大鼠骨髓基质干细胞来源的成骨细胞与血管内皮细胞复合同种异体冻干脱钙骨基质的粘附性研究

发布时间：2018-05-25 02:40

本文选题：组织工程 + 骨髓基质干细胞　；参考：《华北煤炭医学院》2009年硕士论文

【摘要】：目的通过分离大鼠骨髓基质干细胞(BMSCs),检测其在特定培养条件下诱导为血管内皮细胞的生物学特性,并将诱导培养的成骨细胞、血管内皮细胞以及共培养的两种细胞分别接种于I型胶原修饰的同种异体冻干脱钙骨基质支架材料,从而探讨两种细胞与支架材料复合构建组织工程活性骨的可行性,最终为组织工程骨修复骨缺损提供实验依据。方法4周龄SD大鼠,进行BMSCs原代细胞培养。取生长状态良好的第3代BMSCs进行实验。分组如下:1)对照组:普通培养基组;2)实验组:血管内皮条件培养基组。倒置相差显微镜观察细胞形态;CD31 CD34免疫细胞化学染色、透射电镜W-P小体鉴定血管内皮细胞; MTT法检测血管内皮细胞的生长与增殖情况并绘制生长曲线;扫描电镜观察诱导的成骨细胞、血管内皮细胞及两种细胞共培养在I型胶原修饰的支架材料表面粘附、生长情况。结果1. BMSCs接种后随着时间的延长,贴壁细胞增加。细胞形态呈梭形、纤维母细胞样;集落形成,其大小、形状、密度不同,最终融合。细胞无接触性抑制。 2.诱导后的血管内皮细胞具有一定的分化能力,但增殖速度变得缓慢,CD31、CD34免疫细胞化学染色鉴定可见阳性表达;透射电镜观察细胞内可见W-P小体。 3. MTT检测细胞生长、增殖结果显示:第1-2d,细胞数目均无明显变化,为潜伏期;第3天起细胞进入对数生长期,数量开始增加;7天后,细胞进入到生长稳定的平台期。 4.同种异体冻干脱钙骨基质为天然的多孔样结构。诱导后的血管内皮细胞在其表面平铺、伸展、生长状态良好。 5.诱导培养的成骨细胞、血管内皮细胞以及共培养的两种细胞分别接种于I型胶原修饰的同种异体冻干脱钙骨基质,修饰后细胞在支架材料上的粘附率增加。结论1.从骨髓中可分离得到较高纯度BMSCs,体外培养增殖能力活跃,在含有VEGF、bFGF诱导因子诱导培养条件下,能定向分化为具有典型形态学特点的血管内皮细胞,可作为骨组织工程血管化理想的细胞来源。 2.同种异体冻干脱钙骨基质与血管内皮细胞有良好的细胞相容性,同种异体冻干脱钙骨基质有利于细胞的粘附、生长,是较为理想的骨组织工程支架材料。 3.血管内皮细胞与支架材料的粘附率与细胞接种密度密切相关。当细胞接种密度为2×106/ml时,血管内皮细胞与支架材料的粘附率达到最大。提示:细胞接种于支架时有最适接种密度,支架与细胞的粘附能力存在极限即最大细胞粘附数量。 4.支架材料经I型胶原修饰后较修饰前所粘附的细胞数量多。提示:I型胶原是较为理想的支架材料表面修饰物。
[Abstract]:Objective to investigate the biological characteristics of rat bone marrow stromal stem cells (BMSCs) induced into vascular endothelial cells (VEC) under specific culture conditions. Vascular endothelial cells (VECs) and co-cultured cells were inoculated with type I collagen modified allogeneic decalcified bone matrix scaffolds to investigate the feasibility of constructing tissue-engineered active bone by combining two kinds of cells with scaffold materials. Finally, it provides experimental basis for tissue engineering bone to repair bone defect. Methods Primary BMSCs cells were cultured in 4 weeks old SD rats. The third generation BMSCs with good growth state was selected for experiment. The control group was divided as follows: control group: normal medium group 2) experimental group: vascular endothelial conditioned medium group. CD31 CD34 immunocytochemical staining was observed by inverted phase contrast microscope, vascular endothelial cells were identified by transmission electron microscope W-P corpuscles, the growth and proliferation of vascular endothelial cells were detected by MTT method and the growth curve was drawn. The adhesion and growth of osteoblasts, vascular endothelial cells and two kinds of cells co-cultured on the surface of collagen I scaffold were observed by scanning electron microscope (SEM). Result 1. After BMSCs inoculation, adherent cells increased with time. Cell morphology is fusiform, fibroblast-like, colony formation, its size, shape, density is different, finally fusion. There was no contact inhibition. 2. The induced vascular endothelial cells had the ability of differentiation, but the proliferation rate became slow. The positive expression of CD31and CD34 was detected by immunocytochemistry, and W-P corpuscles were observed by transmission electron microscope. 3. The results of MTT showed that the number of cells did not change obviously at the 1st to 2nd day, which was the latent period, and the cells entered the logarithmic growth period on the 3rd day, and the number began to increase 7 days later, the cells entered the stable stage of growth. 4. Allogeneic freeze-dried demineralized bone matrix is a natural porous structure. The induced vascular endothelial cells on the surface of the flat, stretch, good growth state. 5. The osteoblasts, vascular endothelial cells and co-cultured cells were inoculated into the allogeneic freeze-dried decalcified bone matrix modified by type I collagen, and the adhesion rate of the cells on the scaffold material was increased. Conclusion 1. BMSCs with high purity could be isolated from bone marrow. The proliferation of BMSCs was active in vitro. When cultured with VEGF bFGF inducing factors, BMSCs could be differentiated into vascular endothelial cells with typical morphological characteristics. It can be used as an ideal cell source for vascularization of bone tissue engineering. 2. The allogeneic freeze-dried decalcified bone matrix has good cytocompatibility with vascular endothelial cells. The allogeneic freeze-dried decalcified bone matrix is an ideal scaffold material for bone tissue engineering, which is conducive to cell adhesion and growth. 3. The adhesion rate of vascular endothelial cells to scaffold materials is closely related to the density of cell inoculation. When the cell density was 2 脳 106/ml, the adhesion rate of vascular endothelial cells to the scaffold was the highest. The results suggested that the optimal inoculation density was found when the cells were inoculated on the scaffold, and the adhesion capacity of the scaffold to the cells was limited, that is, the maximum number of cell adhesion. 4. The number of cells adhered to the scaffold after type I collagen modification was higher than that before modification. It is suggested that type I collagen is an ideal surface modifier for scaffolds.
【学位授予单位】：华北煤炭医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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