發(fā)布時(shí)間：2018-06-30 10:37

  本文選題：脫細(xì)胞 + 人重組骨形態(tài)發(fā)生蛋白-2�。� 參考：《第二軍醫(yī)大學(xué)》2012年博士論文

【摘要】：一、研究目的 前交叉韌帶斷裂是常見(jiàn)的運(yùn)動(dòng)損傷,斷裂后只有進(jìn)行替代物重建才能恢復(fù)關(guān)節(jié)的穩(wěn)定和功能。目前臨床上常用的韌帶替代物主要為自體韌帶(肌腱)、異體韌帶(肌腱)和人工材料移植物。上述三類(lèi)重建材料都存在著諸多的弊端,無(wú)法成為臨床上理想的選擇。由于無(wú)供區(qū)的繼發(fā)損害,以及保存、取材技術(shù)的改進(jìn),目前同種異體肌腱在臨床上取得了廣泛的應(yīng)用。同種異體肌腱植入體內(nèi),愈合過(guò)程類(lèi)似于自體肌腱,需經(jīng)歷組織壞死、血管化、細(xì)胞爬行替代、重塑四個(gè)階段。與自體肌腱不同,同種異體肌腱移植后,腱細(xì)胞表面的糖蛋白的MHC-1型抗原引起宿主的免疫排斥反應(yīng),免疫反應(yīng)將影響異體肌腱與宿主骨的整合過(guò)程,可以導(dǎo)致腱-骨界面愈合延遲和減弱,甚至移植失敗。肌腱的細(xì)胞外基質(zhì)(ECM)主要由Ⅰ、Ⅲ型膠原蛋白組成,同種異體及異種個(gè)體之間的同一組織,其ECM得成分和結(jié)構(gòu)很相近,其自身的免疫原性低。脫細(xì)胞技術(shù)可以去除肌腱的免疫成分而保留細(xì)胞外成分原有的生物活性及力學(xué)特性。重組人骨形態(tài)發(fā)生蛋白-2具有很強(qiáng)的促進(jìn)成骨細(xì)胞分化和誘導(dǎo)體外成骨的能力,已有試驗(yàn)發(fā)現(xiàn)可促進(jìn)自體半腱肌重建ACL的腱骨愈合。本實(shí)驗(yàn)使用磷酸三丁酯(TBP)進(jìn)行脫細(xì)胞處理去除同種異體肌腱中的細(xì)胞成分,測(cè)定其對(duì)肌腱的組織學(xué)和力學(xué)特性的影響,脫細(xì)胞后的異體肌腱復(fù)合人重組骨形態(tài)發(fā)生蛋白-2重建兔交叉韌帶,定期對(duì)重建的ACL進(jìn)行組織學(xué)觀察、影像學(xué)檢測(cè)及生物力學(xué)的檢測(cè),了解脫細(xì)胞同種異體肌腱復(fù)合rh-BMP-2對(duì)ACL重建的影響。 二、研究方法 (一)同種異體肌腱的脫細(xì)胞處理 取新西蘭兔的趾屈肌腱進(jìn)行脫細(xì)胞處理,使用磷酸三丁酯(TBP)進(jìn)行脫細(xì)胞。TBP法：1、低滲的10mm Tris液(包括絲氨酸蛋白酶抑制劑+金屬蛋白酶抑制劑+青霉素/鏈霉素)保持36h；2、高滲鹽水+50mm Tris液(包含1%辛基含苯氧基聚乙氧基乙醇+蛋白酶抑制劑)保持48h；3、Hank's生理緩沖液進(jìn)行漂洗；4、1%磷酸三丁酯36h+50mm三羥甲基氨基甲烷48h；5、含有抗生素的硫酸鹽緩沖液沖洗+浸泡。 (二)脫細(xì)胞肌腱的組織學(xué)和生物力學(xué)測(cè)定 對(duì)脫細(xì)胞處理的異體肌腱進(jìn)行HE染色、DAPI熒光染色檢測(cè),觀察TBP脫細(xì)胞效果。使用二甲基亞甲藍(lán)(DMMB)法測(cè)定糖胺聚糖(GAG)含量,檢測(cè)脫細(xì)胞前后肌腱內(nèi)的GAG含量；使用動(dòng)物組織基因組DNA小量提取試劑盒測(cè)定DNA含量,檢測(cè)脫細(xì)胞前后肌腱的DNA含量變化；使用羥脯氨酸試劑盒測(cè)定羥脯氨酸含量,根據(jù)羥脯氨酸占膠原蛋白的含量,計(jì)算膠原蛋白含量,觀察脫細(xì)胞前后膠原蛋白的變化；檢測(cè)肌腱的最大張力負(fù)荷和彈性模量,了解脫細(xì)胞處理對(duì)肌腱生物力學(xué)的影響。 (三)脫細(xì)胞肌腱復(fù)合rh-BMP-2重建AGL的影像學(xué)觀察 使用脫細(xì)胞肌腱重建兔前交叉韌帶,腱骨界面注入含rh-BMP-2的纖維蛋白凝膠,術(shù)后1、3、6月分別對(duì)患側(cè)膝關(guān)節(jié)骨隧道段進(jìn)行CT平掃,在矢狀位測(cè)量膝關(guān)節(jié)股骨、脛骨隧道的入口段、出口段及中間段的寬度,并與對(duì)照組進(jìn)行比較,了解脫細(xì)胞肌腱復(fù)合rh-BMP-2對(duì)促進(jìn)ACL重建后骨隧道段的腱骨愈合的影像學(xué)變化。 (四)脫細(xì)胞肌腱復(fù)合rh-BMP-2重建ACL的組織學(xué)觀察和力學(xué)特性檢測(cè) 使用脫細(xì)胞肌腱重建兔前交叉韌帶,腱骨界面注入含rh-BMP-2的纖維蛋白凝膠,術(shù)后1、3、6月分別對(duì)患側(cè)膝關(guān)節(jié)骨隧道段、重建的ACL進(jìn)行硬組織切片、普通HE染色,觀察腱骨界面成骨情況及重建的ACL的炎癥反應(yīng)、細(xì)胞增殖等情況,并與對(duì)照組進(jìn)行比較；使用生物材料測(cè)試機(jī)對(duì)術(shù)后3、6月的膝關(guān)節(jié)標(biāo)本進(jìn)行ACL抗拔出試驗(yàn),記錄拔出的最大抗拉強(qiáng)度及彈性模量,了解脫細(xì)胞肌腱復(fù)合rh-BMP-2對(duì)ACL重建后腱骨愈合的影響。 三、結(jié)果 (一)脫細(xì)胞肌腱的組織學(xué)和生物力學(xué)測(cè)定 經(jīng)磷酸三丁酯(TBP)脫細(xì)胞處理的肌腱,HE染色未見(jiàn)細(xì)胞成分染色,DAPI熒光染色未見(jiàn)陽(yáng)性結(jié)果,提示細(xì)胞成分去除徹底；二甲基亞甲藍(lán)法測(cè)定糖胺聚糖含量,發(fā)現(xiàn)脫細(xì)胞前后對(duì)比無(wú)明顯差異；動(dòng)物組織基因組DNA小量提取試劑盒測(cè)定DNA含量,發(fā)現(xiàn)脫細(xì)胞前后肌腱內(nèi)DNA含量明顯減少；使用羥脯氨酸試劑盒間接測(cè)定膠原蛋白含量,發(fā)現(xiàn)脫細(xì)胞前后膠原含量無(wú)明顯差異；生物力學(xué)測(cè)試發(fā)現(xiàn),脫細(xì)胞前后肌腱的最大張力負(fù)荷和彈性模量無(wú)明顯差異。 (二)脫細(xì)胞肌腱復(fù)合rh-BMP-2重建ACL的影像學(xué)觀察 對(duì)ACL重建后的兔膝關(guān)節(jié)行CT平掃,可以較好地重建兔ACL重建模型的影像學(xué)情況。術(shù)后1月,兩組間骨隧道寬度對(duì)比無(wú)明顯差異；術(shù)后3月,脫細(xì)胞組骨隧道壁不光滑,存在成骨反應(yīng),骨隧道寬度與術(shù)后1月比較無(wú)明顯改變,而對(duì)照組未見(jiàn)成骨反應(yīng),骨隧道寬度與術(shù)后1月相比,有增寬趨勢(shì),但無(wú)統(tǒng)計(jì)學(xué)差異；術(shù)后6月,脫細(xì)胞組骨隧道壁成骨反應(yīng)較前增加,骨隧道寬度與術(shù)后1、3月略有增寬,但無(wú)統(tǒng)計(jì)學(xué)差異；而對(duì)照組仍無(wú)明顯成骨反應(yīng),骨隧道寬度與術(shù)后1、3月相比,出現(xiàn)增寬現(xiàn)象,存在統(tǒng)計(jì)學(xué)差異。 (四)脫細(xì)胞肌腱復(fù)合rh-BMP-2重建ACL的組織學(xué)觀察和生物力學(xué)測(cè)試 骨隧道段硬組織切片發(fā)現(xiàn),術(shù)后1月,脫細(xì)胞組腱骨間隙較小,可見(jiàn)少量Sharpey纖維及軟骨細(xì)胞生成,而對(duì)照組腱骨間隙寬大,無(wú)Sharpey纖維及軟骨細(xì)胞生成；術(shù)后3月,脫細(xì)胞組腱骨間隙進(jìn)一步減小,Sharpey纖維及軟骨細(xì)胞生成增多；而對(duì)照組腱骨間隙較前減小,少量Sharpey纖維及軟骨細(xì)胞生成；術(shù)后6月,脫細(xì)胞組腱骨間隙幾乎消失,Sharpey纖維大量生成排列有序,軟骨細(xì)胞成熟分化,而對(duì)照組腱骨間隙減小但存在,Sharpey纖維及軟骨細(xì)胞生成較前增多,但無(wú)明顯成熟分化趨勢(shì)。重建的ACL,術(shù)后1月,脫細(xì)胞組可見(jiàn)少量淋巴細(xì)胞浸潤(rùn),而對(duì)照組存在大量淋巴細(xì)胞浸潤(rùn)；術(shù)后3月,脫細(xì)胞組膠原纖維排列有序,可見(jiàn)成纖維細(xì)胞及軟骨細(xì)胞增生,而對(duì)照組膠原纖維松散,可見(jiàn)少量成纖維細(xì)胞,未見(jiàn)軟骨細(xì)胞；術(shù)后6月,脫細(xì)胞組軟骨細(xì)胞大量增生并出現(xiàn)成熟分化,膠原纖維排列緊密、有序,而對(duì)照組膠原結(jié)構(gòu)仍較松散,成纖維細(xì)胞及軟骨細(xì)胞數(shù)量較少。術(shù)后3、6月的生物力學(xué)測(cè)試,ACL重建的抗拔出試驗(yàn)中,脫細(xì)胞組在彈性模量、最大載荷等指標(biāo)均優(yōu)于對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義。 四、結(jié)論 (一)、TBP法能有效去除同種異體肌腱內(nèi)部的細(xì)胞成分,去除免疫原性的同時(shí)對(duì)肌腱原有的生物學(xué)活性及力學(xué)特性無(wú)明顯影響。 (二)、CT平掃能較好地重現(xiàn)膝關(guān)節(jié)重建ACL的影像。脫細(xì)胞肌腱復(fù)合rh-BMP-2重建兔ACL后,CT平掃發(fā)現(xiàn),與對(duì)照組相比,術(shù)后3、6月未見(jiàn)骨隧道增寬跡象,腱-骨界面存在成骨反應(yīng)。 (三)、脫細(xì)胞肌腱復(fù)合rh-BMP-2重建兔ACL后,與對(duì)照組相比,術(shù)后1、3、6月腱骨界面愈合良好,界面成骨反應(yīng)逐漸增加,腱骨間隙縮小,異體肌腱細(xì)胞爬行替代速度加快。 (四)、脫細(xì)胞肌腱復(fù)合rh-BMP-2重建兔ACL后,與對(duì)照組相比,術(shù)后3、6月行抗拔出生物力學(xué)測(cè)試,脫細(xì)胞組的彈性模量及最大載荷優(yōu)于對(duì)照組。
[Abstract]:First, the purpose of the study
Rupture of anterior cruciate ligament (ACL) is a common motor injury. Only reconstruction of substitutes can be used to restore the stability and function of the joints. The commonly used substitute ligaments are mainly autogenous ligaments (tendons), allograft ligaments (tendons) and artificial material grafts. These three kinds of reconstruction materials have many disadvantages and can not be used. The clinical ideal choice. Due to the secondary damage without the donor area, and the improvement of preservation and