【摘要】：軟骨缺損修復(fù)是再生醫(yī)學(xué)領(lǐng)域非常有挑戰(zhàn)性的一個課題。組織工程學(xué)的出現(xiàn),將可能使人們從傳統(tǒng)的器官移植和植入這種以傷補傷的方法進入到一個器官制造的新時代,其為軟骨缺損修復(fù)提供了新的思路和方法。軟骨組織的損傷或者缺損往往會伴隨軟骨下骨的改變,近年來,采用雙層支架來模擬關(guān)節(jié)軟骨和軟骨下骨各自內(nèi)在結(jié)構(gòu)和生理功能以實現(xiàn)軟骨和軟骨下骨的同時修復(fù)成為一個新思路,但關(guān)于雙層支架的很多基本問題還有待于進一步探索。 未來生物材料的設(shè)計需要對于材料的細(xì)胞響應(yīng)有深入的認(rèn)識。近幾十年來的相關(guān)研究已經(jīng)大大加深了人們對于“生物材料”涵義的理解,同時也提供了更多探索的方向。材料的諸多因素可以影響細(xì)胞行為,其中,表面形貌的效應(yīng)普遍存在,并且由于形貌改變可不涉及化學(xué)成份的改變,故相應(yīng)的材料改性容易最終獲得批準(zhǔn)應(yīng)用。 本博士論文以聚乳酸—羥基乙酸共聚物(PLGA)這種有應(yīng)用價值的可降解高分子為基質(zhì)材料,開展了生物醫(yī)用材料方面的研究。論文首先從二維平面探索微米拓?fù)湫蚊驳募?xì)胞響應(yīng)行為并得出基本規(guī)律,繼而將其應(yīng)用于三維多孔支架中,最后再用組織工程雙層支架進行關(guān)節(jié)軟骨和軟骨下骨同時修復(fù)的動物實驗。該研究有助于理解材料物理因素對于細(xì)胞的作用,為生物材料設(shè)計提供指導(dǎo)。 本論文的主要創(chuàng)新性工作包括以下幾方面： 1.制備了一系列不同尺寸的微米陣列,研究其細(xì)胞響應(yīng)行為,并發(fā)現(xiàn)適合骨髓基質(zhì)干細(xì)胞黏附的尺寸。本論文在二維PLGA膜表面制備了一系列不同高度(深度)的微柱(微坑)陣列,然后研究骨髓基質(zhì)干細(xì)胞在這些拓?fù)湫蚊脖砻娴纳L情況。根據(jù)實驗結(jié)果,我們發(fā)現(xiàn)1μm高度(或深度)的微柱(或微坑)陣列相對于其它高度(或深度)的陣列更利于細(xì)胞的生長。進一步我們比較了1μm微坑和1μm微柱,發(fā)現(xiàn)1μm微柱比微坑和光滑膜更適合骨髓基質(zhì)干細(xì)胞的黏附。 2.利用材料表面微柱陣列實現(xiàn)了對于細(xì)胞核嚴(yán)重自我變形的調(diào)控。本論文工作過程中,意外發(fā)現(xiàn)新生大鼠骨髓基質(zhì)干細(xì)胞細(xì)胞核在特定高度的PLGA微柱陣列表面能發(fā)生嚴(yán)重自我變形；細(xì)胞核變形并非是由細(xì)胞核的重力作用引起的,而可能是由細(xì)胞鋪展時的內(nèi)應(yīng)力導(dǎo)致；盡管骨髓基質(zhì)干細(xì)胞發(fā)生了如此嚴(yán)重的細(xì)胞核變形,其仍具備增殖和成骨分化的能力。本文通過設(shè)計不同微柱陣列,成功控制了細(xì)胞核的形狀,在國際上首次實現(xiàn)了啞鈴形、方形、十字交叉等異常細(xì)胞核形狀。此外進一步研究了另外五種細(xì)胞(Hela、PC12、NIH3T3、MC3T3、 Chondrocyte),發(fā)現(xiàn)細(xì)胞在微柱陣列表面的細(xì)胞核變形現(xiàn)象具有普適性；同時其響應(yīng)程度具有細(xì)胞類型依賴性。 3.通過設(shè)計新型致孔劑實現(xiàn)了三維PLGA多孔支架內(nèi)部孔結(jié)構(gòu)以及孔壁拓?fù)湫蚊驳目刂啤；诹Ｗ咏黾夹g(shù),首次通過對石蠟球致孔劑表面物理修飾的辦法調(diào)控了組織工程支架內(nèi)表面微米拓?fù)湫蚊病Ｔ谑炃蜻@種致孔劑粒子表面通過氯化鈉粒子的撞擊制備出微米尺寸的凹坑,然后再把這些微米拓?fù)湫蚊厕D(zhuǎn)移到三維多孔支架孔表面,從而獲得微米凸起結(jié)構(gòu)。三維多孔支架中的這些微米凸起能促進新生Sprague Dawley (SD)大鼠骨髓基質(zhì)干細(xì)胞的黏附,同時并不影響干細(xì)胞的增殖和分化。此外,還首次發(fā)明“糖粘鹽”這種新型致孔劑,使得制備得到的組織工程支架具有規(guī)則的球形孔形狀以及良好的連通性,并且孔表面具有微米拓?fù)湫蚊病Ｖ苽涞玫降慕M織工程多孔支架利于SD大鼠骨髓基質(zhì)干細(xì)胞的生長。這些研究成果為組織工程和再生醫(yī)學(xué)中的材料設(shè)計提供新的視野和技術(shù)。 4.設(shè)計了PLGA雙層多孔支架,并通過合作研究,探討了支架物理參量對于體內(nèi)關(guān)節(jié)軟骨修復(fù)效果的影響。