FG复合rhBMP-2、bFGF和妥布霉素促进犬骨折愈合的实验研究

发布时间：2018-05-13 21:07

本文选题：FG + bFGF　；参考：《甘肃农业大学》2016年硕士论文

【摘要】：目前,骨折愈合研究的热点已经转向为创口创造良好的生物学环境和有效控制感染,这是骨外科迫切需要解决的问题,也是成功治疗骨折的关键。BMP能诱导未分化的间充质细胞分化形成软骨和新生骨,对骨量、骨的发生和重建具有多向调节作用;bFGF能显著促进细胞的有丝分裂和血管形成,加速软骨的成熟和骨化,可在BMP启动骨重建后协同骨修复;骨外伤感染中的几种主要致病菌对妥布霉素的耐药性低,敏感性较高;并且妥布霉素对BMP的促骨愈合效果没有抑制作用,反而可促进骨折部位的修复。FG作为一种低抗原性的生物大分子材料,符合BMP和bFGF载体的理想条件,因此,本实验将纤维蛋白胶作为一种支架材料和抗生素药物缓释载体,复合可在骨重建过程中发挥重要作用的两种因子rhBMP-2和bFGF以及妥布霉素,制备成一种具有抗生素缓释系统和细胞因子载体作用的复合物,以评估该复合物对骨折愈合的影响,并从病理组织学及分子生物学角度初步探讨该复合物在骨折愈合过程中的作用机理,为骨组织工程提供实验依据和理论基础。本实验选取12只犬作为实验动物,在无菌条件下用骨科摆锯建立标准犬胫骨骨折模型。每只犬以右侧后肢为实验肢,左侧后肢为对照肢。实验肢采用常规内固定方法+FG/rh BMP-2/bFGF/妥布霉素复合物进行骨折整复固定,对照肢仅采用常规内固定方法进行骨折整复固定。于术后4、8、12、16周随机选取三只取骨折处组织样品,制备石蜡切片,HE染色、MASSON三色染色法与甲苯胺蓝染色观察骨折处组织学变化,并采用免疫组织化学方法检测中4种因子的表达情况,实验结果如下:1.组织学变化:实验组骨折愈合早期成骨细胞、血管内皮细胞和成软骨细胞数目增多、增殖分化快,炎性清除期短,较对照组胶原纤维和骨基质的沉积量多,骨小梁形成和改建快,提前进入骨痂改造期,表明FG/rhBMP-2/bFGF/妥布霉素复合物在骨修复期可增强细胞、黏附增殖作用,为骨折愈合创造良好的细胞微环境,可增加细胞与细胞因子相互作用,促进组织生长,提高骨修复及改建质量。2.VEGF阳性率的表达:实验组和对照组VEGF阳性率均呈先升高后降低趋势,第8周最高,实验组第8周与12周差异显著(P0.01),其余时间点差异不显著;对照组第4、8、12周差异均显著(P0.01),术后第4周和第8周,实验组VEGF阳性率显著高于对照组(P0.01),第12周和16周差异性不显著。表明FG/rhBMP-2/bFGF/妥布霉素复合物在骨折愈合早期促进内皮细胞迁移、增殖,毛细血管新生,提高骨折局部的细胞代谢,优化骨折局部细胞微环境。3.PDGF阳性率的表达:术后各时间点,实验组和对照组PDGF阳性率均呈先增长后下降趋势,实验组第8周最高,各相邻时间点间差异均显著(P0.01);对照组第12周最高,第4周和8周、第12周和第16周相比差异显著,其余相邻时间点间差异不显著(P0.01)。术后第4周和第8周实验组PDGF阳性率均显著高于对照组(P0.01),第12周两组差异不显著,第16w对照组阳性率高于实验组,两组差异不显著。表明FG/rhBMP-2/bFGF/妥布霉素复合物提高碱性成纤维细胞的增殖、分化,可提前增强成骨细胞和成纤维细胞的分泌能力,促进胶原纤维生成与沉积。加快骨折愈合。4.IGF、TGF-β1阳性率的表达:IGF、TGF-β1阳性率均呈先增长后下降趋势,均为第12周最高,IGF实验组阳性率各时间点间差异均显著(P0.01),对照组第4周、8周和12周差异均显著(P0.01),实验组与对照组相比,第4周、8周和12周差异均显著(P0.01);TGF-β实验组阳性率各时间点间差异均显著(P0.01),对照组第4周、8周和12周差异均显著(P0.01),实验组与对照组相比,第4周、8周和12周差异均显著(P0.01)。表明FG/rhBMP-2/bFGF/妥布霉素能促进成骨细胞以自分泌的方式增殖分化和增加胶原合成。刺激细胞募集和增殖,启动修复过程。加速骨基质的钙化,同时改建骨小梁,加快愈合进程。结论:FG/rhBMP-2/bFGF/妥布霉素复合物在骨折早期为细胞和细胞因子创建良好微环境,促进多种细胞趋化、黏附、分化、增殖以及骨折端血管的形成和长入,维持药物和细胞因子较高浓度,骨修复作用增加,能协调骨折愈合改建期破骨细胞和成骨细胞相互作用,加快骨改建进程。
[Abstract]:At present, the hot spot of fracture healing research has turned to create a good biological environment and effective control of infection for the wound, which is an urgent problem in bone surgery. It is also the key.BMP for the successful treatment of fracture. It can induce undifferentiated mesenchymal cells to differentiate into cartilage and new bone, and have multi direction for bone mass, bone formation and reconstruction. BFGF can significantly promote mitosis and angiogenesis of cells, accelerate the maturation and ossification of cartilage, and can be combined with bone repair after the BMP start of bone reconstruction; several major pathogens in bone trauma infection have low resistance to tobramycin and high sensitivity; and tobramycin does not inhibit the effect of BMP on bone healing. The repair of.FG as a low antigenicity biological macromolecular material conforms to the ideal conditions for BMP and bFGF carriers. Therefore, this experiment uses fibrin glue as a scaffold material and antibiotic drug sustained-release carrier, combined with two factors, rhBMP-2 and bFGF, which can play an important role in the process of bone reconstruction. In order to evaluate the effect of the compound on fracture healing, a complex of antibiotic sustained-release system and cytokine carrier was prepared to evaluate the effect of the compound on fracture healing. The mechanism of the compound in the process of fracture healing was preliminarily discussed from the histopathological and molecular biological angles, which provided experimental basis and theoretical basis for bone tissue engineering. In this experiment, 12 dogs were selected as experimental animals. In the aseptic condition, a standard canine tibial fracture model was set up in the Department of orthopedics. The right hind limbs were the experimental limbs and the left hind limbs were the control limbs. The experimental limbs were fixed by the routine internal fixation method +FG/rh BMP-2/bFGF/ tobramycin complex, and the control limbs were only used in routine internal fixation. A fixed method was used to fix the fracture and fix. Three tissue samples were selected at random in 4,8,12,16 weeks after the operation. The paraffin section, HE staining, MASSON staining and toluidine blue staining were used to observe the histological changes of the fracture, and the expression of the 4 factors was detected by immunohistochemistry. The experimental results were as follows: 1. tissues Study changes: the early osteoblast of fracture healing in the experimental group increased, the number of