基于细胞膜片技术构建血管化组织工程骨的实验研究

发布时间：2018-05-24 13:37

本文选题：骨髓间充质干细胞 + 内皮祖细胞　；参考：《第四军医大学》2012年博士论文

【摘要】：实验一骨髓间充质干细胞的分离培养及多向诱导分化 [摘要]目的：体外获取兔骨髓间充质干细胞（mesenchymal stem cells，MSCs）、纯化、扩增，同时探讨其生物学特性及多向分化潜能。方法：全骨髓贴壁法体外分离培养MSCs，观察其增殖、生长特性和组织学形态；并向成骨细胞、脂肪细胞和软骨细胞多向诱导分化，分别采用钙结节茜素红染色、油红O染色和甲苯胺蓝染色鉴定。结果：贴壁的骨髓间充质干细胞为纺锤形，呈克隆样生长，性状稳定，碱性磷酸酶（alkaline phosphatase，ALP）染色弱阳性。经成骨、成脂和成软骨诱导培养后，表现出相应细胞的形态学和生物学特征。结论：全骨髓贴壁筛选法取材方便，骨髓间充质干细胞增殖能力强，在不同的条件培养基诱导下能够多向分化。实验二内皮祖细胞的培养和鉴定 [摘要]目的：探索分离纯化及体外培养内皮祖细胞(endothelialprogenitor cells, EPCs)的方法，并进行鉴定。为采用EPCs促进血管化组织工程骨的构建策略提供实验基础。方法：将骨髓单核细胞(mononuclear cells,MNCs)接种至明胶涂层的培养皿内，使用含10%胎牛血清（fetal bovineserum，FBS）、添加50μg/mL ECGS (endothelial cell growth supplements)的M199培养液，置37℃、体积分数为5%的CO2饱和湿度恒温培养箱培养。3天后弃去未贴壁细胞，此后每3天换液1次。形成内皮祖细胞克隆后，0.05%胰酶-EDTA消化传代，2-4代细胞用于实验。通过CD31免疫荧光染色、荆豆凝集素免疫细胞化学染色、毛细血管腔形成能力及透射电子显微镜(transmission electron microscope, TEM)检测进行鉴定。结果：培养5-7天，内皮祖细胞的早期克隆形成，中央为大量圆形细胞。2周后，细胞表现出典型的“鹅卵石”状。培养过程中可形成管腔状结构。可与荆豆凝集素特异性相结合；内皮细胞特异性表面标志CD31荧光染色呈阳性表达；透射电镜观察细胞质内见内皮细胞特有的细胞器-(W-P)小体。结论：特定的培养条件下可以获取足量的EPCs，细胞可迅速体外扩增，并具有内皮细胞特异性标志物。实验三骨髓间充质干细胞膜片的体外构建及成骨分化研究 [摘要]目的：探索体外构建骨髓间充质干细胞膜片的简便方法，分析膜片组织学结构，并评估其成骨分化潜能。方法：将兔MSCs高密度接种于直径10cm培养皿，成骨诱导条件下连续培养2周形成细胞膜片。收获时，使用细胞刮沿皿底小心刮起，或用镊子轻轻提起。通过组织学、组织化学、碱性磷酸酶活性定量检测、透射电镜、扫描电镜(scanning electronmicroscope，SEM)等一系列方法，检测细胞膜片的物理及生物学特性。.结果：高密度连续培养后形成的MSCs膜片，呈半透明薄膜状，有弹性，其内多个白色结节。HE染色证实骨髓间充质细胞膜片是一种由多层细胞和细胞外基质（extracellular matrix，ECM）组成的聚集体。碱性磷酸酶、茜素红及Von kossa等组织化学染色呈阳性；碱性磷酸酶定量分析含量较高；扫描电镜及透射电镜检查均见基质中大量的矿化结节聚集。结论：通过体外简单的连续培养方法和成骨诱导后，可构建具有良好成骨能力MSCs膜片。实验四骨髓间充质干细胞膜片复合内皮祖细胞构建血管化组织工程骨 [摘要]目的：探讨利用MSCs膜片复合EPCs构建血管化的无外支架组织工程骨的可行性,以解决传统接种方法存在的诸多问题。方法：将骨髓细胞行双向诱导：骨髓单核细胞中MSCs连续培养成骨诱导后，细胞增殖、分泌细胞外基质，形成成骨性膜片；另一部分骨髓单核细胞在内皮生长培养基内分化为EPCs。然后将MSCs膜片复合EPCs，折叠、卷曲成圆柱状的复合细胞聚集体。最后，将复合体移植至免疫缺陷的裸鼠背部皮下。单纯MSCs膜片构建组织块作为对照。术后4周，8周分别进行大体观察、显微CT（micro-computed tomography，Micro-CT）、扫描电镜及组织学检查。结果：MSCs膜片复合EPCs构建物植入体内后，形成血管化骨样组织，外观色红、质硬。CT、扫描电镜及组织学检查均证实新骨形成，而且所形成的骨密度和血管密度均高于对照组。结论：首次利用MSCs膜片复合EPCs构建较大体积的血管化组织工程骨，无需外支架。实验证实EPCs的引入不仅能产生功能性血管网，而且能够促进骨形成。实验五骨髓间充质干细胞膜片复合内皮祖细胞构建可注射血管化组织工程骨 [摘要]目的：探讨可注射的血管化组织工程新的构建策略。