口腔颌面组织工程血管的初步构建

Preliminary construction of the tissue-engineered oral and maxillofacial vessel

背景:大型骨缺损因缺乏有效血供,当骨的厚度过大时,骨细胞就会因缺乏营养而坏死,限制了工程骨在大型骨缺损中的应用。目的:分析兔骨髓基质干细胞和脐静脉血管内皮细胞体外培养后与自制兔主动脉脱细胞血管基质构建人工血管的可能性。方法:全血贴壁法培养兔骨髓基质干细胞,体外分离培养人脐静脉内皮细胞,制备兔主动脉脱细胞血管支架。把培养后的干细胞、内皮细胞与脱细胞血管支架复合,观察与载体材料的生物相容性并进行电镜扫描观察。血管支架-细胞复合物分为3组:空白组(空白组支架)、对照组(由骨髓基质干细胞复合血管支架构成)、实验组(由骨髓基质干细胞与内皮细胞复合血管支架),每组各10条。以上3组分别植入兔皮下小血管旁。术后3个月取血管标本,进行大体观察及苏木精-伊红染色组织切片检测。结果与结论:(1)全血贴壁可形成成纤维样细胞克隆,细胞爬片后CD44+、STRO-1+免疫荧光测定显示阳性;钙化结节茜素红染色阳性。内皮细胞经流式细胞仪测CD34+占43.83%,Ⅷ因子免疫荧光检测阳性;(2)血管支架-细胞复合物经电镜扫描观察内细胞在支架表面形成单细胞层。3个月后皮下人工血管生长较好,均形成管状结构;空白组薄层膜样血管形成,对照组仅有内皮样组织形成血管,实验组有8条血液流通样血管形成;(3)结果说明,血管支架-细胞(内皮、干细胞)复合物兔皮下血管旁植入后,可形成血管样结构,细胞间未见免疫反应产生,但人工血管未见肌层形成。

BACKGROUND： Insufficient blood supply after major bone defects leads to osteocyte necrosis; therefore, the tissue-engineered bone cannot meet the clinical requirements. OBJECTIVE： To construct an artificial vessel using in vitro cultured rabbit bone marrow stromal stem cells and human umbilical vein endothelial cells combined with the rabbit aortic decellularized vascular matrix. METHODS： Rabbit bone marrow stromal stem cells were cultured by adherent method, human umbilical vein endothelial cells were cultured in vitro and isolated, and rabbit aortic decellularized vascular stent was prepared. The stent-cell composite was constructed, and its biocompatibility was observed under scanning electron microscope. There were three groups： blank control, control（bone marrow stromal stem cell-stent） and experimental（stent-cell composites） groups（n=10 per group）, followed by implantation around the small vessels. Three months later, the vascular samples were collected for gross observation and hematoxylin-eosin staining. RESULTS AND CONCLUSION： Fibroblast-like cell clones were formed by adherent method, and were positive for CD44^＋ and STRO-1^＋ after climbing; calcified nodule formed in the ossification. Flow cytometry showed CD34^＋ accounting for 43.83%, and visible VIII factor in the endothelial cells. In the experimental group, endothelial cells formed a single cell layer onto the scaffold under scanning electron microscope; the artificial vessel grew well and tubular structure presented and eight angiod structures were found. Thin film-like vessels formed in the blank control group and there were only endothelial-like tissues in the control group. These results suggest that angiod structures can form after the stent-cell composite implantation, causing no immune reaction, but the artificial vessel is visible in absence of muscular layer.