叠氮钠对脱细胞组织工程心脏瓣膜形态学及生物力学的影响

Effect of sodium azide on morphology and biomechanics of the decellular tissue-engineered cardiac valves

目的对比加叠氮钠和传统的去氧胆酸钠法去除细胞后心脏瓣膜形态学及生物力学差别，为构建理想的组织工程心脏瓣膜提供实验依据。方法应用两种方法处理6～7月龄新鲜猪主动脉心脏瓣膜。光学显微镜、透射电镜观察脱细胞基质改变，厚度仪测量组织厚度，行拉力测试观察两种方法处理后力学变化上的差异。结果两种方法处理后组织厚度差异无统计学意义（P〉0．01）。显微镜检和透射电镜见叠氮钠-去氧胆酸钠法对基质破坏较少，处理后生物力学优于传统去氧胆酸钠法，差异有统计学意义（P〈0．01）。结论叠氮钠-去氧胆酸钠法能更好保护去除瓣叶组织后的细胞外基质。

Objective Heart valves made by tissue engineering procedure may be superior to those by conventional procedure. Optimal techniques for decellularzation from pretreated heart valves should preserve maximtun matrix, which is associated directly with the mechanical strength and durability of the valves. The study was designed to compare the morphology and biomechanics of the matrix after deceUularizaflon procedures with conventional sodium-deoxycholate method and the addition of sodium azide to the former method, in order to provide experimental basis for producing ideal tissue-engineered heart valves. Methods Fresh aortic valves from porcine of 6 to 7 month were divided randomly into two groups （60 valve leaflets in each group）. Valves in one group were treated with Triton-X100 and sodium deoxyeholate, and those in the other group were treated with identical chemicals and sodium azide, Changes of the matrix structure and effects of treatment on the collagen and elastic fibers were observed by microscopy（with hematoxylin and eosin stain, Masson＇s trichrome stain and Weigert＇ s stain for elastic fiber） and transmission electron microscope. Tissue thickness （60 specimens） and biomechanics properties （50 specimens） were measured. Biomechanics characteristics measured after decellularization were maximum deflection, elongation rate, max tensile stress and max load. Statistical analysis was performed to compare the thickness and mechanical properties after two decellulaxization procedures. Results No significant difference in tissue thickness was found between two methods（ P 〉 0.01）, but biomeehanies properfies-msaximum deflection, elongation rate, max tensile stress and max loadwere increased significantly in the group with sodium azide （ P 〈 0.01 ）. Same results could be obtained through Tensile deflection- Tensile stress and Tensile deflection-Load curve. Although complete decellularization was achieved, matrix stmctttre was comparative integrity in the group treated with sodium azide. Intact, dense collagen fibers and plush-like fibers were seen in the experimental group, while sparse collagen fibers and less velvet-like fibers were present in the control group. Complete and continuous elastic fibers were preserved in the specimens treated with sodium azide while discontinuous, broken and thin fibers were seen in the control group. The pattern of ultrastmcture in the sodium azide group revealed matrix in high density and more fiber bundles in the field. In the control group, the quality of the matrix decreased significantly, and loose fibers with apparent gap were seen. Conclusion Sodium azide can preserve the matrix structure efficiently during the decellumzation procedure and improve the bio-mechanical properties of tissue engineered cardiac valves.