[An experimental study of mesenchymal stem cells in tissue engineering scaffolds implanted in rabbit corneal lamellae to increase keratoprosthesis biointegration].
[An experimental study of mesenchymal stem cells in tissue engineering scaffolds implanted in rabbit corneal lamellae to increase keratoprosthesis biointegration].
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DOI：
10.3760/cma.j.issn.0412-4081.2016.03.009
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发表时间：
2016-03-01
期刊：
影响因子：
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通讯作者：
Huang, J X
中科院分区：
文献类型：
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作者：
Bai, H;Wang, L L;Huang, J X
目的：初步评价小鼠骨髓间充质干细胞(BMSC)复合组织工程支架植入兔角膜板层的可行性，为人工角膜与受者表面的结合，减少并发症的发生提供解决方案。(1)体外实验：用红色荧光蛋白(RFP)标记小鼠骨髓间充质干细胞，并将其与脱细胞猪关节软骨细胞外基质(ECM)支架复合。在荧光显微镜下观察4周和8周的细胞存活情况。甲苯胺蓝染色检测细胞分布。在扫描电子显微镜下观察其孔结构和细胞黏附情况。(2)体内实验：将小鼠骨髓间充质干细胞与脱细胞支架的复合体植入8只兔眼的板层角膜内，对侧眼作为对照。分别于术后2、4、8周取眼进行光镜下HE染色观察。在荧光显微镜下观察细胞存活情况，用活体成像技术观察8周后角膜内细胞存活情况。结果：(1)在扫描电子显微镜下，ECM支架具有良好的细胞和组织黏附和生长所需的孔隙率，甲苯胺蓝染色可观察到细胞-支架复合体上的细胞分布。(2)免疫荧光显微镜下观察RFP标记的小鼠BMSC与ECM支架体外复合后，细胞在体外和板层间隙内的增殖情况(最长观察时间为8周)。(3)光镜下(HE染色)见各时间点间质细胞增多。角膜基质浅层可见少量单核细胞和少量小鼠骨髓基质细胞，胶原纤维排列稀疏有序，无新生血管形成。所有上皮细胞均呈单核、柱状，未见损伤，与胶原融合的ECM支架形态不清。(4)体内显像发现，小鼠骨髓间充质干细胞与支架复合后植入兔角膜板层间隙后存活8周。(5)细胞-支架复合体植入后未见严重的炎症反应、明显的结膜充血和新生血管形成。受区周围的角膜组织透明。一周后，几乎没有观察到轻微的炎症反应，角膜足够透明，可以观察到基质层中的支架。四周后，支架变得更薄了。8周后，支架变得非常薄，角膜缘血管系统正常。结论：ECM支架是BMSC生长和增殖的固体和生物相容性载体。小鼠骨髓间充质干细胞可以在角膜板层间隙的微环境中生长增殖。
OBJECTIVE: To complete a preliminary evaluation of the feasibility of implanting the complex of mouse bone marrow mesenchymal stem cells (BMSC) and a tissue engineering scaffold into rabbit corneal lamellae, based on which a solution may be proposed to consolidate the keratoprosthesis and the recipient surface, and to reduce the risk of complications.METHODS: This experimental study was composed of two parts. (1) In vitro: some mouse BMSC were marked with red fluorescent proteins (RFP) and integrated with a decellularized pig articular cartilage extracellular matrix (ECM) scaffold. The cell survival was observed under a fluorescence microscope at 4 and 8 weeks. The cell distribution was examined by toluidine blue staining. The pore structure and the cell adhesion were observed under a scanning electron microscope. (2) in vivo: the complex of mouse BMSC and a decellularized scaffold was implanted into the lamellar cornea of 8 rabbit eyes with the fellow eyes as the controls. The eyes were sampled for observation using HE staining under a light microscope at 2, 4 and 8 weeks, respectively. The cell survival was examined under a fluorescence microscope, and the intracorneal cell survival at 8 weeks was observed using in vivo imaging. The conditions of ocular anterior segment of all the experimental animals were recorded.RESULTS: (1) Under the scanning electron microscope, the ECM scaffolds showed satisfactory porosity required for the adhesion and growth of cells and tissues, and the cell distribution over the cell-scaffold complex can be observed by toluidine blue staining. (2) Under the immunofluorescence microscope, cell proliferation was observed in vitro and in the interlamellar space (the maximum observation time was 8 weeks) after the RFP-marked mouse BMSC were integrated in vitro with ECM scaffolds. (3) Under the light microscope (HE staining), the stromal cells were detected to increase at each timepoint. A small number of monocytes and some mouse BMSC were observed in the superficial layer of corneal stroma, with sparsely and orderly arranged collagenous fibers and no neovascularization. All the epithelial cells appeared as mononuclear, columnar and undamaged, and the shape of ECM scaffolds, which were fused with the collagens, became unclear. (4) By in vivo imaging, it was found that the mouse BMSC survived for 8 weeks after being integrated with scaffolds and implanted into the interlamellar space of rabbit cornea. (5) After the implantation of cell-scaffold complex, severe postoperative inflammatory reactions, obvious conjunctival congestion and neovascularization were not observed. The corneal tissues surrounding the recipient area were transparent. One week later, mild inflammatory reactions were barely observed, and the cornea was transparent enough to observe the scaffold in the stromal layers. Four weeks later, the scaffolds became thinner. Eight weeks later, the scaffolds became extremely thin with normal vascular system in the corneal limbus.CONCLUSIONS: The ECM scaffold is a solid and biocompatible carrier for the growth and proliferation of BMSC. The mouse BMSC can grow and proliferate in the microenvironment of the interlamellar space of cornea.