深低温冷冻去细胞肌腱移植的组织学和生物力学研究

发布时间：2018-07-07 20:26

本文选题：肌腱 + 跟腱缺损　；参考：《吉林大学》2015年硕士论文

【摘要】：背景及目的：在临床工作中经常会遇到肌腱缺损需要移植肌腱进行修复的情况，目前的治疗的“金标准”是自体肌腱移植，但自体肌腱来源非常有限，，同时自体肌腱移植还会造成供区功能丧失；而同种异体肌腱移植、异种肌腱移植存在着免疫排斥反应，会导致移植物坏死、局部炎症反应等严重并发症。最终解决这些问题需要依靠肌腱愈合机制的基础研究以及组织工程学肌腱研究的进展来解决。目前学者认为肌腱愈合有两种方式，一种认为肌腱愈合是依靠腱周组织长入形成粘连的外源性愈合，据此推论肌腱移植中移植肌腱仅起到支架和通路作用。另一种是内源性愈合，认为肌腱可依靠肌腱内细胞自身再生增殖和合成胶原直接完成愈合修复。这两种愈肌腱合方式何者、何时、何种情况下在肌腱移植修复中起主要作用尚不清楚。与此同时失去内源性愈合的去细胞肌腱移植物能否达到生物力学要求尚有争议。因此我们设计了本实验，通过对比观察去细胞大鼠跟腱移植和未经处理的大鼠跟腱移植的组织学和生物力学特性，研究去除肌腱内细胞对移植肌腱愈合的影响，探讨肌腱愈合的机制，为组织工程学肌腱研究做准备。方法：实验选用96只健康成年雄性Wistar大白鼠为实验动物,将大鼠随机分成为实验组和对照组，制备跟腱缺损动物模型。实验组大鼠行深低温冻融冷冻去细胞跟腱自体移植，而对照组行自体跟腱移植。在术后1、2、4、8周处死大鼠取移植跟腱，在光学显微镜下进行HE染色形态学观察，I型和III型胶原免疫组化染色观察及图像分析，并测量移植跟腱最大载荷和弹性模量生物力学参数，进行统计学分析。结果：实验结果显示:随着跟腱移植后时间延长，移植跟腱内I型和III型胶原表达逐渐增加，其中1周、2周时对照组表达水平高于实验组（P 0.05),而4周和8周时两组表达无显著差异（P0.05)；移植跟腱生物力学检测结果显示1、2周时对照组跟腱最大载荷和弹性模量均高于实验组（P 0.05），而4、8周时两组差异无显著性（P0.05)。结论：本实验借助深低温去细胞跟腱移植与新鲜跟腱移植做组织学和生物力学特性比较，发现去细胞的肌腱移植物较之未经处理的移植物愈合滞后，同时发现了此种去细胞肌腱移植体在晚期可满足生物力学强度的需要。通过对本实验结果分析证明：深低温处理去细胞肌腱移植和自体移植远期生物学效果近似，去细胞肌腱可用于肌腱缺损的移植治疗；同时也证明了去细胞肌腱移植物用于异体肌腱移植的可行性。为进一步组织工程学肌腱研究做了基础理论准备。
[Abstract]:Background and purpose:
In clinical work, tendon defects are often encountered in the repair of tendon graft. The current "gold standard" is autologous tendon transplantation, but the source of autologous tendon is very limited and the autograft of autologous tendon can cause loss of donor function; allograft tendon transplantation and xenograft tendon transplantation have immune rejection. The reaction, which leads to severe complications such as graft necrosis and local inflammatory response, needs to be solved by the foundation of the mechanism of tendon healing and the progress of the research of tissue engineering tendon. Currently, there are two ways of tendon healing. One thinks that tendon healing depends on the growth of tendon tissue to form adhesion. It is concluded that the transplantation of tendon only plays the role of scaffolding and pathway in tendon transplantation. The other is endogenous healing. It is believed that the tendon can rely on the regeneration and proliferation of the cells in the tendon and synthesize the collagen to complete the healing and repair directly. At the same time, it is still controversial whether the loss of endogenous acellular tendon grafts without endogenous healing can meet the biomechanical requirements. Therefore, we designed this experiment to compare the histological and biomechanical properties of Achilles tendon transplantation and untreated rat Achilles tendon transplantation, and to study the removal of intracellular cells in the tendon. To investigate the mechanism of tendon healing, we will prepare for tendon tissue engineering.
Method:
In the experiment, 96 healthy adult male Wistar rats were selected as experimental animals. Rats were randomly divided into experimental and control groups. The rat model of Achilles tendon defect was prepared. Rats in the experimental group underwent cryopreservation and cryopreservation and autologous transplantation of Achilles tendon in the control group. The Achilles tendon transplantation was performed in the control group. The Achilles tendon was harvested at 1,2,4,8 weeks after the operation. The morphological observation of HE staining, immunohistochemical staining of type I and type III collagen and image analysis were carried out under the microscope, and the biomechanical parameters of the maximum load and modulus of elasticity of the allograft tendon were measured, and the statistical analysis was made.
Result:
The results showed that the expression of type I and type III collagen in the transplanted Achilles tendon increased gradually with the time of Achilles tendon transplantation. The expression level of the control group was higher than that of the experimental group at 1 weeks and at the 2 week (P 0.05), but there was no significant difference between the two groups at the 4 and 8 weeks (P0.05). The biomechanical results of the transplanted Achilles tendon showed the maximum load of the Achilles tendon in the control group at 1,2 weeks. The loading and elastic moduli were higher than those in the experimental group (P 0.05), but there was no significant difference between the two groups at 4,8 weeks (P0.05).
Conclusion:
In this experiment, the histological and biomechanical characteristics of the deep hypothermic Achilles tendon transplantation and the fresh Achilles tendon transplantation were compared. It was found that the tendon grafts in the cell were lagged behind the untreated grafts. At the same time, the need to meet the biomechanical strength of this kind of cell tendon graft at the late stage was found. The analysis shows that the long-term biological effect of deep hypothermia treatment and autologous transplantation is similar. The decellularized tendon can be used for the transplantation of tendon defect, and the feasibility of the allograft of allograft for the acellular tendon graft is also proved. The basic theoretical preparation for further tissue engineering tendon research is made.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R687.2

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