载骨髓间充质干细胞磷酸钙骨水泥的制备
发布时间：2019-07-02 16:00
【摘要】：一、研究背景 随着现代科学的进步，我们的生活步入了现代化，高速铁路、飞机、汽车遍布周围，高能量的损伤越来越多，组织缺损及功能障碍越来越常见。与此同时，人们的需求也在与时俱进，并不单纯的讲究保全生命，而是追求更高的生活品质，追求功能的恢复及重建。随着现代医学的发展，从上世纪80年代中期开始，组织工程——一个新兴的概念开始兴起。根据美国国家科学基金委员会的定义，组织工程是指应用细胞生物学、生物材料和工程学的原理，研究开发用于修复或改善人体病损组织或器官的结构、功能的生物活性替代物的一门科学。对于创伤而言，各种原因导致的骨缺损都严重影响患者生活质量，传统的自体骨移植不但牺牲了自体健康的组织，且由于供区体积限制，很多情况下并不能完全修复骨质缺损。而骨组织工程的出现，为解决这个困扰骨科界数十年的问题展现了曙光。骨组织工程是指应用骨髓间充质干细胞、成骨细胞等相关合适的种子细胞，在各种可吸收的支架材料上种植，辅以具有相关生物活性的细胞因子，如骨形态发生蛋白（Bone morphogeneticprotein,BMP）、转化生长因子（Transforming Growth Factor,TGF）等，修复缺损的骨组织，实现安全、有效、无副作用的医疗境界。在本课题中，我们试图对传统的磷酸钙骨水泥加以改进，使之具有骨诱导性、速代谢性，高强度性，从而研制一种新型的骨替代材料，为大范围骨质缺损的修复提供一种新的选择。 二、研究目的 通过实验将包含大鼠骨髓间充质干细胞（MSCs）的明胶微球载入到磷酸钙骨水泥（CPC）中并使之存活，利用骨髓间充质干细胞的多向分化潜能促进磷酸钙骨水泥的降解及骨诱导活性，从而研制一种新的具有生物活性的骨替代材料。 三、研究内容和方法 1.分离、培养大鼠骨髓间充质干细胞 我们选用大鼠的骨髓间充质干细胞作为种子细胞。通过传统的贴壁培养法培养骨髓间充质干细胞，并对其进行传代、鉴定，确保最终实验所用的为分裂稳定，具有多向分化潜能的干细胞。 2.制备载骨髓间充质干细胞的明胶微球 取1皿第三代骨髓间充质干细胞用胰酶消化备用，将一定量的明胶颗粒溶于DMEM培养基制备成13%的明胶溶液，取5ml溶液缓慢加入1ml细胞悬液，混匀后与橄榄油25ml均预热至50℃。橄榄油中加入0.15mL Span-80后缓慢加入明胶溶液，于磁力搅拌仪上以900r／min搅拌15min后加入0.01g京尼平于室温下继续搅拌。为测试不同交联度对明胶微球稳定性的影响，分别交联12h、24h、48h。后混合物骤冷至4℃，与含5g/L Tween-80的PBS液以1:1比例置于分液漏斗，清洗3次以去除橄榄油获得明胶微球。置于DMEM培养基中于细胞培养箱保存备用。 3.制备载骨髓间充质干细胞的磷酸钙骨水泥 为确定合适的构成比，将载有MSCs的明胶微球与骨水泥分别按质量比2.5%（B）、5%（C）、10%（D）（w/w）充分混合后与磷酸盐缓冲液按固液相2:1的比例混合为半流质糊状物后置入预先备好的圆柱体模具中于37℃孵箱中固化4h后脱模后于DMEM培养基中保存备用。另制备单纯CPC（A）作为空白对照。 4.测定载干细胞明胶微球的理化性质观察明胶微球的外形、分散度，计算其细胞包封率、平均粒径及溶胀率，用茚三酮法测定明胶微球的交联度，观察微球的降解时间。 5.荧光染色观察微球内的骨髓间充质干细胞将明胶微球浸入DMEM中，用钙黄绿素AM和碘化丙啶(PI)染色后分别于2h、12h、24h于荧光显微镜下观察，其中绿色荧光表明细胞存活，红色荧光表明细胞死亡。 6.测定新型骨水泥的理化性质表征新型磷酸钙骨水泥的初凝时间、抗压强度，计算其孔隙率及代谢时间，分析其代谢产物，观察骨髓间充质干细胞在孔隙内的粘附及生长情况。 7.统计学处理数据统计用SPSS13.0进行统计分析，数据以均数±标准差(x±s)表示。采用AVONA方法，SNK-q检验进行两两比较，P0.05为差异有统计学意义。 四、研究结果 1.我们所培养的骨髓间充质干细胞已稳定传至第三代，经流式细胞仪检测，本实验分离、培养的MSCs符合国际标准。MSCs CD90表达为阳性，阳性率达到95.24%，而CD45则表达为阴性。 2.明胶微球的理化性质观察交联时间控制在24h内各组明胶微球的粒径变化不大，，分别为(75±15)μm、(81.58±24.68)μm和(108.64±37.44)μm，24h后微球粒径明显减小为(27.88±7.46) μm，差异具有显著性(P0.05)；交联过的明胶微球降解时间明显延长，较未交联组差异差异具有显著性(P0.05)。 3.磷酸钙骨水泥的理化性质观察在各组CPC中MSCs均生长良好，且与空白对照组比较，随着明胶微球比例的增大，各组CPC的抗压强度逐步降低，初凝时间延长，孔隙率增大，大孔率增高(P0.05)。 五、研究结论 1、本实验分离、培养的骨髓间充质干细胞复合国际标准。 2、通过对明胶微球的理化性质测定，京尼平交联24h后的明胶微球粒径大小适中，降解时间以及载细胞率符合实验要求，微球内携载的MSCs存活良好。 3、通过对磷酸钙骨水泥理化性质的测定，载MSCs的明胶微球与CPC的质量比为5%时，CPC的理化性质最为理想，细胞生长良好。 4、本实验研究成果可为下一步体内实验奠定坚实的基础。
[Abstract]:I. Background of the study With the progress of modern science, our lives have entered the modern, high-speed railway, the plane, the automobile is spread around, the high-energy damage is more and more, the tissue defect and the dysfunction are more and more often See. At the same time, people's needs are advancing with the times, not only for the preservation of life, but for the higher quality of life, the recovery and the weight of the pursuit function. With the development of modern medicine, from the mid-1980s, the organization project _ an emerging concept has started. The tissue engineering, as defined by the National Commission of National Development, refers to the application of cellular biology, biological materials and engineering principles to study the development of a branch of a biological active substitute for the repair or improvement of the structure and function of a body or organ of a human body. In the case of trauma, the bone defect caused by various causes seriously affects the quality of life of the patient, and the traditional autogenous bone grafting not only sacrifices the