脱细胞同种异体肌腱复合重组人骨形态发生蛋白-2重建兔前交叉韧带的实验研究

发布时间：2018-06-30 10:37

本文选题：脱细胞 + 人重组骨形态发生蛋白-2　；参考：《第二军医大学》2012年博士论文

【摘要】：一、研究目的前交叉韧带断裂是常见的运动损伤,断裂后只有进行替代物重建才能恢复关节的稳定和功能。目前临床上常用的韧带替代物主要为自体韧带(肌腱)、异体韧带(肌腱)和人工材料移植物。上述三类重建材料都存在着诸多的弊端,无法成为临床上理想的选择。由于无供区的继发损害,以及保存、取材技术的改进,目前同种异体肌腱在临床上取得了广泛的应用。同种异体肌腱植入体内,愈合过程类似于自体肌腱,需经历组织坏死、血管化、细胞爬行替代、重塑四个阶段。与自体肌腱不同,同种异体肌腱移植后,腱细胞表面的糖蛋白的MHC-1型抗原引起宿主的免疫排斥反应,免疫反应将影响异体肌腱与宿主骨的整合过程,可以导致腱-骨界面愈合延迟和减弱,甚至移植失败。肌腱的细胞外基质(ECM)主要由Ⅰ、Ⅲ型胶原蛋白组成,同种异体及异种个体之间的同一组织,其ECM得成分和结构很相近,其自身的免疫原性低。脱细胞技术可以去除肌腱的免疫成分而保留细胞外成分原有的生物活性及力学特性。重组人骨形态发生蛋白-2具有很强的促进成骨细胞分化和诱导体外成骨的能力,已有试验发现可促进自体半腱肌重建ACL的腱骨愈合。本实验使用磷酸三丁酯(TBP)进行脱细胞处理去除同种异体肌腱中的细胞成分,测定其对肌腱的组织学和力学特性的影响,脱细胞后的异体肌腱复合人重组骨形态发生蛋白-2重建兔交叉韧带,定期对重建的ACL进行组织学观察、影像学检测及生物力学的检测,了解脱细胞同种异体肌腱复合rh-BMP-2对ACL重建的影响。二、研究方法 (一)同种异体肌腱的脱细胞处理取新西兰兔的趾屈肌腱进行脱细胞处理,使用磷酸三丁酯(TBP)进行脱细胞。TBP法：1、低渗的10mm Tris液(包括丝氨酸蛋白酶抑制剂+金属蛋白酶抑制剂+青霉素/链霉素)保持36h；2、高渗盐水+50mm Tris液(包含1%辛基含苯氧基聚乙氧基乙醇+蛋白酶抑制剂)保持48h；3、Hank's生理缓冲液进行漂洗；4、1%磷酸三丁酯36h+50mm三羟甲基氨基甲烷48h；5、含有抗生素的硫酸盐缓冲液冲洗+浸泡。 (二)脱细胞肌腱的组织学和生物力学测定对脱细胞处理的异体肌腱进行HE染色、DAPI荧光染色检测,观察TBP脱细胞效果。使用二甲基亚甲蓝(DMMB)法测定糖胺聚糖(GAG)含量,检测脱细胞前后肌腱内的GAG含量；使用动物组织基因组DNA小量提取试剂盒测定DNA含量,检测脱细胞前后肌腱的DNA含量变化；使用羟脯氨酸试剂盒测定羟脯氨酸含量,根据羟脯氨酸占胶原蛋白的含量,计算胶原蛋白含量,观察脱细胞前后胶原蛋白的变化；检测肌腱的最大张力负荷和弹性模量,了解脱细胞处理对肌腱生物力学的影响。 (三)脱细胞肌腱复合rh-BMP-2重建AGL的影像学观察使用脱细胞肌腱重建兔前交叉韧带,腱骨界面注入含rh-BMP-2的纤维蛋白凝胶,术后1、3、6月分别对患侧膝关节骨隧道段进行CT平扫,在矢状位测量膝关节股骨、胫骨隧道的入口段、出口段及中间段的宽度,并与对照组进行比较,了解脱细胞肌腱复合rh-BMP-2对促进ACL重建后骨隧道段的腱骨愈合的影像学变化。 (四)脱细胞肌腱复合rh-BMP-2重建ACL的组织学观察和力学特性检测使用脱细胞肌腱重建兔前交叉韧带,腱骨界面注入含rh-BMP-2的纤维蛋白凝胶,术后1、3、6月分别对患侧膝关节骨隧道段、重建的ACL进行硬组织切片、普通HE染色,观察腱骨界面成骨情况及重建的ACL的炎症反应、细胞增殖等情况,并与对照组进行比较；使用生物材料测试机对术后3、6月的膝关节标本进行ACL抗拔出试验,记录拔出的最大抗拉强度及弹性模量,了解脱细胞肌腱复合rh-BMP-2对ACL重建后腱骨愈合的影响。