唑来膦酸联合特立帕肽防治兔人工膝关节假体无菌性松动的实验研究

发布时间：2018-04-20 06:37

本文选题：无菌性松动 + 动物模型　；参考：《青岛大学》2017年博士论文

【摘要】：目的:1、成功构建钛颗粒诱导的兔人工膝关节假体无菌性松动模型。2、探讨唑来膦酸联合特立帕肽防治兔人工膝关节假体松动的作用及机制,为进一步研究非手术方法治疗人工膝关节假体无菌性松动建立基础。方法:1、喂养16只成年雄性新西兰大白兔,平均体重2.85kg,随机分为两组,所有动物适应性喂养1周后进行右膝关节手术,取右膝关节前正中切口显露膝关节后,从后交叉韧带股骨侧止点上方开口,将长度2.0cm,直径0.5cm钛棒假体沿股骨纵轴方向植入髓腔。实验组的钛棒假体表面均匀涂抹50μg直径20μm的钛颗粒(试验组),对照组钛棒假体表面不涂抹任何物质。术后采取相同围手术期处理,喂养12周后处死动物,每组动物采集如下资料:处死前2天的膝关节X线片,滑膜组织行苏木精-伊红(H-E)染色,包含假体的骨组织行硬组织切片进行Goldner三色染色和Kossa染色分析,假体周围界膜组织行Real-time PCR和Western Blot检测OCN、OPG、RANKL和TRAP5b的m RNA和蛋白含量,进行两组之间的相关数据的统计学对比,证实制备兔人工关节假体无菌性松动模型的有效性并明确其机制。2、继续喂养24只新西兰大白兔,以同样手术方式从右侧的股骨膝关节面将钛棒假体沿轴线方向植入髓腔,所有钛棒假体表面均匀涂抹50μg直径20μm的钛颗粒,术后即给予相关药物治疗。根据治疗方案不同分为3组,每组8只:分别为唑来膦酸(ZL)组、特立帕肽(TP)组、唑来膦酸+特立帕肽(ZL+TP)组,同样于第12周时处死动物,采集如下资料:处死前2天的膝关节X线片,包含假体的骨组织行硬组织切片进行Goldner三色染色和Kossa染色,假体周围界膜组织行Real-time PCR和Western Blot检测相关特异性指标的m RNA和蛋白含量。并将第一部分实验组作为此部分对照组(CG组)进行统计学对比分析,验证唑来膦酸和特立帕肽的单独或者联合应用对于防治关节人工假体无菌性松动作用和机制。结果:1、实验组动物处死前X线可见明显的假体周围骨溶解迹象;滑膜H-E染色结果显示实验组动物膝关节滑膜组织明显增生并伴有大量的炎性细胞浸润。骨硬组织切片行Goldner三色染色显示实验组动物假体与骨组织存在较大间隙,间隙由纤维结缔组织填充,假体与骨组织的结合较差。而对照组动物假体与骨组织的结合较紧密,周围间隙较小,周围未见明显纤维组织增生填充。实验组骨-假体接触率和骨体积分数低于对照组。硬组织切片的Von Kossa染色结果显示,实验组动物假体周围的钙盐沉积岛主要是呈线状,数量较多,但是面积较小。而对照组动物假体周围的钙盐沉积岛表现为呈星状,虽然数量较少,但是单个面积较大。Real-time PCR和Western Blot检测假体周围OCN、OPG、RANKL、TRAP5b的m RNA和蛋白含量显示,实验组OCN和OPG的m RNA和蛋白含量低于对照组,而RANKL和TRAP5b的m RNA和蛋白含量高与对照组,OPG/RANKL比值明显低于对照组。2、CG组、ZL组、TP组和ZL+TP组的影像学结果均可见明显的假体周围骨溶解迹象,4组对比未见明显差异。骨硬组织切片行Goldner三色染色结果显示应用唑来膦酸和特立帕肽治疗后假体周围骨组织与假体的界面结合情况均好于对照组,骨组织较均匀地分布于假体周围,纤维结缔组织增生较对照组明显减轻,尤其是ZL+TP组更明显。ZL组、TP组和ZL+TP组的骨-假体接触率和骨体积分数均高于CG组,Von Kossa染色结果显示治疗后各组假体周围钙盐沉积岛排布较对照组更均匀,主要呈星点状排列,ZL组、TP组和ZL+TP组的钙盐沉积岛数量少于对照组,而平均面积大于对照组,以ZL+TP组更显著。PCR和Western Blot检测假体周围OCN、OPG、RANKL、TRAP5b的m RNA和蛋白含量显示,ZL组、TP组和ZL+TP组的OCN的m RNA和蛋白含量高于对照组,TP组和ZL+TP组升高的量大于ZL组。ZL组、TP组和ZL+TP组TRAP5b的m RNA和蛋白含量低于对照组,而ZL和ZL+TP组的降低幅度更大;同对照组的m RNA和蛋白含量相比,ZL组、TP组和ZL+TP组的OPG升高,RANKL降低,OPG/RANKL比值明显高于于对照组,尤其以ZL+TP组升高最为显著。结论:1、应用20μm钛颗粒涂抹钛棒假体表面植入兔股骨髓腔使得假体周围产生持续钛颗粒刺激,有效诱导假体周围骨溶解和假体松动产生,主要机制是钛颗粒诱导巨噬细胞产生炎性因子,通过OPG/RANKL/RANK信号途径刺激破骨细胞分化,产生破骨细胞性骨溶解,同时抑制成骨细胞的功能,减少假体周围骨长入。该模型模拟了人工假体植入和松动的过程,经济、有效,相对快速,可重复性强,是研究人工关节假体松动的理想的病因模型,可以用于病因及生物力学研究,也可用于多种方式的无菌性松动防治研究。2、唑来膦酸和特立帕肽可以通过OPG/RANKL/RANK途径抑制钛颗粒诱导的破骨细胞活化,并加强成骨细胞的功能,起到假体周围骨溶解防治假体无菌性松动的作用。唑来膦酸在抑制破骨细胞活化从而抑制假体周围骨溶解方面作用更强,而特立帕肽在抑制破骨细胞活化的同时还可以上调成骨细胞功能,具有更强的促进假体周围骨长入的作用。两种药物联合使用可以起到协同作用,产生更好的抑制骨溶解,促进骨长入的治疗效果。唑来膦酸联合特立帕肽可能成为有效的防治人工关节无菌性松动的药物。
[Abstract]:Objective: 1, successfully constructed a titanium particle induced aseptic loosening model of the rabbit artificial knee joint prosthesis.2, and explored the effect and mechanism of zoledronic acid combined with teramepin in the prevention of artificial knee prosthesis loosening in rabbits. In order to further study the nonsurgical methods for the aseptic loosening of artificial knee prosthesis. Method: 1, feeding 16 adult males. The New Zealand white rabbits, with an average weight of 2.85kg, were randomly divided into two groups. All the animals were operated on the right knee joint after 1 weeks of adaptive feeding. After the knee joint was exposed in the middle of the right knee joint, the mouth was open above the lateral stop point of the posterior cruciate ligament, and the length of 2.0cm and diameter 0.5cm titanium rod were implanted into the medulla cavity along the longitudinal axis of the femur. The titanium in the experimental group was carried out. The surface of the stick prosthesis was evenly coated with 50 u g diameter 20 m titanium particles (test group). The