technology, the allogenic tendon has been widely used clinically. The allograft tendon is implanted in the body, the healing process is similar to the autogenous tendon, which needs to undergo tissue necrosis, blood tube, cell reptile replacement, and remolding four stages. The MHC-1 type antigen of the glycoprotein on the surface of the tendon causes the host immune rejection after the allograft of the allograft tendon. The immune response will affect the integration of the allograft tendon and the host bone, which can lead to the delay and weakening of the tendon bone interface and even the graft failure. The extracellular matrix of the tendon (ECM) is mainly composed of type I, type III collagen. The same tissue of the protein, allograft and xenogeneic individual, whose ECM has very close components and structures, and its own immunogenicity is low. Decellularization technology can remove the immune components of the tendon and retain the original biological and mechanical properties of the extracellular components. Recombinant human bone morphogenetic protein -2 has a strong promotion of osteoblasts. The ability to differentiate and induce osteogenesis in vitro has been found to promote the healing of the tendon bone in the autologous semitendinosus reconstruction of ACL. This experiment uses three butyl phosphate (TBP) to remove the cell components in the allograft tendon and determine its effect on the histology and mechanical properties of the tendon. The allogeneic tendon after dehydration is combined with human weight. The rabbit cruciate ligament was reconstructed by group bone morphogenetic protein -2, and the reconstructed ACL was observed regularly, the imaging detection and biomechanical detection were used to understand the effect of the acellular allograft tendon complex rh-BMP-2 on the reconstruction of ACL.
Two, research methods
(1) acellular disposal of allogenic tendon
The flexor digitorum tendon of New Zealand rabbits was deactivated and acellular.TBP method was performed using three butyl phosphate (TBP) phosphate (TBP). 1, the low permeability 10mm Tris solution (including serine protease inhibitor + metalloproteinase inhibitor + penicillin / streptomycin) maintained 36h; 2, hypertonic saline +50mm Tris solution (including 1% octyl polyoxyl polyethoxy ethanol + protein. Enzyme inhibitor) kept 48h; 3, Hank's physiological buffer solution was rinsed, 4,1% phosphoric acid three butyl ester, three hydroxymethyl amino methylamino methane 48h; 5, sulfate buffer solution containing antibiotics was washed + soaked.
