BMP2诱导豚鼠MSCs分化成骨细胞制备组织工程人工听骨的实验研究

发布时间：2019-04-30 07:07

【摘要】：目的：观察豚鼠骨髓间充质干细胞(MSCs)与纳米羟基磷灰石(nHA)材料体外复合培养的结合程度,并分析其构建组织工程人工听骨的可行性。方法：(1)采用细胞差速贴壁法分离培养、纯化豚鼠骨髓间充质干细胞,使用流式细胞仪检测CD29、CD45、CD44进行间充质干细胞表面标记物鉴定。采用骨形态发生蛋白2(BMP2)将第三代的MSCs诱导分化为成骨细胞；倒置显微镜下观察细胞形态学变化,诱导后免疫组化染色和成骨诱导并检测其成骨细胞分化能力；(2)将骨髓间充质干细胞与纳米羟基磷灰石共培养1天,3天,5天,7天,10天,电镜观察体外培养的骨髓间充质干细胞与此种支架材料的复合情况；(3)将复合培养5天的支架材料植入豚鼠听泡模型,分别在2周、4周、8周、10周、12周取出组织工程人工听骨进行病理学观察及扫描电镜观察材料的成骨能力。结果：(1)细胞培养的检测：①MSCs的检测：第三代的MSCs形态均一,呈单层生长,细胞多为长梭形,呈旋涡状排列；其表面标记物CD29、CD44和CD45表达分别为95.7%、70%、0.7%；②成骨细胞检测：诱导后倒置显微镜下可见细胞具有成骨细胞形态特征；免疫组化I型胶原检测可观察到细胞内有黄褐色颗粒沉淀,碱性磷酸酶染色见细胞胞浆内出现蓝紫色颗粒,茜素红染色时可见被染成红色的诱导成骨细胞形成的钙结节；(2)体外复合培养3天后,可见大量骨髓间充质干细胞贴附在材料表层,并且形态稳定,生长旺盛,增殖力强,具有极佳的延伸性；培养5天后可见材料表面己全部覆盖骨髓间充质干细胞,细胞结合紧密,细胞表面可见大量分泌颗粒,胞体明显增大,边缘完整,呈纤维样伸长；7天后细胞逐渐从材料表面脱落,仍附在材料表而的细胞出现“星形”改变,“伪足”增多；10天后细胞呈片状脱落；(3)病理学观察到回植10周后的组织工程人工听骨被大量纤维结缔组织包裹,与周围组织相融合；回植12周的人工听骨能谱分析及扫描电镜观察可见材料表面形成了骨陷窝,微量元素含量测定与正常骨组织几乎相同。结论：说明纳米羟基磷灰石材料保持了它良好的生物相容性,利于细胞黏附、增殖,可结合大量的骨髓间充质干细胞,用于构建组织工程人工听骨。
[Abstract]:Aim: to observe the degree of co-culture of guinea pig bone marrow mesenchymal stem cells (MSCs) and nano-hydroxyapatite (nHA) materials in vitro, and to analyze the feasibility of constructing tissue-engineered artificial ossicles. Methods: (1) the bone marrow mesenchymal stem cells of guinea pig were isolated and cultured by differential cell adhesion method. The surface markers of mesenchymal stem cells (MSCs) were identified by flow cytometry (FCM). The CD29,CD45,CD44 of mesenchymal stem cells was detected by flow cytometry (FCM). Bone morphogenetic protein 2 (BMP2) was used to induce the third generation of MSCs to differentiate into osteoblasts, the morphological changes of the cells were observed under inverted microscope, and the differentiation ability of osteoblasts was detected by immunohistochemical staining and osteogenic induction after induction of bone morphogenetic protein 2 (BMP-2). (2) Bone marrow mesenchymal stem cells were co-cultured with nano-hydroxyapatite for 1 day, 3 days, 5 days, 7 days and 10 days. (3) the scaffold material was implanted into the guinea pig auditory vesicle model for 5 days. The tissue engineering artificial ossicles were taken out at 2 weeks, 4 weeks, 8 weeks, 10 weeks and 12 weeks respectively for pathological observation and scanning electron microscope (SEM) observation of the osteogenic ability of the materials. Results: (1) Detection of cell culture: detection of 1MSCs: the MSCs of the third generation was homogeneous and grew in monolayer, and the cells were spindle-shaped and vortex-like. The expression of CD29,CD44 and CD45 were 95.7%, 70% and 0.7% respectively. The morphological characteristics of osteoblasts were observed under inverted microscope after induction. There were yellow-brown granules precipitated in the cells by immunohistochemical staining of collagen I, blue-purple granules appeared in the cytoplasm of the cells by alkaline phosphatase staining, and calcium nodules of osteoblasts were stained red with alizarin red staining. (2) after 3 days of co-culture in vitro, a large number of bone marrow mesenchymal stem cells were attached to the surface of the material, and the morphology was stable, the growth was vigorous, the proliferation power was strong, and the bone marrow mesenchymal stem cells had excellent extensibility; After 5 days of culture, bone marrow mesenchymal stem cells were covered with bone marrow mesenchymal stem cells on the surface of the materials. The cells were closely bound, and a large number of secretory granules were seen on the surface of the cells. The cell bodies were obviously enlarged and the edges were intact, showing fiber-like elongation. After 7 days, the cells gradually exfoliated from the surface of the material, the cells still attached to the material surface appeared "star" change, "pseudopod" increased, after 10 days, the cells showed lamellar shedding. (3) the tissue engineering artificial ossicles were wrapped in a large amount of fibrous connective tissue and fused with the surrounding tissues 10 weeks after reimplantation. Energy spectrum analysis and scanning electron microscopy (SEM) of artificial ossicular ossicles at 12 weeks showed that bone lacunae were formed on the surface of the materials, and the content of trace elements was almost the same as that of normal bone tissues. Conclusion: nano-hydroxyapatite materials maintain good biocompatibility, facilitate cell adhesion and proliferation, and can combine with a large number of bone marrow mesenchymal stem cells for the construction of tissue engineering artificial ossicular bone.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764

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