仿生控释FGF-2和BMP-2的组织工程骨膜联合骨膜来源干细胞修复颅骨极量骨缺损的实验研究

发布时间：2018-07-06 19:38

本文选题：骨膜来源干细胞 + 成纤维细胞生长因子-2　；参考：《南方医科大学》2017年硕士论文

【摘要】：骨组织工程的目标是找一个更好的方案解决大段骨缺损、骨延迟愈合或不愈合的难题。由于越来越多的学者认识到骨膜在骨损伤修复过程中的重要作用,目前组织工程骨膜在不断被研发,研究者将组织工程的理念融入到组织工程骨膜中,制备了材料、细胞及细胞因子为三要素的组织工程膜,希望能从结构或者功能上模拟自然的骨膜,并利用其促进骨损伤处的修复。从骨膜中提取到的细胞并被广大学者习惯称之为骨膜来源干细胞(periosteum derived stem cell,PDSC),已被证实其表面表达间充质干细胞标记物,并具有成骨、成软骨、成脂分化的潜能。所以PDSC可能是组织工程骨膜的优秀的种子细胞。基于种子细胞的组织工程中仍然面临一个关键问题,即在体外扩增过程中种子细胞的成骨能力逐步下降,这可能与成骨祖细胞干性下降有关,而在细胞移植过程中通过模拟内环境提供关键性因子有望解决这个难题,骨折的自然愈合早期,成纤维细胞生长因子-2(FGF-2)显著增加,但在随后的骨生成阶段低表达,而骨形态发生蛋白-2(BMP-2)则在成骨分化阶段大量表达。本课题制备了一种能仿生释放FGF-2和BMP-2的壳聚糖-胶原支架膜,并联合PDSC,进行了颅骨极量骨缺损修复实验,证明了 PDSC在体内外具有成骨分化能力,该仿生控释膜材料能支持PDSC的体内增殖、分化成骨及促进骨缺损处新骨形成。本课题采用主要方法如下:1.按以往方法制备胶原-壳聚糖支架及仿生控释FGF-2和BMP-2的微囊,冻干后备用。2.提取PDSC,通过流式细胞仪检测其表面干细胞标记物,并进行三系诱导分化实验,鉴定其多能干性。PDSC与胶原-壳聚糖支架共培养,检测细胞在支架上的粘附、增殖、分化能力。3.制备组织工程膜支架,进行颅骨缺损造模并移植手术,MicroCT和组织学检查评价新骨生成,用EdU法检测PDSC在移植部位的存活。4.统计学分析:应用SPSS 20.0统计软件进行数据分析,各组数值以Mean±SD表示,组间比较用单因素方差分析(one-wayANOVA),若方差齐用LSD检验、方差不齐则用DunnettT3检验作两两比较。设p值0.05具有统计学差异。主要结果:1.本实验PDSC经过流式鉴定,其表面表达间充质干细胞标记物,并经过诱导可向成骨、成软骨、成脂等多向分化,证明其在该组织工程膜支架上具有增殖、成骨分化能力。2.MicroCT和组织学检测证明,PDSC联合仿生释放的组织工程膜具有更优秀的骨缺损修复能力。结论:骨膜来源的干细胞会是组织工程骨膜的优秀种子细胞,并序贯释放FGF-2和BMP-2为PDSC提供更好的增殖分化环境,从而提高了仿生组织工程骨膜的骨修复效能。
[Abstract]:The goal of bone tissue engineering is to find a better solution to the problem of large bone defect, delayed union or nonunion. As more and more scholars realize the important role of periosteum in the repair of bone injury, tissue engineering periosteum has been developed constantly. Researchers have integrated the concept of tissue engineering into tissue engineering periosteum and prepared materials. Cells and cytokines are three elements of tissue engineering membrane, hoping to simulate the natural periosteum structurally or functionally, and use it to promote the repair of bone injury. The cells extracted from periosteum and often referred to as periosteum derived stem cells (periosteum derived stem cells) have been confirmed to express mesenchymal stem cell markers on the surface and have the potential of osteogenesis, cartilage formation and adipogenic differentiation. So PDSC may be an excellent seed cell for tissue engineering periosteum. One of the key problems in tissue engineering based on seed cells is that the osteogenic ability of seed cells decreases gradually during the process of expansion in vitro, which may be related to the decline in dryness of osteoblast progenitor cells. In the process of cell transplantation, it is expected to solve this problem by providing key factors through simulating the internal environment. In the early stage of natural healing of fracture, fibroblast growth factor 2 (FGF-2) is increased significantly, but the expression of FGF-2 is low in the later stage of bone formation. Bone morphogenetic protein-2 (BMP-2) was highly expressed during osteogenic differentiation. In this paper, a chitosan collagen scaffold membrane which can release FGF-2 and BMP-2 by bionic method was prepared, and PDSC-PDSC was used to repair the maximal bone defect of the skull. It was proved that PDSC has the ability of osteogenic differentiation in vivo and in vitro. The biomimetic controlled-release membrane can support PDSC proliferation, differentiation and osteogenesis and promote the formation of new bone at bone defect. The main methods of this subject are as follows: 1. Collagen chitosan scaffolds and biomimetic controlled release microcapsules of FGF-2 and BMP-2 were prepared according to the previous methods. The PDSCs were extracted, the surface stem cell markers were detected by flow cytometry, and the differentiation was induced by three lines. The pluripotency. PDSC and collagen-chitosan scaffold co-culture were identified. The adhesion, proliferation and differentiation ability of the cells on the scaffold were detected. Tissue engineering membrane scaffolds were prepared to model the skull defect, and microCT and histological examination were performed to evaluate the new bone formation. The survival of PDSC in the graft site was detected by EdU method. Statistical analysis: SPSS 20.0 statistical software was used to analyze the data. The values of each group were expressed as mean 卤SD. The comparison between groups was done by one-way ANOVA. If the variance was tested by LSD, the variance was compared by DunnettT3 test. Let p value 0.05 have statistical difference. The main result is 1: 1. In this experiment, PDSC expressed mesenchymal stem cell markers on the surface of PDSC by flow cytometry, and was induced to differentiate into osteogenesis, cartilage formation and lipid formation, which proved that PDSC could proliferate on the scaffold of tissue engineering membrane. 2. Osteogenic differentiation ability. 2. MicroCT and histological examination showed that PDSC combined with biomimetic release of tissue engineering membrane had better repair ability of bone defect. Conclusion: the stem cells derived from periosteum may be excellent seed cells for tissue engineering periosteum, and the sequential release of FGF-2 and BMP-2 provides a better environment for PDSC to proliferate and differentiate, thus improving the bone repair efficiency of biomimetic tissue engineered periosteum.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.08;R68

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