(two) histological and biomechanical determination of acellular tendons
The acellular allograft tendon was stained with HE, DAPI fluorescence staining was used to detect the decellular effect of TBP. The content of glycosaminoglycan (GAG) was measured by two methylene blue (DMMB) method and the content of GAG in the tendon was detected before and after the dehydration. The content of DNA was measured by the genomic DNA small extraction kit of animal tissue, and the tendon was detected before and after the dehydration. The content of hydroxyproline was measured with hydroxyproline kit and the content of hydroxyproline was measured by hydroxyproline. The collagen content was calculated and the changes of collagen before and after cell removal were observed. The maximum tension load and modulus of elasticity of tendon were detected and the effect of define cell treatment on tendon biomechanics was understood.
(three) imaging study of acellular tendon combined with rh-BMP-2 for AGL reconstruction
The rabbit anterior cruciate ligament was reconstructed with acellular tendon, and fibrin gel containing rh-BMP-2 was injected into the tendon bone interface. CT plain scan was carried out on the tunnel segment of the knee joint at 1,3,6 months after the operation. The width of the knee joint, the entrance section of the tibial tunnel, the exit section and the middle segment were measured in the sagittal position, and compared with the control group, the acellular muscle was understood. Radiographic changes of tendon bone healing after ACL reconstruction by rh-BMP-2 combined with tendon.
(four) histological observation and mechanical properties of ACL reconstructed with acellular tendon combined with rh-BMP-2
The rabbit anterior cruciate ligament was rebuilt with acellular tendon, and fibrin gel containing rh-BMP-2 was injected into the tendon bone interface. After 1,3,6 months, the bone tunnel segment of the knee joint and the reconstructed ACL were sectioning, the normal HE staining, the osteogenesis of the tendon and bone interface and the inflammatory reaction of the reconstructed ACL, the cell proliferation and so on were observed and compared with the control group. By using a biomaterial test machine, a ACL anti pull-out test was performed on the knee joint specimens of 3,6 months after operation to record the maximum tensile strength and modulus of elasticity, and to understand the effect of the acellular tendon complex rh-BMP-2 on the healing of the tendon bone after ACL reconstruction.