self-healthy tissue, but also can not completely repair the bone defect due to the volume limitation of the donor area. The appearance of bone tissue engineering has shown a great deal of problems to solve the problem that has plagued the orthopedic industry for decades. Bone tissue engineering refers to the use of appropriate seed cells, such as bone marrow mesenchymal stem cells, osteoblasts and the like, to be grown on various absorbable scaffold materials, supplemented by cytokines with related biological activity, such as bone morphogenetic protein (BMP), transformation growth factor (BMP), and transformation growth factor (BMP). r, TGF) and the like, the bone tissue of the defect is repaired, the safe, effective and side-effect medical condition is realized, In this subject, we try to improve the traditional calcium phosphate cement to have bone-induced, rapid metabolic and high-strength, so as to develop a new kind of bone substitute material, which provides a new choice for the repair of large-scale bone defect. alternative. two, The purpose of this study was to load the gelatin microspheres containing the rat bone marrow mesenchymal stem cells (MSCs) into the calcium phosphate bone cement (CPC) and to make it survive, and to promote the degradation of the calcium phosphate bone cement by the multi-directional differentiation potential of the bone marrow mesenchymal stem cells. osteoinductive activity, thereby developing a new biological activity bone substitute material Study content and method 1. Separation, culture Rat Bone Marrow Mesenchymal Stem Cells: The Rat Bone Marrow Mesenchymal Stem Cells The mesenchymal stem cells are used as seed cells, bone marrow mesenchymal stem cells are cultured by a traditional adherent culture method, A stem cell with multi-directional differentiation potential. the gelatin microspheres carrying the bone marrow mesenchymal stem cells are used for digesting the third generation bone marrow mesenchymal stem cells with the pancreatin for standby, and a certain amount of the gelatin particles are dissolved in the DMEM medium to be prepared into 13 percent of the gelatin solution, and the gelatin microspheres 1 ml of cell suspension is slowly added to the solution, and then the solution is uniformly mixed. preheating to 50 & deg; C with 25 ml of olive oil, adding 0.15 mL of Span-80 into olive oil, slowly adding gelatin solution, stirring at 900 r/ min on a magnetic stirrer for 15min, and adding 0 in order to test the effect of different cross-linking degree on the stability of the gelatin microspheres, The mixture was quenched to 4 & deg; C for 12 h,24 h, and 48 h, respectively, and the PBS solution containing 5 g/ L Tween-80 was placed in a separatory funnel at a ratio of 1:1, 3 times to remove olive oil to obtain gelatin microspheres. The reserve was stored in the cell incubator in M medium. Preparation of Bone Marrow Mesenchymal Stem The calcium phosphate bone cement of the cells is used to determine the appropriate component ratio, and the gelatin microspheres loaded with MSCs and the bone cement are respectively mixed with the bone cement according to the mass ratio of 2.5% (B),5% (C),10% (D) (w/ w) and then mixed with the phosphate buffer solution according to the solid-liquid phase 2:1 to form a semi-fluid paste, and then the calcium phosphate bone cement is put into a