人骨髓基质细胞定向培养为内皮细胞应用于心脏组织工程瓣膜

Application of endothelial cells cultured from human bone marrow stromal cells in tissueengineered heart valve

目的:观察在脱细胞生物瓣支架上种植自体内皮细胞后完成体外构建生物组织工程瓣的可行性。方法:实验于2002-12/2004-12在中国医学科学院阜外心血管病医院先心病研究室和解放军军事医学科学院全军造血干细胞中心实验室完成。①骨髓样品取自军事医学科学院附属医院全军造血干细胞移植中心的健康成人献髓员,从正常成人骨髓中分离骨髓基质细胞进行定向培养和扩增其中的内皮细胞。②十二烷基硫酸钠脱去成人主动脉带瓣管道表面的内皮细胞后作为支架材料;脱细胞的同种生物瓣支架在无菌条件下,剪切成0.8cm×0.8cm的大小,种子细胞选择人骨髓基质细胞体外定向培养的内皮细胞,高密度(>105cm-2)种植于瓣膜支架上静态培养20d。③分别在静态培养的第7,14,20天结束时取瓣膜片用0.5%硝酸银染色行立体显微镜和扫描电镜观察、摄片。瓣膜被内皮细胞覆盖的面积用百分数表示,以确定其再内皮化率。结果:①5.0×105个骨髓基质细胞体外定向培养的内皮细胞共传8代,最后获得了6.0×109个内皮细胞,扩增了约1.2×104倍,可以满足高密度种植内皮细胞的需求。②0.03%十二烷基硫酸钠完全脱去了同种生物瓣表面的内皮细胞,而瓣膜的细胞外基质成份在脱细胞前后均无明显变化,其形态学结构保持良好。③内皮细胞与支架复合体静态培养第7,14,20天,再内皮化率分别为65%、85%和90%。结论:骨髓基质细胞定向培养的内皮细胞,经扩增后是瓣膜组织工程研究中种子细胞的又一来源;静态培养条件下基本实现了生物组织工程瓣体外构建的目标,为进一步脉动流培养以及动物实验提供了材料基础。

AIM： To observe the.feasibility of constructing tissue-engineered heart valve in vitro by implanting endothelial cells （ECs） onto the decellularized scaffold of homograft bioprosthetic valve （HBV）. METHODS： The experiment was carded out in the laboratory of Congenital Heart Diseases, Cardiovascular Institute of Fuwai Hospital, Chinese Academy of Medical Sciences, and the laboratory of the Hemopoietic Stem Cell Transplantation Center, affiliated hospital of China Military Medical Academy of Science between December 2002 and December 2004. ①Human bone marrow stromal cells （BMSCs） were Obtained from the health adult bone marrow donators of the laboratory of the Hemopoietic Stem Cell Transplantation Center affiliated hospital of China Military Medical Academy of Science. The BMSCs were separated from normal human bone marrow and orientational cultured, and the endothelial cells （ECs） were amplified. ②ECs on the surface of the aortic tube valve were removed with sodium dodecyl suffate （SDS） to prepare the scaffold material. The decelluladzed homograft bioprosthetic valve scaffold was cut into 0.8 cm×0.8 cm. Orientational cultured ECs of hBMSCs were selected as the seed cells and implanted onto the decellularized scaffold with high-density seeding （〉 10^5 cm^-2） under static culture In vitro for 20 days. ③The morphologic structure of valve sections was observed and photographed by scanning electron microscope and stereomicroscope with 0.5% silver nitrate （AgNO3） staining at days 7, 14 and 20 of culture. The retention rate of the ECs overlaid onto the constructs was evaluated with percentage （%） to identify the recellulanzation level. RESULTS：①in the orientational cultivation, 5.0×10^5 of BMSCs in the primary culture was increased to 6.0×10^9 of ECs, that was increased 1.2×10^4 times after 8 generations incubated, which could satisfy the requirement of high density seeding. ②The ECs of the HBV were not only removed completely with 0.03% SDS, but also reserved the main components of extracellular matrix. ③The recellularization levels of culture of ECs and HBV scaffold on the 7^th, 14^th and 20= days ware 65%, 85% and 90% respectively. CONCLUSION： Orientational cultured ECs of BMSCs is another source of seed cells for the valvular tissue,engineered after amplification in vitro, tissue-engineered heart valve is fabricated preliminarily under static culture, which provides basic material for pulsatile-fiow-cultivation and the animal experment in the future.