灭菌处理牛心包制备脱细胞支架的内皮化

Endothelialization of acellular scaffold from sterilized bovine pericardium

背景:组织工程学研究常用的动物组织不可避免地存在各种微生物附着,而无菌是组织工程材料临床应用的一项基本要求。目的:观察体积分数75%乙醇灭菌对牛心包性能及生物相容性的影响。方法:将牛心包组织分别用无菌PBS(对照组)、含1%抗生素(青霉素/链霉素/两性霉素B溶液)的PBS、氯己定及体积分数75%乙醇进行灭菌处理。采用LB固体培养基评价4种方法的杀菌效果;采用VB染色评估4组处理对牛心包组织结构的影响;通过CCK-8实验测定4种处理抽提液的细胞毒性。将体积分数75%乙醇灭菌处理的牛心包制作为脱细胞支架,与人脐静脉内皮细胞共培养,观察细胞的黏附与内皮化效果。结果与结论:①体积分数75%乙醇和氯己定处理24 h的牛心包满足完全灭菌的要求,1%抗生素处理组和对照组可见明显菌落形成;②VB染色显示,体积分数75%乙醇、氯己定和1%抗生素处理的牛心包胶原纤维呈波浪状排列整齐,结构紧凑,弹性纤维含量较少但结构清晰;③体积分数75%乙醇灭菌处理的牛心包不影响L929细胞的增殖活性,培养1-3 d内的细胞存活率均在100%以上;氯己定灭菌处理的牛心包有很强的细胞毒性,导致细胞死亡;④人脐静脉内皮细胞可在脱细胞支架表面正常生长与黏附;在20 d的种植期内,第8-12天脱细胞支架表面黏附的细胞最多;⑤结果说明,体积分数75%乙醇能够有效消灭附着在牛心包上的所有微生物,不会影响牛心包的组织学完整性与生物相容性。

BACKGROUND: In the research of tissue engineering, there are inevitably various kinds of microorganisms attached to animal tissues, and asepsis is a basic requirement of clinical application of tissue engineering materials. OBJECTIVE: To investigate the effect of 75% ethanol sterilization on the properties and biocompatibility of bovine pericardium.METHODS: Bovine pericardial tissue was sterilized with sterile PBS(control group), PBS containing 1% antibiotic(penicillin/streptomycin/amphotericin B solution), chlorhexidine and 75% ethanol. LB solid medium was used to evaluate the bactericidal effect of four methods. VB staining was used to evaluate the effect of four sterilization treatments on the tissue structure of bovine pericardium. The cytotoxicity of four sterilized extracts was determined by the CCK-8 assay. The bovine pericardium was sterilized with 75% ethanol and then used to make acellular scaffold, which was co-cultured with human umbilical vein endothelial cells to observe the effect of cell adhesion and endothelialization. RESULTS AND CONCLUSION:(1) The bovine pericardium treated with 75% ethanol and chlorhexidine for 24 hours met the requirements of complete sterilization, and significant colony formation was observed in 1% antibiotic-treated and control groups.(2) VB staining revealed that the collagen fibers of bovine pericardium treated with 75% ethanol, chlorhexidine and 1% antibiotic were arranged in wavy pattern, with compact structure, less elastic fiber content but clear structure.(3) Bovine pericardium treated with 75% ethanol did not affect the proliferation activity of L929 cells, and the cell survival rate within 1-3 days was more than 100%. Chlorhexidine-sterilized bovine pericardium had strong cytotoxicity, leading to cell death.(4) Human umbilical vein endothelial cells grew and adhered normally on the surface of acellular scaffold. During the 20-day implantation period, the highest number of cells adhering to acellular scaffold appeared on days 8-12. These results suggest that 75% ethanol could effectively eliminate all microorganisms attached to the bovine pericardium without affecting the histological integrity and biocompatibility.