本文關(guān)鍵詞： 聚對(duì)苯二甲酸乙二醇酯 腱骨愈合 人工韌帶 前交叉韌帶 多肽 生物相容性　出處：《第二軍醫(yī)大學(xué)》2013年博士論文　論文類型：學(xué)位論文

【摘要】：【背景】 對(duì)于重建膝關(guān)節(jié)交叉韌帶損傷的材料有多種，但各自均有優(yōu)缺點(diǎn)。聚對(duì)苯二甲酸乙二醇酯（PET）人工韌帶具有力學(xué)性能良好，取材方便，手術(shù)簡單，術(shù)后康復(fù)快等優(yōu)點(diǎn)，是一種具有良好應(yīng)用前景的移植材料。但PET人工韌帶在骨隧道內(nèi)能否達(dá)到“腱骨愈合”可能影響交叉韌帶重建的遠(yuǎn)期療效。雖然文獻(xiàn)報(bào)道應(yīng)用LARS人工韌帶重建術(shù)后的近期療效良好，但遠(yuǎn)期療效尚不明確。LARS人工韌帶為PET惰性材料，PET材料與骨界面是否能最終達(dá)到生物性愈合，對(duì)于應(yīng)用PET人工韌帶重建交叉韌帶術(shù)后的遠(yuǎn)期療效具有重要意義。本研究通過應(yīng)用多肽表面修飾PET材料，促進(jìn)PET材料與細(xì)胞的組織相容性，進(jìn)一步觀察其在體內(nèi)與骨界面的生物性愈合情況，初步探討PET人工韌帶與骨界面實(shí)現(xiàn)生物性愈合的可行性。 【目的】 1.通過回顧性分析應(yīng)用LARS人工韌帶重建前交叉韌帶術(shù)后患者的骨隧道變化特點(diǎn)及可能的影響因素，初步探討人工韌帶與骨界面的結(jié)合情況，為下一步研究人工韌帶“腱骨愈合”打下基礎(chǔ)。2.應(yīng)用GRGDSPC多肽表面修飾PET材料，初步探討實(shí)現(xiàn)PET材料與GRGDSPC多肽實(shí)現(xiàn)高效共價(jià)結(jié)合的簡單易行的操作方法。3.將表面修飾后的PET材料與骨髓間充值干細(xì)胞體外共培養(yǎng)，探討GRGDSPC表面修飾PET材料后促進(jìn)其生物相容性的有效性。4.將GRGDSPC表面修飾后的PET材料植入體內(nèi)，初步觀察PET材料與骨界面的生物性愈合情況，為將來實(shí)現(xiàn)臨床應(yīng)用提供依據(jù)。 【方法】 1．2004年8月至2007年4月對(duì)43例應(yīng)用LARS人工韌帶重建ACL患者（其中男31例，女12例；左膝19例，右膝24例；平均年齡為27.5歲）進(jìn)行3年的隨訪，于術(shù)后1、3、6、12、24、36個(gè)月分別進(jìn)行X線和CT檢查，記錄各個(gè)時(shí)間段脛骨和股骨骨道的寬度。以Lysholm評(píng)分測定膝關(guān)節(jié)功能，以KT-1000測量膝關(guān)節(jié)的松弛度。 2．分別應(yīng)用NaOH堿性水解方法處理PET材料及應(yīng)用H2SO4和KMnO4處理PET材料。將處理后的PET材料及未經(jīng)處理的PET材料分別與不同溶度的GRGDSPC多肽溶液進(jìn)行結(jié)合，采用傅里葉變紅外光譜分析及X線衍射分析技術(shù)，分析其共價(jià)結(jié)合效果和多肽結(jié)合含量。 3．分別將表面修飾的PET材料和未經(jīng)處理的PET材料與骨髓間充質(zhì)干細(xì)胞共培養(yǎng)，掃描電鏡下分別觀察細(xì)胞的粘附和增殖情況，細(xì)胞血球板計(jì)數(shù)法測定細(xì)胞的粘附率。同時(shí)應(yīng)用細(xì)胞茜素紅染色法及堿性磷酸酶法鑒定骨髓間充質(zhì)干細(xì)胞的成骨誘導(dǎo)分化。 4.選用新西蘭兔建立前交叉韌帶損傷動(dòng)物模型，分別將表面修飾的PET材料和未經(jīng)處理的PET材料植入動(dòng)物體內(nèi)，在不同時(shí)間節(jié)點(diǎn)上采用HE染色法和掃描電鏡觀察PET材料與骨界面的結(jié)合情況。 【結(jié)果】 1．隨訪時(shí)間為36～49個(gè)月，平均39.5個(gè)月。按照Peyrache等提出的分級(jí)方法（0級(jí)2mm，1級(jí)2～4mm，2級(jí)4～6mm，3級(jí)≥6mm），僅3例出現(xiàn)了骨道1級(jí)擴(kuò)大，均為術(shù)后6個(gè)月發(fā)生在股骨隧道，未發(fā)現(xiàn)2級(jí)和3級(jí)骨道擴(kuò)大，3例骨道擴(kuò)大部分均位于股骨隧道近關(guān)節(jié)面水平，平均擴(kuò)大（2.5±0.3） mm。