新型脱细胞动脉基质的制备及理化性能评估

Preparation of the new acellular artery matrix and evaluation of its physicochemical properties

目的探索新型脱细胞动脉基质的制备方法,并分析评估其各项理化性能。方法采用自主设计的三联脱细胞程序,逐步去除动脉血管中的细胞和抗原成分。通过甲苯胺蓝、HE及天狼猩红染色和羟脯氨酸定量分析,对比脱细胞前后血管基质成分与天然半月板之间的差异;并采用Hoechst33258荧光染色法观察脱细胞前后动脉中DNA残留情况;通过MTT法考察材料经脱细胞程序后的毒性强弱以及对细胞活性的影响;将兔半月板细胞接种到脱细胞血管基质后培养,观察两者的相容性。结果三联脱细胞方法制备的牛大动脉基质的组织学染色结果与正常半月板类似,进一步胶原定量证实,材料经脱细胞后胶原含量无明显统计学差异;通过Hoechst33258染色,未发现血管基质中细胞核和DNA残留;甲苯胺蓝及天狼猩红染色证实脱细胞血管基质支架可以很好地保留胶原成分;MTT结果显示脱细胞动脉基质无细胞毒性,具有良好的生物相容性。半月板细胞接种材料后扫描电镜结果显示,脱细胞血管基质能很好促进细胞黏附,并诱导细胞的增殖。结论自主设计的脱细胞程序能完全去除主要抗原成分,无DNA及细胞碎片的残留,并保留了大部分细胞外基质成分、完整组织结构和良好的力学性能,同时具有良好的细胞生物相容性。对比性分析研究得出,在结构和成分上,脱细胞血管基质与半月板具有高度相似性,是未来非常有应用潜力的组织工程半月板支架之一。

Objective To explore the preparation method of new acellular arterial matrixes,and to analyze and evaluate its physicochemical properties.Methods Using self-designed triple linked acellular procedure,cells and antigen components in the arterial wall were gradually removed.Qualitative analysis was performed by toluidine blue staining,HE staining and picrosirius red staining.Furtherly,hydroxyproline quantification was used to analyze the difference between vascular matrix components and native meniscus before and after decellularization.Hoechst33258 fluorescence staining was used to observe the DNA residue in the artery before and after decellularization.Rabbit meniscus cells were seeded into acellular vascular matrix and cultured to observe the compatibility of meniscal cells and acellular vascular matrix through the level of OD value(MTT method)determining the material toxicity and the effect on cell viability after the acellular procedure.Results The results of histological staining of bovine aorta matrices prepared by triple decellularization procedure were similar to that of normal meniscus.Further collagen quantitation confirmed that there was no significant difference in collagen content after decellularization.There was no evidence of nuclear and DNA residues in the vascular stroma with Hoechst33258 staining.Toluidine blue and picrosirius red staining confirmed that the acellular vascular matrix scaffolds retained collagen components.The results of MTT assay showed that the acellular arterial matrix showed no cytotoxicity and possessed good biocompatibility.Scanning electron microscopy results of meniscus inoculated materials showed that acellular vascular matrix well promoted cell adhesion and induced cell proliferation.Conclusion The self-designed acellular procedure can completely remove the major antigenic components without DNA and cell debris residues,and retain most of the extracellular matrix components,complete tissue structure,good mechanical properties,and good cell biocompatibility.The comparative analysis of the study shows that the acellular vascular matrix and the meniscus have a high degree of similarity in terms of structure and composition.It is one of the most promising scaffolds for tissue engineering meniscal scaffolds in the future.