pre-prepared cylindrical mould to be cured in a 37 DEG C cold box After 4 h, the preparation was stored in DMEM medium. and 4. determining the physical and chemical properties of the loaded stem cell gelatin microspheres to observe the appearance and the dispersion degree of the gelatin microspheres, and calculating the cell encapsulation rate, the average particle diameter and the swelling rate of the gelatin microspheres, The cross-linking degree of the gelatin microspheres was measured and the degradation time of the microspheres was observed. 6. The physical and chemical properties of the new bone cement were used to characterize the initial setting time and the compressive strength of the new type of calcium phosphate bone cement. The porosity and the metabolic time of the new bone cement were calculated, and its metabolism was analyzed. The adhesion and growth of bone marrow-derived mesenchymal stem cells in the pores were observed. 13.0 Statistical analysis is performed and data is expressed in the mean square standard deviation (x% s). The AVONA method, S SNK-q妫
本文编号：2509068
[Abstract]:I. Background of the study With the progress of modern science, our lives have entered the modern, high-speed railway, the plane, the automobile is spread around, the high-energy damage is more and more, the tissue defect and the dysfunction are more and more often See. At the same time, people's needs are advancing with the times, not only for the preservation of life, but for the higher quality of life, the recovery and the weight of the pursuit function. With the development of modern medicine, from the mid-1980s, the organization project _ an emerging concept has started. The tissue engineering, as defined by the National Commission of National Development, refers to the application of cellular biology, biological materials and engineering principles to study the development of a branch of a biological active substitute for the repair or improvement of the structure and function of a body or organ of a human body. In the case of trauma, the bone defect caused by various causes seriously affects the quality of life of the patient, and the traditional autogenous bone grafting not only sacrifices the self-healthy tissue, but also can not completely repair the bone defect due to the volume limitation of the donor area. The appearance of bone tissue engineering has shown a great deal of problems to solve the problem that has plagued the orthopedic industry for decades. Bone tissue engineering refers to the use of appropriate seed cells, such as bone marrow mesenchymal stem cells, osteoblasts and the like, to be grown on various absorbable scaffold materials, supplemented by cytokines with related biological activity, such as bone morphogenetic protein (BMP), transformation growth factor (BMP), and transformation growth factor (BMP). r, TGF) and the like, the bone tissue of the defect is repaired, the safe, effective and side-effect medical condition is realized, In this subject, we try to improve the traditional calcium phosphate cement to have bone-induced, rapid metabolic and high-strength, so as to develop a new kind of bone substitute material, which provides a new choice for the repair of large-scale bone defect. alternative. two, The purpose of this study was to load the gelatin microspheres containing the rat bone marrow