方法：利用MSCs膜片复合EPCs，形成具有双重细胞成分的聚集体。复合种子细胞附着于自身分泌的细胞外基质支架上，形成具有骨组织三维结构和生理活性的复合体。再注射至无免疫动物背部皮下，术后4周，8周分别进行大体观察、显微CT、扫描电镜及组织学检查，观察其在体内发育成骨样组织的能力。结果：MSCs膜片/EPCs复合体，可以顺利通过注射针头至皮下。8周后形成鲜红骨样组织，呈扁圆形，外周密布一层血管网，质地硬，最大长度近2.0cm。Micro-CT影像显示高密度矿化组织形成。横断面扫描电镜观察呈现松质骨样结构，其内大量片状骨小梁排列，交织为多孔的网格样结构。组织学检查证实新生的骨组织和丰富的血管形成。此外，新骨的形成存在两种方式：组织块外周以膜内骨化为主，骨基质内可见骨细胞，骨小梁边缘成行排列矮柱状的成骨细胞；组织块内部还可见肥大的软骨细胞和钙化软骨，提示软骨内骨化方式。结论：本研究，首次验证了将EPCs复合MSCs膜片，构建可注射的血管化组织工程骨的可行性，无需外源性载体。为构建可注射的血管化组织工程骨提供了全新的思路。实验六骨髓间充质干细胞膜片/内皮祖细胞/珊瑚构建大块管状骨 [摘要]目的：利用细胞膜片技术，辅以管状形态的内支撑体，首次探索移植自体体内构建大块的具备特定形态的组织工程骨。方法：构建兔来源MSCs膜片-EPCs，与珊瑚内支撑体卷层样复合，形成管样结构。体外实验，置灌注式生物反应器动态培养8周，观察细胞生长和组织的形成；体内实验，移植自体背部皮下，利用自体的微环境和自身分泌的各种生长因子调控来完成骨组织的构建。体内、外标本取材，进行大体观察、显微CT、扫描电镜及组织学检查，分析新骨形成。结果：体外，，随着灌注式反应器动态培养时间的增长，细胞基质分泌旺盛，细胞与细胞产生连接，各层聚集体之间逐渐融合、增厚，质地变硬，并与珊瑚材料紧密结合，呈现出白色的骨样组织外观。体内培养8周后，构建了长度近5.0cm大块管状骨。显微CT、扫描电镜及组织学检查均证实，珊瑚表面、材料间隙及深部均有新骨形成。结论：MSCs膜片-EPCs与珊瑚自体体内可构建大块的管状骨。
[Abstract]:Isolation and multidirectional differentiation of bone marrow mesenchymal stem cells in Experiment 1
[Abstract] Objective: to obtain mesenchymal stem cells (MSCs) in vitro, to purify and amplify, and to explore its biological characteristics and pluripotent differentiation potential. Methods: the whole bone marrow adherent method was used to isolate and culture MSCs in vitro, to observe its proliferation, growth and histology, and to osteoblasts, adipocytes and chondrocytes. Multidirectional differentiation, using alizarin red staining with calcium nodules, oil red O staining and toluidine blue staining. Results: the adherent bone marrow mesenchymal stem cells were spindle shaped, cloned and stable, and alkaline phosphatase (alkaline phosphatase, ALP) dyed weak Yang. After induction of osteogenesis, fat formation and cartilage formation, the phase was displayed. The morphological and biological characteristics of the cells. Conclusion: all bone marrow adherent screening method is convenient, bone marrow mesenchymal stem cells have strong proliferation ability and can be multidifferentiated under the induction of different conditions.
