BMP2誘導豚鼠MSCs分化成骨細胞制備組織工程人工聽骨的實驗研究
發(fā)布時間：2019-04-30 07:07
【摘要】：目的：觀察豚鼠骨髓間充質(zhì)干細胞(MSCs)與納米羥基磷灰石(nHA)材料體外復合培養(yǎng)的結(jié)合程度,并分析其構(gòu)建組織工程人工聽骨的可行性。方法：(1)采用細胞差速貼壁法分離培養(yǎng)、純化豚鼠骨髓間充質(zhì)干細胞,使用流式細胞儀檢測CD29、CD45、CD44進行間充質(zhì)干細胞表面標記物鑒定。采用骨形態(tài)發(fā)生蛋白2(BMP2)將第三代的MSCs誘導分化為成骨細胞；倒置顯微鏡下觀察細胞形態(tài)學變化,誘導后免疫組化染色和成骨誘導并檢測其成骨細胞分化能力；(2)將骨髓間充質(zhì)干細胞與納米羥基磷灰石共培養(yǎng)1天,3天,5天,7天,10天,電鏡觀察體外培養(yǎng)的骨髓間充質(zhì)干細胞與此種支架材料的復合情況；(3)將復合培養(yǎng)5天的支架材料植入豚鼠聽泡模型,分別在2周、4周、8周、10周、12周取出組織工程人工聽骨進行病理學觀察及掃描電鏡觀察材料的成骨能力。結(jié)果：(1)細胞培養(yǎng)的檢測：①MSCs的檢測：第三代的MSCs形態(tài)均一,呈單層生長,細胞多為長梭形,呈旋渦狀排列；其表面標記物CD29、CD44和CD45表達分別為95.7%、70%、0.7%；②成骨細胞檢測：誘導后倒置顯微鏡下可見細胞具有成骨細胞形態(tài)特征；免疫組化I型膠原檢測可觀察到細胞內(nèi)有黃褐色顆粒沉淀,堿性磷酸酶染色見細胞胞漿內(nèi)出現(xiàn)藍紫色顆粒,茜素紅染色時可見被染成紅色的誘導成骨細胞形成的鈣結(jié)節(jié)；(2)體外復合培養(yǎng)3天后,可見大量骨髓間充質(zhì)干細胞貼附在材料表層,并且形態(tài)穩(wěn)定,生長旺盛,增殖力強,具有極佳的延伸性；培養(yǎng)5天后可見材料表面己全部覆蓋骨髓間充質(zhì)干細胞,細胞結(jié)合緊密,細胞表面可見大量分泌顆粒,胞體明顯增大,邊緣完整,呈纖維樣伸長；7天后細胞逐漸從材料表面脫落,仍附在材料表而的細胞出現(xiàn)“星形”改變,“偽足”增多；10天后細胞呈片狀脫落；(3)病理學觀察到回植10周后的組織工程人工聽骨被大量纖維結(jié)締組織包裹,與周圍組織相融合；回植12周的人工聽骨能譜分析及掃描電鏡觀察可見材料表面形成了骨陷窩,微量元素含量測定與正常骨組織幾乎相同。結(jié)論：說明納米羥基磷灰石材料保持了它良好的生物相容性,利于細胞黏附、增殖,可結(jié)合大量的骨髓間充質(zhì)干細胞,用于構(gòu)建組織工程人工聽骨。
[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.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R764
本文編號：2468577
[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.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R764
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