【作者】 吴凯；

【导师】 邓展生；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一章兔骨髓间充质干细胞的分离、培养、鉴定及诱导分化成骨细胞目的：探讨兔BMSCs的分离、培养、鉴定及向成骨细胞诱导分化的方法,探讨BMSCs在体外增殖,定向分化能力。方法：取兔股骨的骨髓冲洗液,采用Percoll分离液密度梯度离心法结合贴壁筛选法分离、培养、纯化兔BMSCs,光镜下观察传代细胞生长状况和形态变化；MTT法检测细胞增殖情况,绘制生长动力学曲线；HE染色形态观察和流式细胞仪抗体鉴定兔BMSCs表面标记物；体外诱导P3BMSCs向成骨细胞分化,对诱导分化为骨细胞进行Gomori钙钴法ALP染色和茜素红钙矿化结节染色鉴定向成骨分化的能力。结果：所分离培养的细胞形态均一,沿胞体长轴生长,呈旋涡状、网状、梭状、放射状,形成较典型的BMSCs。P3BMSCs的生长曲线大致呈S型,第7-11天细胞处于高速增长期,细胞数量迅速增加；HE染色BMSCs形态呈长梭形或长三角形,细胞核浆比例大,核蓝染,胞浆丰富红染。流式检测细胞表面标记物显示BMSCsCD44表达百分率为98.63%,呈阳性,而CD45表达呈阴性。P3BMSCs在体外经成骨诱导3周后,Gomori钙钴法染色ALP呈阳性,胞浆显示有灰黑色颗粒或棕黑色或黑色沉淀。茜素红染色呈阳性,细胞间出现致密的圆形或者卵圆形矿化结节。结论：应用Percoll分离液密度梯度离心法和贴壁筛选法结合能分离、培养出兔BMSCs,是一种比较理想获取兔BMSCs的方法；兔P3BMSCs纯度较高和良好的增殖能力,具有向成骨细胞定向分化的生物学特性,是组织工程理想的种子细胞,也为后续进行骨缺损修复实验奠定了坚实的基础。第二章n-HA/n-LDIG复合材料的制备及与BMSCs体外复合培养的细胞生物学评价目的：以赖氨酸盐,甘油及n-HA为主要原料,制备出一种新型n-HA/n-LDIG复合材料,分析其与BMSCs体外复合培养的生物相容性,探讨其作为组织工程支架的可行性。方法：选择赖氨酸盐和甘油为原材料,通过有机合成、聚合反应、纳米化、超临界抗溶剂结晶、超声分散等技术制备出n-HA/n-LDIG复合材料。电镜观察其结构及计算孔隙率,细胞计数法检测细胞对复合材料的黏附能力,MTT法检测复合材料对细胞增殖的影响,ALP活性和钙含量测定观察复合材料对细胞分化的影响。结果：n-HA/n-LDIG复合材料肉眼为白色散在,为比较均匀的粉状固体。扫描电镜可见类似天然松质骨的三维孔洞网络结构,透射电镜可见排列不规则的晶须和空穴。共培养从2h至10h,随时间细胞的黏附率有所增加,其中6h时细胞黏附率明显高于4h时,8h时明显高于6h时,其差异均有统计学意义(P<0.05)。MTT实验结果显示共培养第2天到第16天,细胞在复合材料上数量随时间而增加,特别是从第6天开始复合材料上的细胞数量迅速增加,细胞处于对数生长期,12天后过渡到生长平台期,增殖缓慢。从第4天开始,黏附细胞的ALP相对活性和钙相对含量迅速上升；第8天时ALP相对活性和钙相对含量明显高于第4天时,第12天明显高于第8天时,其差异均有统计学意义(P<0.05)；12天后ALP相对活性和钙相对含量增加不明显,差异无统计学意义。结论：制备的n-HA/n-LDIG复合材料具备适宜的孔隙率和孔径,组成性质相似于天然骨结构,具有良好的生物学特性,是一种新型的纳米骨修复材料。n-HA/n-LDIG复合材料具备良好的细胞相容性,能为成骨细胞在其表面生长、增殖、分泌基质提供良好的微环境,是一种良好的骨修复替代材料,为其作为骨的快速修复和骨组织工程支架材料奠定了基础。第三章BMP-2和BMSCs结合n-HA/n-LDIG复合材料修复兔桡骨缺损的实验研究目的：构建兔桡骨缺损模型,探讨BMP-2和BMSCs结合n-HA/n-LDIG复合材料修复兔桡骨缺损的效果。方法：取新西兰大白兔构建兔桡骨缺损模型,实验组植入BMP-2和BMSCs结合n-HA/n-LDIG复合材料,对照组植入BMSCs结合n-HA/n-LDIG复合材料,空白组不植入任何材料。分别于2、4、8、12周随机处死家兔,进行动物大体观察,X线检查和Lane-Sandhu放射学评分观察骨缺损处愈合情况,通过组织学检查和Lane-Sandhu组织学评分观察骨缺损处的修复情况。结果：所有实验动物桡骨骨缺损模型构建均成功,未见皮下和肌肉组织充血、水肿、渗出及感染等现象。从大体观察,X线检查和组织学检查等发现随着时间的延长,实验组修复效果优于对照组,对照组优于空白组。实验组术后3、6、9周新生骨所占骨缺损面积百分比,Lane-Sandhu放射学评分,新生骨所占骨缺损面积百分比及Lane-Sandhu组织学评分均明显高于对照组,对照组均明显高于空白组,其差异均具有统计学意义(P<0.05)。结论：BMP-2明显够促进早期成骨,缩短成骨时间,加速骨缺损区骨组织的再生和钙化；BMP-2和BMSCs结合n-HA/n-LDIG复合材料具有良好的骨缺损修复效果,能明显够促进早期成骨,缩短成骨时间,加速骨缺损区骨组织的再生和钙化,能为临床应用提供了理论基础和新思路,具有良好的临床应用前景。更多还原

【Abstract】 Chapter1Separation, Culture, Identification of Rabbit Bone Marrow Mesenchymal Stem Cells and Their Induced Differentiation to OsteoblastsObjective:To explore the separation, culture, identification of rabbit bone marrow mesenchymal stem cells (BMSCs) and their induced differentiation to osteoblasts, as well as the capacity of BMSCs in proliferation in vitro and directed differentiation.Method:The bone marrow washing fluid of rabbit femur was taken for separation, culture, purified rabbit of BMSCs by Percoll separation medium density gradient centrifugation and adherence screening method, the growth conditions and morphological changes of passaged cells were observed under light microscope; the cell proliferation was determined by MTT assay so as to fit growth kinetic curves; the rabbit BMSCs surface markers were observed by HE staining morphology and identified by flow cytometry antibodies; the P3BMSCs were induced in vitro to differentiate into the osteoblasts, the induced BMSCs were stained by Gomori Gomori ALP staining and alizarin Red calcium mineralized nodules to identify their capacity to differentiate into osteoblasts.Results:The