GRGDSPC多肽表面修饰PET人工韧带促进腱骨愈合的实验研究

发布时间：2018-02-15 11:33

  本文关键词： 聚对苯二甲酸乙二醇酯 腱骨愈合 人工韧带 前交叉韧带 多肽 生物相容性　出处：《第二军医大学》2013年博士论文　论文类型：学位论文

【摘要】：【背景】 对于重建膝关节交叉韧带损伤的材料有多种，但各自均有优缺点。聚对苯二甲酸乙二醇酯（PET）人工韧带具有力学性能良好，取材方便，手术简单，术后康复快等优点，是一种具有良好应用前景的移植材料。但PET人工韧带在骨隧道内能否达到“腱骨愈合”可能影响交叉韧带重建的远期疗效。虽然文献报道应用LARS人工韧带重建术后的近期疗效良好，但远期疗效尚不明确。LARS人工韧带为PET惰性材料，PET材料与骨界面是否能最终达到生物性愈合，对于应用PET人工韧带重建交叉韧带术后的远期疗效具有重要意义。本研究通过应用多肽表面修饰PET材料，促进PET材料与细胞的组织相容性，进一步观察其在体内与骨界面的生物性愈合情况，初步探讨PET人工韧带与骨界面实现生物性愈合的可行性。 【目的】 1.通过回顾性分析应用LARS人工韧带重建前交叉韧带术后患者的骨隧道变化特点及可能的影响因素，初步探讨人工韧带与骨界面的结合情况，为下一步研究人工韧带“腱骨愈合”打下基础。2.应用GRGDSPC多肽表面修饰PET材料，初步探讨实现PET材料与GRGDSPC多肽实现高效共价结合的简单易行的操作方法。3.将表面修饰后的PET材料与骨髓间充值干细胞体外共培养，探讨GRGDSPC表面修饰PET材料后促进其生物相容性的有效性。4.将GRGDSPC表面修饰后的PET材料植入体内，初步观察PET材料与骨界面的生物性愈合情况，为将来实现临床应用提供依据。 【方法】 1．2004年8月至2007年4月对43例应用LARS人工韧带重建ACL患者（其中男31例，女12例；左膝19例，右膝24例；平均年龄为27.5岁）进行3年的随访，于术后1、3、6、12、24、36个月分别进行X线和CT检查，记录各个时间段胫骨和股骨骨道的宽度。以Lysholm评分测定膝关节功能，以KT-1000测量膝关节的松弛度。 2．分别应用NaOH碱性水解方法处理PET材料及应用H2SO4和KMnO4处理PET材料。将处理后的PET材料及未经处理的PET材料分别与不同溶度的GRGDSPC多肽溶液进行结合，采用傅里叶变红外光谱分析及X线衍射分析技术，分析其共价结合效果和多肽结合含量。 3．分别将表面修饰的PET材料和未经处理的PET材料与骨髓间充质干细胞共培养，扫描电镜下分别观察细胞的粘附和增殖情况，细胞血球板计数法测定细胞的粘附率。同时应用细胞茜素红染色法及碱性磷酸酶法鉴定骨髓间充质干细胞的成骨诱导分化。 4.选用新西兰兔建立前交叉韧带损伤动物模型，分别将表面修饰的PET材料和未经处理的PET材料植入动物体内，在不同时间节点上采用HE染色法和扫描电镜观察PET材料与骨界面的结合情况。 【结果】 1．随访时间为36～49个月，平均39.5个月。按照Peyrache等提出的分级方法（0级2mm，1级2～4mm，2级4～6mm，3级≥6mm），仅3例出现了骨道1级扩大，均为术后6个月发生在股骨隧道，未发现2级和3级骨道扩大，3例骨道扩大部分均位于股骨隧道近关节面水平，平均扩大（2.5±0.3） mm。40例0级扩大患者至末次随访时，胫骨骨道平均增宽为（0.8±0.3） mm，股骨骨道平均增宽为（1.1±0.3） mm。不同时间段骨道宽度的差异无统计学意义（P0.05）。X线与CT测量结果基本一致。末次随访时，3例骨道1级扩大病例Lysholm评分平均为（94.5±4.6）分，KT-1000前移差值平均为（1.5±1.1）mm；40例0级扩大病例Lysholm评分平均为（95.6±4.8）分，KT-1000前移差值平均为（1.4±1.5）mm。 2．采用NaOH碱性水解方法处理PET材料方法简便可靠，效率较高。应用H2SO4和KMnO4处理PET材料及单纯将PET材料与GRGDSPC多肽混合，并无法实现与GRGDSPC多肽共价结合。碱性水解法处理的PET材料与GRGDSPC多肽的结合效果最佳。FTIR提示7.5mg/ml的GRGDSPC多肽浓度与材料共价结合的效率较好，增加GRGDSPC多肽浓度后，其共价结合效率提高并不明显。XPS提示：7.5mg/ml的GRGDSPC多肽表面修饰的PET材料氮元素含量达2.43%，而未经处理的PET材料分别与7.5mg/ml，12mg/ml和3mg/ml的GRGDSPC多肽混合后的氮元素含量分别为1.99%，1.96%，1.42%。 3．新西兰兔骨髓间充质干细胞实现成功的分离、传代培养、冷冻保存、复苏及诱导分化为成骨细胞。骨髓间充质干细胞与PET材料共培养后实现了早期粘附和增殖，随着时间的增长，各组材料的细胞粘附率均提高，培养48小时后，GRGDSPC多肽表面修饰的PET材料对细胞的粘附率显著增高，，其次为GRGDSPC多肽混合PET材料组，最次为未处理PET材料组。 4.新西兰兔膝关节交叉韧带损伤模型建立后，将各种处理条件的PET材料植入体内。随着造模时间的延长，应用GRGDSPC多肽表面修饰的PET组效果最好，PET材料与组织结合紧密，且结合量很多。其次为GRGDSPC多肽混合PET材料组，PET材料与组织结合部分紧密，材料空隙间结合量少；最次为未处理PET材料组，仅少量PET与组织结合。HE染色提示表面修饰后的PET材料表面有大量的成纤维细胞粘附增殖，而未处理PET材料材料表面仅有少量的细胞粘附生长。 【结论】 LARS人工韧带重建前交叉韧带术后，骨道扩大并不明显，这种变化特点间接提示了人工韧带在骨道内并没有经历类似自体移植物那样的组织坏死及对骨道壁的再塑形过程，虽然人工韧带与骨道的结合情况尚不清楚，但进一步研究促进人工韧带与骨界面的生物性愈合具有重要意义。应用NaOH碱性水解方法处理PET材料方法简便可靠，效率较高，碱性水解法处理后的PET材料与GRGDSPC多肽实现了共价结合，GRGDSPC多肽表面修饰的PET材料可明显提高材料与细胞的粘附和增殖，体内试验证明表面修饰后的PET材料与组织结合更紧密，成纤维细胞的含量更多。但PET人工韧带与骨界面的结合情况仍需延长动物体内实验时间，以进一步观察其是否最终实现生物性结合。同时，临床病例的随访研究仍需继续，特别是LARS人工韧带重建术后10年的随访结果值得期待。
[Abstract]:[background]
There are many for the reconstruction of knee joint cruciate ligament injury, but have their respective advantages and disadvantages. Polyethylene terephthalate (PET) artificial ligament has good mechanical properties, convenient, simple operation, quick recovery after operation, has a good application prospect of the transplantation material. But PET artificial ligament in bone tunnel can achieve "tendon bone healing" may affect the long-term curative effect of anterior cruciate ligament reconstruction. Although the recent curative effect reported the application of LARS artificial ligament reconstruction is good, but the long-term effect is not clear.LARS