制备脱细胞全喉-下咽-食管复合支架用于咽喉重建的实验研究

Development of an acellular laryngo-esophagopharynx composite scaffold for laryngopharyngeal reconstruction

摘要:

目的 制备新型脱细胞全喉-下咽-食管复合支架，并探讨其作为咽喉重建替代材料的可行性。方法 通过成年新西兰大白兔颈总动脉灌注去离子剂制备脱细胞全喉-下咽-食管复合支架，并行HE染色、扫描电镜（SEM）、免疫原性检测及生物相容性分析；采用密度梯度离心法分离培养兔骨髓间充质干细胞（BMSCs），CCK8法分析支架浸提液的毒性作用，并将细胞复合于脱细胞支架上，体内埋植1周后观察细胞在支架上的粘附及增殖情况。结果 HE染色显示脱细胞全喉-下咽-食管支架除软骨以外的其他组织均仅剩细胞外基质（ECM），无细胞成分及结构残留，SEM电镜显示ECM的胶原纤维束，呈蜂窝状，无细胞结构，CCK8提示支架浸提液对细胞生长无明显毒性作用，且细胞可在支架上成功粘附并生长。结论 通过灌注法制备低免疫原性的保留ECM的脱细胞全喉-下咽-食管支架具有可行性。

关键词:头颈肿瘤;脱细胞;细胞外基质;全喉-下咽-食管复合支架;器官重建

Abstract:

Objective To prepare an acellular laryngo-esophagopharynx composite scaffold and explore its feasibility as an alternative material for laryngopharynx reconstruction. Methods The acellular laryngo-esophagohypopharynx composite scaffolds were prepared by perfusing detergent into the common carotid artery of adult New Zealand white rabbits and observed by HE staining, scanning electron microscope (SEM), immunofluorescence staining and biocompatibility analysis. Bone marrow mesenchyml stem cells (BMSCs) were isolated by density gradient centrifugation and seeded in the acellular scaffold. The toxicity of acellular scaffold extract was analyzed by CCK8. The scaffold with BMSCs was implanted in vivo for 1 week to observe the adhesion and proliferation of the cells in the scaffold. Results HE staining showed that there was only extracellular matrix (ECM) and no cellular components in the scaffold except cartilage. SEM revealed collagen fiber bundles of ECM, which were honeycomb without cell structure. CCK-8 assays indicated that the acellular scaffolds were not cytotoxic and BMSCs could adhere and grow in the scaffold. Conclusion It is feasible to prepare an acellular laryngo-esophagohypopharyngeal scaffold with native ECM of low immunogenicity by perfusion method.

Key words:Head and neck tumor;Acellularization;Extracellular matrix;Laryngo-esophagopharynx composite scaffold;Organ reconstruction