cells for separated culture were highly homogeneous, and grew along the long axis of cell body, presented swirly, mesh, fusiform, radial, and formed typical BMSCs. The growth curve of P3BMSCs was roughly S-shaped, their7～11d cells were in a period of rapid growth, the number of cells here rapidly increased; the HE staining BMSCs morphology presented long fusiform or long triangle, the nucleus cytoplasm ratio was high, the nuclear was blue-stained, the cytoplasm was abundantly red-stained. The flow cytometry detection for the cell surface markers showed the expression percentage of the BMSCs CD44was98.63%, positive, while the expression of CD45presented negative. At3weeks after the P3BMSCs were induced to osteoblasts in vitro, the Gomori calcium cobalt staining ALP was positive, the cytoplasm showed gray and black particles or brown-black or black precipitates. Alizarin Red staining was positive, the intercellular space appeared round or oval dense mineralized nodules.Conclusion:The rabbit BMSCs can be separated and cultured through the combination of Percoll separated medium density gradient centrifugation and adherence screening method which is an ideal method to obtain rabbit BMSCs; rabbit P3BMSCs have good purity, good proliferative capacity, and biological characteristics of directed differentiation to osteoblasts, which is a kind of ideal seed cells for tissue engineering and also lays a solid foundation for follow-up bone defect repair experiments. Chapter2Rreparation of n-HA/n-LDIG composites and biology assessment for the cells compoundly cultured from bmscs in vitroObjective:The lysine salt, glycerine and n-HA were selected as the main raw materials to prepare a new n-HA/n-LDIG composite, analyzing its compatibility to the cells compoundly cultured from BMSCs in vitro so as to explore its feasibility to be a tissue engineering scaffold.Methods:The lysine salt and glycerol were selected as raw materials, through organic synthesis, polymerization, nanotechnology, supercritical anti-solvent crystallization, ultrasonic dispersion technologies, they were processed to prepare a n-HA/n-LDIG composite. By electron microscopy, its structure and porosity was observed and calculated, then the cell adhesion of it was determined by cell counting method, and its influence on cell proliferation was determined by MTT assay, next its influence on cell differentiation was determined by ALP activity and calcium content determination.Results:The n-HA/n-LDIG composite visually likes scatteredly white, relatively uniform powdery solid. The3D porous network structure similar to natural cancellous bone was observed under the scanning of electron microscopy, and its irregular arrangement of whiskers and pores could be found under transmission electron microscopy. The cells were cultured within2h to10h, with the time increased, the cell adhesion also increased, and the cell adhesion rate at6h was significantly higher than that at4h, the cell adhesion rate at8h was significantly higher than that at6h, their differences were statistically significant (P<0.05). MTT assay results showed:from2d to16d of culture, with the time increased, the cell counts on composite also increased, especially rapidly increased from6d (the cells were in the exponential growth phase), on12d, the cells developed to a growth plateau with slow