本文選題：無菌性松動 + 動物模型　； 參考：《青島大學(xué)》2017年博士論文

【摘要】：目的:1、成功構(gòu)建鈦顆粒誘導(dǎo)的兔人工膝關(guān)節(jié)假體無菌性松動模型。2、探討唑來膦酸聯(lián)合特立帕肽防治兔人工膝關(guān)節(jié)假體松動的作用及機制,為進一步研究非手術(shù)方法治療人工膝關(guān)節(jié)假體無菌性松動建立基礎(chǔ)。方法:1、喂養(yǎng)16只成年雄性新西蘭大白兔,平均體重2.85kg,隨機分為兩組,所有動物適應(yīng)性喂養(yǎng)1周后進行右膝關(guān)節(jié)手術(shù),取右膝關(guān)節(jié)前正中切口顯露膝關(guān)節(jié)后,從后交叉韌帶股骨側(cè)止點上方開口,將長度2.0cm,直徑0.5cm鈦棒假體沿股骨縱軸方向植入髓腔。實驗組的鈦棒假體表面均勻涂抹50μg直徑20μm的鈦顆粒(試驗組),對照組鈦棒假體表面不涂抹任何物質(zhì)。術(shù)后采取相同圍手術(shù)期處理,喂養(yǎng)12周后處死動物,每組動物采集如下資料:處死前2天的膝關(guān)節(jié)X線片,滑膜組織行蘇木精-伊紅(H-E)染色,包含假體的骨組織行硬組織切片進行Goldner三色染色和Kossa染色分析,假體周圍界膜組織行Real-time PCR和Western Blot檢測OCN、OPG、RANKL和TRAP5b的m RNA和蛋白含量,進行兩組之間的相關(guān)數(shù)據(jù)的統(tǒng)計學(xué)對比,證實制備兔人工關(guān)節(jié)假體無菌性松動模型的有效性并明確其機制。2、繼續(xù)喂養(yǎng)24只新西蘭大白兔,以同樣手術(shù)方式從右側(cè)的股骨膝關(guān)節(jié)面將鈦棒假體沿軸線方向植入髓腔,所有鈦棒假體表面均勻涂抹50μg直徑20μm的鈦顆粒,術(shù)后即給予相關(guān)藥物治療。根據(jù)治療方案不同分為3組,每組8只:分別為唑來膦酸(ZL)組、特立帕肽(TP)組、唑來膦酸+特立帕肽(ZL+TP)組,同樣于第12周時處死動物,采集如下資料:處死前2天的膝關(guān)節(jié)X線片,包含假體的骨組織行硬組織切片進行Goldner三色染色和Kossa染色,假體周圍界膜組織行Real-time PCR和Western Blot檢測相關(guān)特異性指標(biāo)的m RNA和蛋白含量。并將第一部分實驗組作為此部分對照組(CG組)進行統(tǒng)計學(xué)對比分析,驗證唑來膦酸和特立帕肽的單獨或者聯(lián)合應(yīng)用對于防治關(guān)節(jié)人工假體無菌性松動作用和機制。結(jié)果:1、實驗組動物處死前X線可見明顯的假體周圍骨溶解跡象;滑膜H-E染色結(jié)果顯示實驗組動物膝關(guān)節(jié)滑膜組織明顯增生并伴有大量的炎性細胞浸潤。骨硬組織切片行Goldner三色染色顯示實驗組動物假體與骨組織存在較大間隙,間隙由纖維結(jié)締組織填充,假體與骨組織的結(jié)合較差。而對照組動物假體與骨組織的結(jié)合較緊密,周圍間隙較小,周圍未見明顯纖維組織增生填充。實驗組骨-假體接觸率和骨體積分數(shù)低于對照組。硬組織切片的Von Kossa染色結(jié)果顯示,實驗組動物假體周圍的鈣鹽沉積島主要是呈線狀,數(shù)量較多,但是面積較小。而對照組動物假體周圍的鈣鹽沉積島表現(xiàn)為呈星狀,雖然數(shù)量較少,但是單個面積較大。Real-time PCR和Western Blot檢測假體周圍OCN、OPG、RANKL、TRAP5b的m RNA和蛋白含量顯示,實驗組OCN和OPG的m RNA和蛋白含量低于對照組,而RANKL和TRAP5b的m RNA和蛋白含量高與對照組,OPG/RANKL比值明顯低于對照組。2、CG組、ZL組、TP組和ZL+TP組的影像學(xué)結(jié)果均可見明顯的假體周圍骨溶解跡象,4組對比未見明顯差異。骨硬組織切片行Goldner三色染色結(jié)果顯示應(yīng)用唑來膦酸和特立帕肽治療后假體周圍骨組織與假體的界面結(jié)合情況均好于對照組,骨組織較均勻地分布于假體周圍,纖維結(jié)締組織增生較對照組明顯減輕,尤其是ZL+TP組更明顯。ZL組、TP組和ZL+TP組的骨-假體接觸率和骨體積分數(shù)均高于CG組,Von Kossa染色結(jié)果顯示治療后各組假體周圍鈣鹽沉積島排布較對照組更均勻,主要呈星點狀排列,ZL組、TP組和ZL+TP組的鈣鹽沉積島數(shù)量少于對照組,而平均面積大于對照組,以ZL+TP組更顯著。PCR和Western Blot檢測假體周圍OCN、OPG、RANKL、TRAP5b的m RNA和蛋白含量顯示,ZL組、TP組和ZL+TP組的OCN的m RNA和蛋白含量高于對照組,TP組和ZL+TP組升高的量大于ZL組。ZL組、TP組和ZL+TP組TRAP5b的m RNA和蛋白含量低于對照組,而ZL和ZL+TP組的降低幅度更大;同對照組的m RNA和蛋白含量相比,ZL組、TP組和ZL+TP組的OPG升高,RANKL降低,OPG/RANKL比值明顯高于于對照組,尤其以ZL+TP組升高最為顯著。結(jié)論:1、應(yīng)用20μm鈦顆粒涂抹鈦棒假體表面植入兔股骨髓腔使得假體周圍產(chǎn)生持續(xù)鈦顆粒刺激,有效誘導(dǎo)假體周圍骨溶解和假體松動產(chǎn)生,主要機制是鈦顆粒誘導(dǎo)巨噬細胞產(chǎn)生炎性因子,通過OPG/RANKL/RANK信號途徑刺激破骨細胞分化,產(chǎn)生破骨細胞性骨溶解,同時抑制成骨細胞的功能,減少假體周圍骨長入。該模型模擬了人工假體植入和松動的過程,經(jīng)濟、有效,相對快速,可重復(fù)性強,是研究人工關(guān)節(jié)假體松動的理想的病因模型,可以用于病因及生物力學(xué)研究,也可用于多種方式的無菌性松動防治研究。2、唑來膦酸和特立帕肽可以通過OPG/RANKL/RANK途徑抑制鈦顆粒誘導(dǎo)的破骨細胞活化,并加強成骨細胞的功能,起到假體周圍骨溶解防治假體無菌性松動的作用。唑來膦酸在抑制破骨細胞活化從而抑制假體周圍骨溶解方面作用更強,而特立帕肽在抑制破骨細胞活化的同時還可以上調(diào)成骨細胞功能,具有更強的促進假體周圍骨長入的作用。兩種藥物聯(lián)合使用可以起到協(xié)同作用,產(chǎn)生更好的抑制骨溶解,促進骨長入的治療效果。唑來膦酸聯(lián)合特立帕肽可能成為有效的防治人工關(guān)節(jié)無菌性松動的藥物。
[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.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R687.4

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