control group was not smeared with any material on the surface of the titanium rod prosthesis. After the same perioperative period, the animals were sacrificed for 12 weeks. Each group of animals collected the following data: the knee joint X ray of the first 2 days before death, the synovial tissue of the hematoxylin eosin (H-E) staining, and the prosthesis. Goldner tricolor staining and Kossa staining were performed in the hard tissue section of the bone tissue. The periprosthetic periprosthetic membrane tissues were examined by Real-time PCR and Western Blot to detect the m RNA and protein content of OCN, OPG, RANKL and TRAP5b. The statistical comparison of the related data between the two groups proved to be effective for the preparation of the aseptic loosening model of the rabbit artificial joint prosthesis. 24 New Zealand white rabbits were fed, and 24 New Zealand white rabbits were continuously fed. Titanium rods were implanted into the marrow cavity along the knee joint of the right side of the femur with the same operation. All titanium rods were evenly coated with 50 g diameter 20 u m titanium particles. After the operation, the related drugs were given. According to the treatment scheme, there were 3 groups of 8 rats in each group. A group of zoledronic acid (ZL), tramopirin (TP), zoledronic acid + tralamopride (ZL+TP), and animals were killed at twelfth weeks. The following data were collected: 2 days before the death of the knee joint x, the bone tissue of the prosthesis was stained with Goldner trichrome and Kossa, and the peripheral membrane tissues of the prosthesis were treated with Real-time PCR and West. Ern Blot detected the m RNA and protein content of related specific indicators. The first part of the experimental group was used as a control group (group CG) to perform statistical comparison and analysis to verify the aseptic loosening action and mechanism of zoledronic acid and teramopeptide for the prevention and treatment of joint prosthesis. Results: 1, before the death of the experimental group, the animals were executed. The X-ray showed obvious signs of osteolysis around the prosthesis, and the results of synovial H-E staining showed that the synovial tissue of the knee joint in the experimental group was obviously proliferated and accompanied by a large number of inflammatory cell infiltration. The Goldner tricolor staining of the bone hard tissue section showed that there was a large gap between the animal prosthesis and the bone tissue in the experimental group, and the space was filled with fibrous connective tissue and the prosthesis was filled. The combination of bone tissue and bone tissue in the control group was relatively tight, and the surrounding space was smaller and no obvious fibrous proliferation was filled around. The bone prosthesis contact rate and bone volume fraction in the experimental group were lower than that of the control group. The Von Kossa staining results of the hard tissue sections showed that the calcium salt deposition island around the animal prosthesis was found in the experimental group. The calcium salt deposition island around the prosthesis of the control group showed star shape, although the number was small, but the single area larger.Real-time PCR and Western Blot detected the OCN, OPG, RANKL, TRAP5b's M RNA and protein content, and the experimental group of OCN and OPG and protein content Compared with the control group, the m RNA and protein