proliferation. From4d, the ALP relative activity and calcium relative content quickly raised; the ALP relative activity and calcium relative content on8d were significantly higher than those on4d, and the ALP relative activity and calcium relative content on12d were significantly higher than those on8d, their differences were statistically significant (P<0.05); from12d, the growth rate of ALP relative activity and calcium relative content slowed down, their difference was not statistically significant (P>0.05).Conclusions:The prepared n-HA/n-LDIG composite has suitable porosity and pore size, and its composition properties are similar to those of natural bone structures, having good biological characteristics, so it is a new nano-bone repair material. The n-HA/n-LDIG composite have good cell compatibility and can act as microenvironment for the growth, proliferation, matrix secretion of cells on its surface. As a premium bone repair alternative material, it laid a foundation for rapid bone repair and bone tissue engineering scaffold material development. Chapter3Experimental study on repairmen of rabbit radial bone defects using BMP-2and BMSCs combined with n-HA/n-LDIG composite materialObjective:To build the rabbit radial bone defect model, and explore the repair effects of rabbit radial bone defects using BMP-2and BMSCscombined with n-HA/n-LDIG composite material.Method:The New Zealand white rabbits were used to build bone defects models, the experiment group was implanted BMP-2and BMSCs combined with n-HA/n-LDIG composite material, and the control group was implanted the BMSCs combined with n-HA/n-LDIG composite material, and the blank group was not implanted. The rabbits were randomly sacrificed after2,4,8and12weeks and taken the general observation, X-ray examination and Lane-Sandhu scoring to observe the healing of the bone defect, and through the histological examination and Lane-Sandhu histological scoring to observe the repair of the bone defect.Results:All experimental animal radius bone defect models were successfully built, subcutaneous and muscle tissue congestion, edema, exudate and infection were not observed. From the general observation, X-ray examination and histological examination, we can found that, with the time going, the repair effects of the experiment group were better than that of the control group, and the repair effects of the control group were better than that of the blank group. The area percentage of newborn bone of bone defect, Lane-Sandhu radiological scores and the Lane-Sandhu histological scores after3,6and9weeks of the experiment group were significantly higher than of the control group, and the control group were better than the blank group, and the differences were statistically significant (P<0.05).Conclusion:The BMP-2can significantly promote the early osteogenesis, shorten the osteogenesis time and accelerate the regeneration and calcification of the bone tissue at bone defect area; the BMP-2and BMSCs combined with n-HA/n-LDIG composite material has good bone defect repair effects, can significantly promote the early osteogenesis, shorten the osteogenesis time and accelerate the regeneration and calcification of the bone tissue at bone defects area, provides a theoretical basis and new idea for clinical application, and has good prospects for clinical application.更多还原