發(fā)布時間：2018-02-10 20:39

  本文關(guān)鍵詞： 半月板 脫細(xì)胞基質(zhì)支架 山羊　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：半月板對于維持膝關(guān)節(jié)的生理功能是很重要的,半月板損傷與膝關(guān)節(jié)骨關(guān)節(jié)炎(OA)的發(fā)生密切相關(guān)。由于半月板的自我修復(fù)能力有限,半月板部分或全部切除是治療半月板損傷的常用手段。隨著醫(yī)學(xué)的發(fā)展,組織工程對損傷半月板缺損的再生修復(fù)帶來了希望,而找尋合適的半月板支架在半月板組織工程中起著至關(guān)重要的作用。脫細(xì)胞半月板支架不僅具有良好的三維結(jié)構(gòu)、生物相容性和生物力學(xué)性能。本文旨在研究山羊半月板脫細(xì)胞基質(zhì)支架的制備方法、觀察它們的形態(tài)結(jié)構(gòu)、測定其生物力學(xué)性能,并且進(jìn)一步研究擴(kuò)大支架孔徑地方法,為構(gòu)建組織工程半月板研究打下基礎(chǔ)。方法:取淘汰山羊半月板,采用不同脫細(xì)胞方法進(jìn)行山羊半月板脫細(xì)胞基質(zhì)支架的制備。方法一:采用高滲-低滲反復(fù)凍融、Tris-HCl溶液聯(lián)合胰蛋白酶溶液進(jìn)行半月板基質(zhì)的脫細(xì)胞處理;方法二:用高滲-低滲反復(fù)凍融、雙氧水和胰蛋白酶溶液輪流浸泡后,再用化學(xué)去垢劑TrionX-100溶液和脫氧膽酸鈉溶液交替使用進(jìn)行半月板基質(zhì)的脫細(xì)胞處理。組織學(xué)觀察:用HE染色、天狼猩紅染色、甲苯胺藍(lán)染色、番紅O染色和番紅-固綠染色方法對兩種不同方法制備的脫細(xì)胞基質(zhì)進(jìn)行組織學(xué)檢測,并將無細(xì)胞殘留組與天然半月板進(jìn)行掃描電鏡觀察和生物力學(xué)測定。最后分別用甲酸、I型膠原酶、胰蛋白酶+I型膠原酶對制備好的山羊半月板脫細(xì)胞基質(zhì)支架用進(jìn)行處理,在掃描電鏡下觀察并進(jìn)行生物力學(xué)測定。結(jié)果:方法一制備的脫細(xì)胞支架組織學(xué)觀察有細(xì)胞殘留,方法二制備的脫細(xì)胞支架組織學(xué)檢測無任何細(xì)胞殘留,基質(zhì)中保存了大量的膠原纖維。掃描電鏡結(jié)果顯示脫細(xì)胞支架組的孔徑和孔隙率比天然半月板略大,生物力學(xué)性能上二者無顯著性差異。擴(kuò)大孔徑研究結(jié)果顯示:甲酸組、I型膠原酶組和胰蛋白酶+I型膠原酶組的孔徑和孔隙率較脫細(xì)胞基質(zhì)支架的大,生物力學(xué)測定顯示甲酸組、I型膠原酶組和胰蛋白酶+I型膠原酶組與脫細(xì)胞基質(zhì)支架組均存在顯著性差異。結(jié)論:1.用蒸餾水-10%NaCl溶液反復(fù)凍融、0.1%雙氧水和0.5%的胰蛋白酶輪流浸泡后,再用化學(xué)去垢劑TrionX-100溶液和脫氧膽酸鈉溶液交替使用進(jìn)行半月板基質(zhì)脫細(xì)胞方法可以將羊半月板中的細(xì)胞成分除去,并保留了半月板的三維立體結(jié)構(gòu),生物力學(xué)特性與正常半月板接近,可以作為半月板支架材料;2.甲酸、I型膠原酶、胰蛋白酶+I型膠原酶對山羊脫細(xì)胞基質(zhì)支架處理結(jié)果顯示,支架的孔徑和孔隙率均有所提升,但會造成生物力學(xué)性能的下降。
[Abstract]:Meniscus is very important to maintain the physiological function of knee joint. Meniscus injury is closely related to the occurrence of osteoarthritis of knee joint (OAA). Partial or total meniscal excision is a common method in the treatment of meniscus injury. With the development of medicine, tissue engineering brings hope to the regeneration and repair of injured meniscus defect. Finding suitable meniscus scaffolds plays an important role in meniscus tissue engineering. Acellular meniscus scaffolds not only have a good three-dimensional structure. The purpose of this paper is to study the preparation methods of goat meniscus acellular matrix scaffolds, observe their morphological structure, determine their biomechanical properties, and further study the method of expanding the pore size of the scaffolds. Methods: goat meniscus was eliminated. Preparation of goat meniscus acellular matrix scaffolds with different acellular methods. Methods: 1. Hyperosmotic and hypotonic thawing Tris-HCl solution combined with trypsin solution were used to acellular the meniscus matrix. Method two: after repeated freezing and thawing with hyperosmotic and hypotonic solution, the solution of hydrogen peroxide and trypsin alternately soaked, Then acellular treatment of meniscus matrix was carried out with chemical defouling agent TrionX-100 solution and sodium deoxycholate solution alternately. Histological observation: stained with HE, Sirius red, toluidine blue. The acellular substrates prepared by two different methods were detected by the methods of phannin O staining and fast green staining. Scanning electron microscopy and biomechanical analysis were carried out between the cells free residual group and the natural meniscus. Finally, the prepared scaffolds of goat meniscus acellular matrix were treated with formic acid type I collagenase and trypsin type I collagenase, respectively. Results: methods: cell residues were observed in the acellular scaffolds, and no cell residues were observed in the acellular stents prepared in the second method. A large number of collagen fibers were preserved in the matrix. SEM results showed that the pore size and porosity of the acellular scaffolds were slightly larger than those of the natural meniscus. There was no significant difference in biomechanical properties between the two groups. The results of expanded pore size study showed that the pore size and porosity of formic acid group and trypsin type I collagenase group were larger than those of acellular matrix scaffold. Biomechanical measurements showed that there were significant differences between formic acid group and trypsin type I collagenase group and acellular matrix scaffold group. Conclusion: 1. Frozen and thawed with distilled water -10 NaCl solution, 0.1% hydrogen peroxide and 0.5% pancreatic eggs were repeatedly frozen and thawed with distilled water. After the white enzyme was soaked in turns, Using the chemical descaling agent TrionX-100 solution and sodium deoxycholate solution alternately to carry on the meniscus matrix acellular method can remove the cell composition from the sheep meniscus and retain the three-dimensional structure of the meniscus. The biomechanical properties were close to the normal meniscus and could be used as meniscus scaffolds. The results of treatment of goat acellular matrix scaffolds with formate type I collagenase and trypsin type I collagenase showed that the pore size and porosity of the scaffolds were increased. But the biomechanical properties will decrease.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.0

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