Three, the result
(1) histological and biomechanical determination of acellular tendon
Three butyl phosphate (TBP) phosphate (TBP) was removed from the tendon, no staining of cell components was found in HE staining, and no positive results were found in DAPI fluorescent staining. The content of glycosaminoglycan was determined by two methylene blue method, and there was no significant difference before and after cell removal; genomic DNA small extraction kits of animal tissues were used to determine DNA content. It was found that the content of DNA in the tendon was significantly reduced before and after the dehydration, and the content of collagen was measured indirectly by the hydroxyproline kit. There was no significant difference between the collagen content before and after the cell removal. The biomechanical test found that the maximum tension load and modulus of elasticity before and after the dehydration were not significantly different.
(two) imaging study of acellular tendon combined with rh-BMP-2 for ACL reconstruction
ACL reconstruction of rabbit knee joint CT plain scan can better reconstruct the image of rabbit ACL reconstruction model. In January after operation, there was no significant difference in bone tunnel width between the two groups. In March after operation, the bone tunnel wall was not smooth and there was bone formation reaction, the width of bone tunnel was not significantly changed after the operation in January, but no osteogenesis in the control group was found in the control group. The width of the bone tunnel was wider than that of January, but there was no statistical difference. In June, the osteogenesis of the bone tunnel wall increased and the width of the bone tunnel was widened slightly with the 1,3 month after the operation, but there was no significant difference in the width of the bone tunnel, but there was no obvious osteogenic reaction in the control group, and the width of the bone tunnel was wider than that of the 1,3 month after the operation. There are statistical differences in the image.
(four) histological observation and biomechanical test of acellular tendon combined with rh-BMP-2 for ACL reconstruction
In the hard tissue section of the bone tunnel section, it was found that in January after the operation, a small number of Sharpey fibers and chondrocytes were found in the acellular group, but the interspace of the tendon bone in the control group was wide, and no Sharpey fibers and chondrocytes were generated. In March, the interspace of the tendon and bone in the deacellular group was further reduced, and the growth of Sharpey fibers and chondrocytes increased; and the control was compared. In the group of tendon and bone, a small amount of Sharpey fibers and chondrocytes were generated. In June, the interspace of tendon and bone almost disappeared, the Sharpey fibers were arranged in order, the cartilage cells were mature and differentiated, while the control group had a decrease in the interspace of tendon and bone, and the growth of Sharpey fibers and soft bone cells increased, but there was no obvious trend of maturation and differentiation. Rebuilt ACL, in January after operation, a small amount of lymphocyte infiltration was found in the acellular group, while a large number of lymphocytes were infiltrated in the control group. In March, the collagenous fibers in the decellular group were arranged orderly, and the fibroblasts and chondrocytes proliferated, while the control group was loose, a small amount of fibroblasts and no chondrocytes were seen. In June after operation, the cells were removed. In the cell group, the chondrocytes proliferated and formed mature differentiation, and the collagen fibers were arranged closely and orderly. The collagen structure of the control group was still loose and the number of fibroblasts and chondrocytes was less. The biomechanical test of 3,6 months after operation and the anti pullout test of ACL reconstruction were superior to the control group in the modulus of elasticity and the maximum load. The difference is statistically significant.
Four. Conclusion
(1) the TBP method can effectively remove the cell components in the allograft tendon and remove the immunogenicity and have no obvious effect on the original biological and mechanical properties of the tendon.
(two) CT scan can better reproduce the image of the knee joint reconstruction of ACL. After the acellular tendon combined with rh-BMP-2 to reconstruct the rabbit ACL, CT plain scan was found. Compared with the control group, there was no sign of bone tunnel widening in 3,6 month after operation, and the tendon bone interface had osteogenic reaction.
(three) after reconstructing rabbit ACL with acellular tendon complex rh-BMP-2, the bone interface of 1,3,6 month tendon healed well, bone formation reaction gradually increased, the interspace of tendon bone narrowed, and the rate of substitutes of allogenic tendon cells was quicker than the control group.
(four) after the rabbit ACL was rebuilt with the acellular tendon complex rh-BMP-2, the biomechanical test was performed at 3,6 month after the operation compared with the control group. The modulus of elasticity and the maximum load of the decellular group were better than the control group.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R686.5;R-332

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相關(guān)期刊論文 前5條

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