mesenchymal stem cells (MSCs) into the calcium phosphate bone cement (CPC) and to make it survive, and to promote the degradation of the calcium phosphate bone cement by the multi-directional differentiation potential of the bone marrow mesenchymal stem cells. osteoinductive activity, thereby developing a new biological activity bone substitute material Study content and method 1. Separation, culture Rat Bone Marrow Mesenchymal Stem Cells: The Rat Bone Marrow Mesenchymal Stem Cells The mesenchymal stem cells are used as seed cells, bone marrow mesenchymal stem cells are cultured by a traditional adherent culture method, A stem cell with multi-directional differentiation potential. the gelatin microspheres carrying the bone marrow mesenchymal stem cells are used for digesting the third generation bone marrow mesenchymal stem cells with the pancreatin for standby, and a certain amount of the gelatin particles are dissolved in the DMEM medium to be prepared into 13 percent of the gelatin solution, and the gelatin microspheres 1 ml of cell suspension is slowly added to the solution, and then the solution is uniformly mixed. preheating to 50 & deg; C with 25 ml of olive oil, adding 0.15 mL of Span-80 into olive oil, slowly adding gelatin solution, stirring at 900 r/ min on a magnetic stirrer for 15min, and adding 0 in order to test the effect of different cross-linking degree on the stability of the gelatin microspheres, The mixture was quenched to 4 & deg; C for 12 h,24 h, and 48 h, respectively, and the PBS solution containing 5 g/ L Tween-80 was placed in a separatory funnel at a ratio of 1:1, 3 times to remove olive oil to obtain gelatin microspheres. The reserve was stored in the cell incubator in M medium. Preparation of Bone Marrow Mesenchymal Stem The calcium phosphate bone cement of the cells is used to determine the appropriate component ratio, and the gelatin microspheres loaded with MSCs and the bone cement are respectively mixed with the bone cement according to the mass ratio of 2.5% (B),5% (C),10% (D) (w/ w) and then mixed with the phosphate buffer solution according to the solid-liquid phase 2:1 to form a semi-fluid paste, and then the calcium phosphate bone cement is put into a pre-prepared cylindrical mould to be cured in a 37 DEG C cold box After 4 h, the preparation was stored in DMEM medium. and 4. determining the physical and chemical properties of the loaded stem cell gelatin microspheres to observe the appearance and the dispersion degree of the gelatin microspheres, and calculating the cell encapsulation rate, the average particle diameter and the swelling rate of the gelatin microspheres, The cross-linking degree of the gelatin microspheres was measured and the degradation time of the microspheres was observed. 6. The physical and chemical properties of the new bone cement were used to characterize the initial setting time and the compressive strength of the new type of calcium phosphate bone cement. The porosity and the metabolic time of the new bone cement were calculated, and its metabolism was analyzed. The adhesion and growth of bone marrow-derived mesenchymal stem cells in the pores were observed. 13.0 Statistical analysis is performed and data is expressed in the mean square standard deviation (x% s). The AVONA method, S SNK-q妫
本文编号：2509068
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