eosin, collagen fibers or elastic fibers for observation and photographs by light microscope or by scanning electron microscope (SEM) after decellularized. Results When the leaflets of HBTV were incubated together with 0.03% SDS or 0.5% DOA of Tris buffer respectively for 48 hours, the active endothelial cells (ECs) in the leaflets were not only decellularized completely, but also reserved the collagen fibers or elastic fibers integrally, which is two of the main components of extracellular matrix (ECM). A part of fibroblast in the center leaflets was reserved. The morphologic structure of leaflets after decellularized was not significantly different from that before decellularized. The concentration of SDS was increased to 0.1% when decellularized the cells of aortic wall, but DOA was still kept 0.5%. Conclusion The better decellularized scaffold of HBTV obtained was disposed by 0.03%-0.1% SDS or 0.5% DOA, which was advantageous to adhesiveness and amplification of implantation cells on the decellularized scaffold of HBTV in order that HBV reendothelialized or for the TEHV fabricated in vitro.