人骨髓间充质干细胞体外应力培养构建组织工程心脏瓣膜

Construction of tissue engineering heart valve in vitro by autologous human bone mesenchymal stem cells under pulsatile flow condition

目的:以人体骨髓间充质干细胞(BMSCs)为种子细胞,以去细胞猪主动脉瓣为支架,体外三维构建组织工程心脏瓣膜(TEHV)。方法:以胸外科术中切除的肋骨为骨髓来源,采用1.073 g/ml的Percoll梯度分离液离心法获取BMSCs,体外培养扩增、鉴定;以0.05%胰酶+0.02%EDTA消化法制备去细胞猪主动脉瓣叶支架,将体外培养扩增的BMSCs种植于支架上,体外三维培养,应力环境下构建TEHV, 观察TEHV细胞分泌的一氧化氮(NO)及内皮素(ET 1)水平、免疫组化特征及光镜和电镜下的结构。结果:人BMSCs为梭形,αSMA及Vimentin染色阳性,CD 31 及Ⅷ因子染色阴性,其分泌NO及ET 1分别为(104.1±5.9)μmol/L及(40.9±7.6) ng/L,明显较正常人血管内皮细胞低(P<0.01);猪去细胞瓣膜支架去细胞完全,弹力纤维及胶原纤维保持完整;应力条件下体外三维构建的TEHV上活细胞数较静态下构建明显增多(P<0.01),H E染色示人BMSCs在支架上生长良好,形成完整的细胞层, 扫描电镜示瓣膜表面细胞层完整,细胞呈梭形生长。结论:人BM SCs作为种子细胞已初步具有内皮细胞功能,在去细胞猪主动脉瓣膜支架上生长良好,可作为一种有前途的种子细胞构建自体TEHV;体外三维应力条件下构建TEHV效果明显优于静态培养。

Objective:To develop tissue engineering heart valve (TEHV) in vitro by seeding autologous human bone mesenchymal stem cells on acellular porcine aortic valve matrices under pulsatile flow condition. Methods: Human bone marrow was obtained from the rib excised during thoracic operations and then the mesenchymal stem cells (BMSCs) were isolated by Percoll gradient centrifugation, cultured and expanded in vitro and characterized by immunohistochemistry. The secretion levels of nitric oxide (NO) and endothelin 1(ET-1) by the BMSCs were measured; decellularized porcine aortic valve matrices were created by 0.05% trypsin+0.02%EDTA digestion procedure; TEHVs were constructed by reseeding the human BMSCs on the acellular porcine aortic valve matrices in a bioreactor designed by our laboratory and the histological structures were studied by light microscopy, scanning electron microscopy (SEM) and immunohistochemistry. MTT assay was used to evaluate the viable cells on the porcine acellular scaffolds. Results: Human BMSCs were spindle-shaped with positive staining of α-SMA and Vimentin and negative staining of CD-31 and vWF, the levels of NO and ET-1 being (104.1±5.9) μmol/L and (40.9±7.6) ng/L, respectively, which were significantly lower than those of vascular endothelial cells( P<0.01). Porcine cells could no longer be detected on porcine aortic valve by H-E staining after decellularization, with elastic fibers and collagenous fibers well-preserved. MTT assay showed that there were much more viable seeding BMSCs on the TEHVs under pulsatile flow condition than that under static condition (P<0.01). SEM demonstrated a confluent seeding of BMSCs on the surface of TEHV. Conclusion: In vitro human BMSCs can function as vascular endothelial cells and may be a potential cell source for TEHV. It is feasible to construct human autologous TEHVs by reseeding BMSCs on porcine acellular scaffolds under pulsatile flow condition,which is better than that under static condition.