The culture and identification of experiment two inner skin progenitor cells
[Abstract] Objective: To explore the method of isolation and purification and in vitro culture of endothelialprogenitor cells (EPCs), and to identify it. It provides an experimental basis for the use of EPCs to promote the construction of vascularized tissue engineering bone. Methods: bone marrow mononuclear cells (mononuclear cells, MNCs) are inoculated into the culture dish of gelatin coating, so that the bone marrow mononuclear cells (MNCs) are inoculated into the culture dish of gelatin coating. A M199 culture containing 10% fetal bovine serum (fetal BovineSerum, FBS) and 50 g/mL ECGS (endothelial cell growth supplements) was added to the incubator at 37 degrees C and 5% in the volume fraction of CO2 saturated humidity and constant temperature incubator. After.3 days, the non adherent cells were abandoned, and then 1 times every 3 days. After the formation of endothelial progenitor cells, 0.05% pancreatin was digested and passed to 2. 2 -4 cells were used for experiments. By CD31 immunofluorescence staining, immunocytochemical staining of agglutinin, capillary cavity formation and transmission electron microscopy (transmission electron microscope, TEM) detection. Results: 5-7 days of culture, the early cloning of endothelial progenitor cells was formed in the center of a large number of round cells after.2 weeks, The cells show a typical "cobblestone" shape. In the process of culture, a lumen structure can be formed. It can be combined with the specificity of the bean agglutinin; the specific surface of the endothelial cell mark CD31 fluorescent staining is positive; transmission electron microscopy is used to observe the specific organelles - (W-P) corpuscles in the cytoplasm of the cytoplasm. Conclusion: specific culture conditions A sufficient amount of EPCs can be obtained under this condition. The cells can be amplified in vitro and have specific markers of endothelial cells.
Experiment three the construction and osteogenic differentiation of bone marrow mesenchymal stem cell membrane in vitro
[Abstract] Objective: To explore a simple method of constructing bone marrow mesenchymal stem cell membrane in vitro, analyze the histological structure of the diaphragm and evaluate its osteogenic differentiation potential. Methods: the rabbit MSCs was inoculated at high density in the diameter 10cm culture dish, and the cell membrane was formed continuously for 2 weeks under the induction of osteogenesis, and the cell was scraped along the dish bottom carefully. Or gently lift it with tweezers. Through a series of methods such as histology, histochemistry, quantitative detection of alkaline phosphatase activity, transmission electron microscopy, scanning electron microscopy (scanning electronmicroscope, SEM), and so on, the physical and biological properties of the cell diaphragms are detected. Results: the MSCs diaphragm formed after high density continuous culture, is translucent thin film and elastic, The.HE staining of multiple white nodules confirmed that the bone marrow mesenchymal cell membrane was a group of multi-layer cells and extracellular matrix (extracellular matrix, ECM). The histochemical staining of alkaline phosphatase, alizarin red and Von Kossa was positive; the quantitative analysis of alkaline phosphatase was higher; scanning electron microscope and transmission electron microscope examination A large number of mineralized nodules were found in the matrix. Conclusion: MSCs film with good osteogenesis ability can be constructed by a simple continuous culture method and osteogenic induction in vitro.
