脱细胞支架复合犬骨髓源内皮祖细胞构建组织工程血管

Tissue-engineered blood vessels constructed by bone marrow-derived endothelial progenitor cells and decellularized vascular scaffolds

背景:目前临床使用的小口径(<6cm)人工血管因生物相容性差、远期通畅率低,效果并不理想。目的:将犬骨髓源内皮祖细胞与脱细胞血管支架动态复合培养,尝试构建一种全新的组织工程血管代用品。设计、时间及地点:随机对照实验,细胞学、组织病理学体外观察,于2005-12/2007-12在南京大学医学院附属鼓楼医院实验室完成。材料:通过去污剂-酶消化法制备犬颈动脉脱细胞支架;采用密度梯度离心法和内皮系条件培养法,分离扩增犬骨髓源内皮祖细胞,将扩增后的内皮祖细胞种植于脱细胞支架并置于生物反应器中动态构建组织工程血管。方法:20只犬均暴露两侧颈总动脉及一侧股动脉,每只犬在3段血管随机移植组织工程血管、脱细胞血管支架及自体静脉,分别为组织工程血管组、脱细胞血管支架组及自体静脉组,每组的移植血管数量均为20例。主要观察指标:对培养的内皮祖细胞进行免疫组化鉴定;血管移植术后6个月行数字减影血管造影、病理切片、扫描电镜等观察移植效果。结果:犬骨髓单个核细胞在体外培养10d后形成"铺路石样"细胞,免疫组化结果符合内皮祖细胞表型特征;将内皮祖细胞与脱细胞支架置于生物反应器培养10d后,种子细胞在血管腔内黏附生长;犬动脉移植术6个月后,组织工程血管及自体静脉通畅率分别为85%,90%,均优于脱细胞支架移植组25%。结论:犬骨髓源内皮祖细胞复合脱细胞血管支架,可获得一种具有良好生物相容性和通畅率的生物人工血管。

BACKGROUND： At present, the commercial artificial small diameter （〈 6 mm） vascular grafts are still unsatisfactory, due to poor biocompatibility and low long-term patency rate. OBJECTIVE： This study was to design a novel tissue engineering blood vessel constructed by dog bone marrow derived endothelial progenitor cells and decellularized allogeneic vascular scaffolds. DESIGN, TIME AND SETTING： From December 2005 to December 2007, the experiment, a randomized control trial of cells and histopathology in vitro, was completed in the Affiliated Drum Tower Hospital of Nanjing University Medical School. MATERIALS： The decellularized canine common carotids were obtained by a detergent-enzymatic procedure; Bone marrow mononuclear cells from mongrel adult dogs were isolated using density gradient centrifugation method and cultured in culture flasks coated with fibronecfin. Subsequently, the expanded endothelial progenitor cells were seeded on the decellularized scaffolds, and then co-cultured in the self-made bioreactor to construct the tissue engineering blood vessels. METHODS： Bilateral common carotid artery and lateral femoral artery in twenty dogs were all exposed. Three vascular segments from each dog were harvested and then randomly divided into three groups according to the transplant with the tissue engineering blood vessels, decellularized allogeneic vascular scaffolds or autogenous vein. Twenty vessels were implanted into the dogs of each group. MAIN OUTCOME MEASURES： Immunocytochemical staining was used to identify the cultured endothelial progenitor cells; After 6 months of transplantation, the grafts were retrieved for digital subtraction angiography, pathological test and scanning electron microscope examination. RESULTS： Dog bone marrow mononuclear cells presented a cobblestone-like appearance at 10 days of the culture. The immunocytochemistry results were consistent with the phenotype of endothelial progenitor cells; After highly proliferative endothelial progenitor cells were seeded onto the decellularized scaffold for 10 days, seed cells attached well in the lumen of blood vessels; Six months after implantation, the tissue engineering blood vessels were 85% patent and autogenous vein grafts were 90 patent, which was superior to decellularized allogeneic vascular scaffolds （25%）. CONCLUSION： This study provides a new strategy to develop a tissue engineering blood vessel with excellent biocompatibility and high patency rate by the co-culture of dog bone marrow derived endothelial progenitor cells with decellularized vascular scaffolds.