vascular endothelial cells and chondrocytes increased, the proliferation and differentiation were fast, the inflammatory clearance period was short, the deposition of collagen fibers and bone matrix in the control group was more, the formation and reconstruction of bone trabeculae were fast, and the FG/rhBMP-2/bFGF/ tobramycin complex was repaired in the bone repair. It can enhance cell and adhesion and proliferate, create a good cell microenvironment for fracture healing, increase the interaction of cell and cytokine, promote tissue growth, improve the expression of.2.VEGF positive rate of bone repair and remodeling: the positive rate of VEGF in the experimental group and the control group all increased first and then decreased, the highest in eighth weeks and eighth weeks in the experimental group. The difference between the 12 weeks was significant (P0.01) and the difference of other time points was not significant. The difference of the control group at week 4,8,12 was significant (P0.01). The positive rate of VEGF in the experimental group was significantly higher than that of the control group (P0.01) at the fourth and eighth weeks postoperatively, and the difference between the twelfth and the 16 weeks was not significant. It showed that the FG/rhBMP-2/ bFGF/ tobramycin complex promoted endothelial cell migration early in the fracture healing. Shift, proliferation, capillary neovascularization, improve the cell metabolism in the part of the fracture, and optimize the expression of.3.PDGF positive rate in the microenvironment of the fracture. The positive rates of PDGF in the experimental group and the control group all increased first after the operation, the highest in the experimental group eighth weeks, and the difference between the adjacent time points (P0.01), and the highest in the control group for Twelfth weeks, Fourth weeks and 8 weeks, compared with twelfth weeks and sixteenth weeks, the difference was not significant (P0.01). The positive rate of PDGF in the experimental group was significantly higher than that of the control group (P0.01) at fourth weeks and eighth weeks after the operation, and the difference of two groups in twelfth weeks was not significant. The positive rate of the 16W control group was higher than that of the experimental group, and the difference of the two groups was not significant. It showed that the FG/rhBMP-2/bFGF/ was duly distributed. Mycomycin complex enhanced the proliferation of basic fibroblasts, enhanced the secretion of osteoblasts and fibroblasts in advance, promoted the formation and deposition of collagen fibers, accelerated the.4.IGF and TGF- beta 1 positive rate of fracture healing: the positive rates of IGF, TGF- beta 1 were all first increased and then decreased, both were the highest in twelfth weeks, and the positive rate of IGF experimental group. The difference in each time point was significant (P0.01), the difference was significant (P0.01) in the control group for fourth weeks and at the 8 and 12 weeks. Compared with the control group, the difference was significant (P0.01) at fourth weeks, 8 and 12 weeks in the experimental group, and the difference in the positive rate of the TGF- beta test group was significant (P0.01), the control group was fourth weeks, and the difference between the 8 weeks and the 12 weeks was significant (P0.01). The experimental group and the control group were all significantly different (P0.01). The difference between fourth weeks, 8 and 12 weeks was significant (P0.01). It showed that FG/rhBMP-2/bFGF/ tobramycin could promote the proliferation and differentiation of osteoblasts in the autocrine way and increase collagen synthesis, stimulate cell recruitment and proliferation, start the repair process, accelerate calcification of bone matrix, reconstruct trabecular bone and accelerate the healing process. Conclusion: FG/rhBMP-2/bFGF/ tob. Mycin complex creates a good microenvironment for cells and cytokines at the early stage of fracture to promote the chemotaxis, adhesion, differentiation, proliferation, and formation and growth of the blood vessels at the end of the fracture, maintaining a high concentration of drugs and cytokines, increasing the effect of bone repair, and coordinating the interaction of osteoclasts and osteoblasts in the healing period of fracture healing and accelerating the interaction of bone cells and osteoblasts. Bone remodeling process.

【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.292

【参考文献】