三、结果 (一)脱细胞肌腱的组织学和生物力学测定经磷酸三丁酯(TBP)脱细胞处理的肌腱,HE染色未见细胞成分染色,DAPI荧光染色未见阳性结果,提示细胞成分去除彻底；二甲基亚甲蓝法测定糖胺聚糖含量,发现脱细胞前后对比无明显差异；动物组织基因组DNA小量提取试剂盒测定DNA含量,发现脱细胞前后肌腱内DNA含量明显减少；使用羟脯氨酸试剂盒间接测定胶原蛋白含量,发现脱细胞前后胶原含量无明显差异；生物力学测试发现,脱细胞前后肌腱的最大张力负荷和弹性模量无明显差异。 (二)脱细胞肌腱复合rh-BMP-2重建ACL的影像学观察对ACL重建后的兔膝关节行CT平扫,可以较好地重建兔ACL重建模型的影像学情况。术后1月,两组间骨隧道宽度对比无明显差异；术后3月,脱细胞组骨隧道壁不光滑,存在成骨反应,骨隧道宽度与术后1月比较无明显改变,而对照组未见成骨反应,骨隧道宽度与术后1月相比,有增宽趋势,但无统计学差异；术后6月,脱细胞组骨隧道壁成骨反应较前增加,骨隧道宽度与术后1、3月略有增宽,但无统计学差异；而对照组仍无明显成骨反应,骨隧道宽度与术后1、3月相比,出现增宽现象,存在统计学差异。 (四)脱细胞肌腱复合rh-BMP-2重建ACL的组织学观察和生物力学测试骨隧道段硬组织切片发现,术后1月,脱细胞组腱骨间隙较小,可见少量Sharpey纤维及软骨细胞生成,而对照组腱骨间隙宽大,无Sharpey纤维及软骨细胞生成；术后3月,脱细胞组腱骨间隙进一步减小,Sharpey纤维及软骨细胞生成增多；而对照组腱骨间隙较前减小,少量Sharpey纤维及软骨细胞生成；术后6月,脱细胞组腱骨间隙几乎消失,Sharpey纤维大量生成排列有序,软骨细胞成熟分化,而对照组腱骨间隙减小但存在,Sharpey纤维及软骨细胞生成较前增多,但无明显成熟分化趋势。重建的ACL,术后1月,脱细胞组可见少量淋巴细胞浸润,而对照组存在大量淋巴细胞浸润；术后3月,脱细胞组胶原纤维排列有序,可见成纤维细胞及软骨细胞增生,而对照组胶原纤维松散,可见少量成纤维细胞,未见软骨细胞；术后6月,脱细胞组软骨细胞大量增生并出现成熟分化,胶原纤维排列紧密、有序,而对照组胶原结构仍较松散,成纤维细胞及软骨细胞数量较少。术后3、6月的生物力学测试,ACL重建的抗拔出试验中,脱细胞组在弹性模量、最大载荷等指标均优于对照组,差异具有统计学意义。四、结论 (一)、TBP法能有效去除同种异体肌腱内部的细胞成分,去除免疫原性的同时对肌腱原有的生物学活性及力学特性无明显影响。 (二)、CT平扫能较好地重现膝关节重建ACL的影像。脱细胞肌腱复合rh-BMP-2重建兔ACL后,CT平扫发现,与对照组相比,术后3、6月未见骨隧道增宽迹象,腱-骨界面存在成骨反应。 (三)、脱细胞肌腱复合rh-BMP-2重建兔ACL后,与对照组相比,术后1、3、6月腱骨界面愈合良好,界面成骨反应逐渐增加,腱骨间隙缩小,异体肌腱细胞爬行替代速度加快。 (四)、脱细胞肌腱复合rh-BMP-2重建兔ACL后,与对照组相比,术后3、6月行抗拔出生物力学测试,脱细胞组的弹性模量及最大载荷优于对照组。
[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.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R686.5;R-332

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