content of RANKL and TRAP5b was higher than that of the control group, and the ratio of OPG/RANKL was significantly lower than that of the control group.2. The imaging results of the CG group, ZL, TP and ZL+TP groups were all obvious signs of periprosthetic osteolysis, and there was no significant difference between the 4 groups. The interface of bone tissue around the prosthesis was better than that of the control group after the treatment of acid and trabepin. The bone tissue was more evenly distributed around the prosthesis, and the fibrous connective tissue proliferation was significantly reduced than that of the control group, especially in group ZL+TP.ZL. The bone prosthesis contact rate and bone volume fraction in group TP and ZL+TP were higher than that in group CG, Von Ko The results of SSA staining showed that the calcium salt deposition Island arrangement around the prosthesis was more uniform than the control group. The number of calcium salt deposition islands in group ZL, group TP and ZL+TP was less than that of the control group, and the average area was larger than the control group, and the ZL+TP group was more significant.PCR and Western Blot to detect OCN, OPG, RANKL, TRAP5b. The content of protein content showed that the content of M RNA and protein in group ZL, TP and ZL+TP group was higher than that of the control group. The increase in TP group and ZL+TP group was greater than that in the ZL group.ZL group. PG increased, RANKL decreased, and the ratio of OPG/RANKL was significantly higher than that in the control group, especially in the ZL+TP group. Conclusion: 1, the implantation of rabbit femoral marrow cavity on the surface of the titanium rod with 20 m titanium particles on the surface of the prosthesis produces continuous titanium particle stimulation around the prosthesis, which can effectively induce periprosthetic osteolysis and prosthesis loosening. The main mechanism is titanium particle lure. The macrophage produces inflammatory factors, which stimulates osteoclast differentiation through OPG/RANKL/RANK signal pathway, produces osteoclast osteolysis, and inhibits osteoblast function and reduces bone growth around the prosthesis. This model simulates the process of prosthesis implantation and loosening, economic, effective, relatively fast and repeatable. It is a study of artificial artificial prosthesis. The ideal etiological model for joint prosthesis loosening can be used in etiological and biomechanical studies, and can also be used in a variety of ways of aseptic loosening prevention and treatment of.2. Zoledronic acid and teramepin can inhibit the activation of osteoclast induced by titanium particles through the OPG/RANKL/RANK pathway, and strengthen the function of osteoblasts and play an osteolysis around the prosthesis. Zoleponic acid plays a stronger role in inhibiting osteoclast activation and inhibiting osteolysis around the prosthesis, while teramepin can increase the osteoblast function while inhibiting osteoclast activation. It has a stronger role in promoting bone growth around the prosthesis. The combination of two drugs can be used. Synergistic effects produce better inhibition of osteolysis and promote the treatment of bone growth. Zoledronic acid combined with teramepine may be an effective drug to prevent aseptic loosening of artificial joints.

【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R687.4

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