Experiment four bone marrow mesenchymal stem cell membrane combined with endothelial progenitor cells to construct vascularized tissue-engineered bone
[Abstract] Objective: To explore the feasibility of constructing vascularized non stenting tissue engineered bone with MSCs film combined with EPCs in order to solve the problems existing in traditional inoculation methods. Methods: bone marrow cells were induced by bidirectional induction: bone marrow mononuclear cells were continuously cultured and cultured for bone induction, cell proliferation, extracellular matrix, and formation of osteogenesis. The other bone marrow mononuclear cells were differentiated into EPCs. in the endothelial growth culture base and then the MSCs diaphragm was combined with EPCs, folded and curled into a cylindrical composite cell aggregate. Finally, the complex was transplanted subcutaneously into the back of the immune deficient nude mice. The simple MSCs diaphragm was used as the control. 4 weeks after the operation, the mass was carried out, respectively. Body observation, microscopic CT (micro-computed tomography, Micro-CT), scanning electron microscope and histological examination. Results: after the MSCs diaphragm composite EPCs construction was implanted in the body, the vascularized osteoid tissue was formed. The appearance of the tissue was red and hard, the scanning electron microscope and the histological examination confirmed the formation of the new bone, and the bone density and the density of the blood vessel were higher than that of the pair. Conclusion: a large volume of vascularized tissue engineering bone was constructed with MSCs film combined with EPCs for the first time without external scaffold. It was proved that the introduction of EPCs could not only produce functional vascular network, but also promote bone formation.
Experiment five bone marrow mesenchymal stem cells and endothelial progenitor cells were used to construct injectable tissue-engineered bone.
[Abstract] Objective: To explore a new construction strategy for injectable vascularized tissue engineering. Methods: MSCs membrane composite EPCs was used to form a polymer with double cell components. Compound seed cells attached to the self secreted extracellular matrix scaffold to form a complex of three-dimensional structure and physiological activity of bone tissue. The immune animals were subcutaneously on the back, 4 weeks and 8 weeks after the operation. Microscopical CT, scanning electron microscopy and histological examination were used to observe the ability to develop osteoid tissue in the body. Results: the MSCs diaphragm /EPCs complex could successfully form a red bone like tissue through the injection of the needle to the subcutaneous.8 week, a flat circle, and a layer of blood vessels in the outer circumference. Web, texture hard, the maximum length near 2.0cm.Micro-CT images showed high density mineralized tissue. Cross section scanning electron microscopy showed a loose bone like structure, a large number of trabecular bone trabeculae arranged and interwoven into a porous mesh like structure. Histological examination confirmed the formation of new bone tissue and rich vascularization. In addition, the formation of new bone was two The tissue mass was mainly in the outer membrane of the tissue, bone cells were visible in the bone matrix, the bone trabecular edge was arranged in a row of short columnar osteoblasts, and the hypertrophic chondrocytes and calcified cartilage were also visible within the tissue. Conclusion: the EPCs composite MSCs diaphragm was first verified and the injectable blood was constructed for the first time. The feasibility of tubular tissue-engineered bone does not require exogenous vectors. It provides a new idea for the construction of injectable vascularized tissue-engineered bone.
Experiment six bone marrow mesenchymal stem cells / endothelial progenitor cells / corals construct large tubular bone.
[Abstract] Objective: To explore the construction of a large block of tissue engineered bone with a tubular form of internal support with cell membrane technique and tubular form for the first time. Method: to construct a rabbit source MSCs film -EPCs, combined with the reel sample of the corals, to form a tube like structure. In vitro experiment, the perfusion bioreactor was moved. The growth of cells and the formation of tissue were observed for 8 weeks. In vivo experiments, transplantation of autologous back subcutaneous, autologous microenvironment and the regulation of various growth factors which were secreted by themselves to complete the construction of bone tissue. The body and external specimens were taken for general observation, microscopical CT, scanning electron microscopy and histological examination, and analysis of new bone formation. Results: body results: body results: body In addition, with the growth of the dynamic culture time of the perfusion reactor, the cell matrix was exuberant and the cells were connected with the cells. The aggregates of each layer were gradually fused, thickened and hardened, and the appearance of the white bone like tissue was presented closely with the coral material. After 8 weeks of culture, the length of the tubular bone with a length of nearly 5.0cm was constructed. Microscopical CT, Scanning electron microscopy and histological examination confirmed that there were new bone formation in the coral surface, the material gap and the deep part. Conclusion: the MSCs diaphragm -EPCs and the coral autologous body can build large tubular bones.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

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