比较两种方法制备脱细胞小血管支架

Fabrication of decellularized scaffold of small diameter vessels with two kinds of cell detergents

目的:比较0.5%Triton X-100+0.05%NH4OH和酶法制备脱细胞小血管支架的效果。方法:分别用含0.5%Triton X-100+0.05%NH4OH处理3 d或1.0%Triton X-100+0.125%胰蛋白酶+DNase+RNase孵育48 h,脱去犬股静脉中的细胞成分,60Co辐照消毒,血清浸泡24 h,将平滑肌细胞和内皮细胞接种到脱细胞支架中;进行H-E染色、胶原纤维和弹力纤维染色,扫描电镜观察及力学检测。结果:0.5%Triton X-100+0.05%NH4OH法完全地脱去了血管细胞;细胞外基质较完整地保留下来,其形态结构与脱细胞前无明显改变;见种植细胞在支架内生长良好,连成片,支架具有良好的生物相容性;力学结果显示其弹性回复率和最大断裂强度好于酶组。结论:两种方法比较,0.5%Triton X-100+0.05%NH4OH法简便易行,成本低,脱细胞效果好,组织相容性佳,对力学性状影响小,是比较理想的制备脱细胞小血管支架的方法。

Objective.. To compare the effect of fabricating decellularized scaffold of small diameter vessels with two kinds of cell detergents and to provide a homograft bioprosthetic scaffold for creating tissue-engineering small diameter vessel. Meth- ods： Fresh canine femoral veins were dipped in 0.5% Triton X-100-1-0.05%NH4OH for 3 days under 4℃, then washed by ion-free water for another 3 days,or incubated in 1.0% Triton X-100-1-0. 125%Trypsin＋DNase＋RNase for 48 h, and final- ly irradianted with Co60 （15 K） and fabricated grafts with seeding smooth muscle cells and endothelial cells. The small diameter vessels scaffold and the grafts were fixed with fixative and stained with hematoxylin and eosin, collagen fibers or elastic fibers for observation and photographs by light microscope and scanning electron microscope . Results.. These 2 methods could effectively removed the cells in fresh tissues because there was no visible nuclear stain. A series of biomechanical ana- lyses revealed that these vessels scaffolds had nearly the same mechanical properties as fresh tissues. Also, these vessels scaffolds showed good cell compatibility, and their surfaces were suitable for endothelial cells and smooth muscle cells to grow on. The active endothelial cells in the vessels were not only decellularized completely, but also reserved the collagen fi- bers or elastic fibers integrally, which are two of the main components of extracellular matrix. The morphologic structure of vessels after decellularization was not significantly different from that before being decellularized. The scaffolds mechanical property was better in Group 1 than Group 2. Conclusion： The better seeding scaffolds of vessels is by 0.5%Triton X-100-1- 0.05%NH4OH, which was benefit for adhesion and proliferation of seeding cells on the decellularized scaffolds of small diameter vessels in order to treat tissue engineering small diameter vessels in vitro.