40例0級(jí)擴(kuò)大患者至末次隨訪時(shí)，脛骨骨道平均增寬為（0.8±0.3） mm，股骨骨道平均增寬為（1.1±0.3） mm。不同時(shí)間段骨道寬度的差異無統(tǒng)計(jì)學(xué)意義（P0.05）。X線與CT測量結(jié)果基本一致。末次隨訪時(shí)，3例骨道1級(jí)擴(kuò)大病例Lysholm評(píng)分平均為（94.5±4.6）分，KT-1000前移差值平均為（1.5±1.1）mm；40例0級(jí)擴(kuò)大病例Lysholm評(píng)分平均為（95.6±4.8）分，KT-1000前移差值平均為（1.4±1.5）mm。 2．采用NaOH堿性水解方法處理PET材料方法簡便可靠，效率較高。應(yīng)用H2SO4和KMnO4處理PET材料及單純將PET材料與GRGDSPC多肽混合，并無法實(shí)現(xiàn)與GRGDSPC多肽共價(jià)結(jié)合。堿性水解法處理的PET材料與GRGDSPC多肽的結(jié)合效果最佳。FTIR提示7.5mg/ml的GRGDSPC多肽濃度與材料共價(jià)結(jié)合的效率較好，增加GRGDSPC多肽濃度后，其共價(jià)結(jié)合效率提高并不明顯。XPS提示：7.5mg/ml的GRGDSPC多肽表面修飾的PET材料氮元素含量達(dá)2.43%，而未經(jīng)處理的PET材料分別與7.5mg/ml，12mg/ml和3mg/ml的GRGDSPC多肽混合后的氮元素含量分別為1.99%，1.96%，1.42%。 3．新西蘭兔骨髓間充質(zhì)干細(xì)胞實(shí)現(xiàn)成功的分離、傳代培養(yǎng)、冷凍保存、復(fù)蘇及誘導(dǎo)分化為成骨細(xì)胞。骨髓間充質(zhì)干細(xì)胞與PET材料共培養(yǎng)后實(shí)現(xiàn)了早期粘附和增殖，隨著時(shí)間的增長，各組材料的細(xì)胞粘附率均提高，培養(yǎng)48小時(shí)后，GRGDSPC多肽表面修飾的PET材料對(duì)細(xì)胞的粘附率顯著增高，，其次為GRGDSPC多肽混合PET材料組，最次為未處理PET材料組。 4.新西蘭兔膝關(guān)節(jié)交叉韌帶損傷模型建立后，將各種處理?xiàng)l件的PET材料植入體內(nèi)。隨著造模時(shí)間的延長，應(yīng)用GRGDSPC多肽表面修飾的PET組效果最好，PET材料與組織結(jié)合緊密，且結(jié)合量很多。其次為GRGDSPC多肽混合PET材料組，PET材料與組織結(jié)合部分緊密，材料空隙間結(jié)合量少；最次為未處理PET材料組，僅少量PET與組織結(jié)合。HE染色提示表面修飾后的PET材料表面有大量的成纖維細(xì)胞粘附增殖，而未處理PET材料材料表面僅有少量的細(xì)胞粘附生長。 【結(jié)論】 LARS人工韌帶重建前交叉韌帶術(shù)后，骨道擴(kuò)大并不明顯，這種變化特點(diǎn)間接提示了人工韌帶在骨道內(nèi)并沒有經(jīng)歷類似自體移植物那樣的組織壞死及對(duì)骨道壁的再塑形過程，雖然人工韌帶與骨道的結(jié)合情況尚不清楚，但進(jìn)一步研究促進(jìn)人工韌帶與骨界面的生物性愈合具有重要意義。應(yīng)用NaOH堿性水解方法處理PET材料方法簡便可靠，效率較高，堿性水解法處理后的PET材料與GRGDSPC多肽實(shí)現(xiàn)了共價(jià)結(jié)合，GRGDSPC多肽表面修飾的PET材料可明顯提高材料與細(xì)胞的粘附和增殖，體內(nèi)試驗(yàn)證明表面修飾后的PET材料與組織結(jié)合更緊密，成纖維細(xì)胞的含量更多。但PET人工韌帶與骨界面的結(jié)合情況仍需延長動(dòng)物體內(nèi)實(shí)驗(yàn)時(shí)間，以進(jìn)一步觀察其是否最終實(shí)現(xiàn)生物性結(jié)合。同時(shí)，臨床病例的隨訪研究仍需繼續(xù)，特別是LARS人工韌帶重建術(shù)后10年的隨訪結(jié)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]
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