artificial ligament of PET inert material, whether PET material and bone interface can reach the biological healing, has important significance for the long-term curative effect of cruciate ligament reconstruction using PET artificial ligament. This study through the application of peptide surface modification of PET material, PET material and promote cell biocompatibility and further outlook To investigate the biological healing of the bone interface between the body and the body, the feasibility of PET artificial ligament and bone interface to achieve biological healing was preliminarily investigated.
[Objective]
1. through the retrospective analysis of the bone tunnel changes the characteristics of the patients with cruciate ligament after operation and the possible factors affecting the application of LARS artificial ligament reconstruction, preliminary study on combination of artificial ligament and bone interface, the next step for the research of artificial ligament tendon bone healing "lay the foundation for.2. application of GRGDSPC peptide modified PET surface materials, preliminary study the.3. operation method is simple and easy to realize PET materials and GRGDSPC peptide to achieve efficient covalent binding of PET material and surface modified bone marrow mesenchymal stem cells co cultured in vitro, to investigate the GRGDSPC surface modification of PET material after the promotion of.4. effectiveness of the biocompatibility of PET material implanted GRGDSPC after surface modification, the preliminary observation of biological PET materials and bone interface healing, provide the basis for future clinical application.
[method]
From August 1.2004 to April 2007 43 cases of application of LARS artificial ligament reconstruction ACL patients (male 31 cases, female 12 cases; 19 cases of left, right knee in 24 cases; the average age was 27.5 years) to 3 years of follow-up, in 1,3,6,12,24,36 months after operation respectively by X-ray and CT, the width of each time segment of tibia record and the femoral tunnel. Determination of knee function Lysholm score, measured by KT-1000 of the knee joint laxity.
2. respectively using NaOH alkaline hydrolysis method for processing PET material and application of H2SO4 and KMnO4 PET materials. After treatment of PET materials and PET materials were untreated with GRGDSPC peptide solution with different solubility are combined, using Fu Liye infrared spectrum analysis and X-ray diffraction analysis technology, analysis of the covalent binding effect and peptide content.
3. were surface modified PET materials and PET materials without treatment with bone marrow mesenchymal stem cells were cultured under scanning electron microscope were used to observe the adhesion and proliferation of cells, cell adhesion cell count method to determine cell rate. At the same time the application of alizarin red staining and alkaline phosphatase method for identification of bone marrow mesenchymal osteogenic differentiation of stem cells.
4. New Zealand rabbits were used to establish animal models of anterior cruciate ligament injury. The surface modified PET material and untreated PET material were implanted into the animal body. HE staining and scanning electron microscopy were used to observe the combination of PET material and bone interface at different time points.
[results]
1锛庨殢璁挎椂闂翠负36锝

本文编号：1513168