动物血管脱细胞方法及细胞外基质材料评价研究

采用独立设计的反复冻融方法,去除兔颈总动脉中的血管细胞,对所获得的细胞外基质进行组织、生化、力学等分析。在分离兔颈总动脉后,首先用低渗缓冲液处理,然后经低温反复冻融,最后用非离子型去污剂处理。所得的支架行组织染色和扫描电镜分析,显示基质的微观结构;荧光染色和基因组DNAPCR分析,检测细胞DNA残留;分别行羟脯氨酸定量分析和轴向拉伸强度分析,检测胶原蛋白和血管生物力学性能的变化;支架没有明显的细胞毒性,溶血率低于医用材料的标准;支架随时间缓慢降解。种植兔的骨髓干细胞,研究细胞在支架上的粘附生长能力。应用本方法能彻底去除血管细胞,没有细胞碎片和DNA的残留,并保留了较完整的细胞外基质和力学性能,具有开放的大孔径结构,细胞生物相容性好,是一种理想的组织工程血管支架材料。

Decellularization of Arteries and Evaluation of Extracellular Matrix as Scaffolds

To establish an efficient method for preparing decellularized rabbit carotid arteries used as scaffolds in small vascular engineering. Repeated frozen/thawing method was established to decellularize the rabbit carotid arteries. The arteries were treated with hypotonic solution, repeat frozen/thawing and non-ionic detergent. The obtained extracellular matrix (ECM) was examined with histochemical, biochemical and mechanical methods. Histological staining and SEM were performed to examine the micro architecture of the acellular scaffolds. The cellular and DNA remains were detected by Hoechst33258 staining and PCR of genomic DNA. The collagen retention was quantified by L-hydroxyproline assay. To examine whether the decellularization process impaired the mechanical properties of the vessels, the uniaxial tensile testing was carried out. Rabbit bone marrow mesenchymal stem cells (MSCs) were seeded onto the acellular scaffolds for evaluation of cellular attachment and growth. The vascular cells, cellular fragments and genomic DNA were removed completely by the established approach. The collagen content and the mechanical properties of the artery were not significantly changed after decellularization. SEM observation demonstrated excellent porous micro architecture of the scaffolds. The scaffold shows negligible hemolysis and cell toxicity, slow degradation and good biocompatibility to MSCs. This acellular artery matrix could be ideal scaffolds for tissue engineering of blood vessels.