去细胞喉支架的制备及喉肌细胞再生的可行性研究

摘要

Objective To prepare a deeelhilarized whole laryngeal scaffold by utilizing a perfusion-decellularized technique, reseed cells on it, and construct recellularized laryngeal muscles. Methods Perfusion decelluarized larynxes were obtained by common carotid arterious perfusion with detergents. Then they were performed by macroscopic view, histological examination, scanning electron microscopy (SEM) and cartilage viability. Decellularized laryngeal scaffold were then reseeded with inducted mesenchymal stem cells (MSCs). Composites were transferred into greater omentums of rabbits after one day' s adherence and harvested after eight weeks. Macroscopic view, histological examination and immunohistochemistry were performed. Results Perfusion larynxes became transparent after two hours. Histology and SEM indicated that perfnsion method shewed better deculluarized effect. More ventages and collagen fibers but no intact cell or anclei were retained in the decellularized martrix. Porosity measured by Image pro plus 6. 0 was 80. 4% ± 3.2% (x ± s). Chondrocyte vitality assay indicated chondrocyte vitality rate in the perfusion group was 86. 9% ± 1.5% . After eight weeks, vascularization formed and integrated cartilage frameworks still remained. Histological examination could clearly show the presence of muscle bundles and vessels. Immunohistoehemical examination indicated that sarcomeric-α actin expressed positively in corresponding areas. Conclusions It is feasible to reseed MSCs into the decellularized laryngeal muscle matrix for constructing tissue-engineered laryngeal muscles. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.%目的 探讨灌注法去细胞技术制备去细胞全喉支架及利用骨髓间充质干细胞使喉肌细胞再生的可行性.方法 新西兰大白兔作为喉支架供体,通过双侧颈总动脉顺行灌注去离子剂,构建出喉肌去细胞外基质及活性软骨的全喉支架,取部分灌注后标本进行观察,其余用于下一步实验.青紫蓝兔为受体兔,取其骨髓问充质干细胞诱导培养为骨骼肌细胞后注入全喉支架的去细胞基质中.体外培养1 d后异位移植入受体兔大网膜.8周取出标本进行观察.结果 大体观察发现喉在经去离子剂灌注后2 h已经呈透明状态,组织学及扫描电镜显示喉肌去细胞基质内无细胞残留物,纤维网状结构存在,孔隙率80.4%±3.2%(x±s,下同);软骨细胞存活率86.9%±1.5%.移植至大网膜的喉体4周和8周取出时大体观察发现喉支架完整,其表面有血管形成.组织学显示肌束存在.免疫组织化学检测α-肌动蛋白表达阳性.结论诱导后的骨髓间充质干细胞注入经去细胞技术处理的全喉支架可以构建出保留软骨支架并含有再生肌肉组织的喉,植人大网膜内生长有助于其血管化.