在動物實驗之前,本論文按照國家標(biāo)準(zhǔn)“醫(yī)療器械生物學(xué)評價”的相關(guān)要求設(shè)計了PLGA支架材料體內(nèi)外的生物相容性實驗,并研究了其在新西蘭大白兔體內(nèi)的降解行為。通過實驗,發(fā)現(xiàn)PLGA支架材料細(xì)胞毒性小,血液相容性好,且不會在骨/軟骨缺損處引起明顯的炎癥反應(yīng),同時具有合適的降解速率。另外,制備了雙層支架以修復(fù)新西蘭大白兔骨/軟骨缺損,并首次實現(xiàn)對雙層支架中各層最優(yōu)孔隙率、孔徑探索。設(shè)計并制備好雙層支架后,通過合作研究,將PLGA雙層支架植入新西蘭大白兔骨/軟骨缺損處(直徑4mm,深度5mm),以檢驗各組支架對其修復(fù)效果。術(shù)后12周,從大體觀、HE染色、甲苯胺藍染色、免疫組化染色、組織學(xué)評分和相關(guān)基因的表達這些結(jié)果中我們得出：軟骨段支架孔隙率為92%,骨段支架孔隙率為77%的雙層支架及軟骨段孔徑為100-200μm,骨段孔徑為300-450μm的雙層支架能實現(xiàn)比其它孔隙率或孔徑組合更好的修復(fù)效果。 除了上述工作以外,在“復(fù)旦大學(xué)博士生短期國際訪學(xué)資助計劃”資助下赴美國密歇根大學(xué)進行為期4個月短期交流期間,我還合成了兩組分的聚酸酐,并研究了其表面溶蝕性能、制備了藥物釋放裝置。
[Abstract]:The repair of cartilage defect is a very challenging topic in the field of regenerative medicine. The emergence of tissue engineering will make it possible for people to enter into a new era of organ manufacture from traditional organ transplantation and implantation, which provides a new way of thinking and method for the repair of cartilage defects. The damage or defect of the cartilage is often accompanied by the change of the subchondral bone. In recent years, a double-layer stent is used to simulate the internal structure and physiological function of the articular cartilage and the subchondral bone to realize the simultaneous repair of the subchondral bone and the subchondral bone. But many of the basic problems about the double-layer stand still need to be further explored. The design of the future biological materials requires in-depth recognition of the cell response of the material The research in recent decades has greatly increased people's understanding of the meaning of the "biological material", and also provided more exploration The various factors of the material can affect the behavior of the cells, wherein the effect of the surface morphology is common, and due to the change of the morphology, the change of the chemical components can not be involved, so that the corresponding material modification is easy to finally obtain the approval. In this dissertation, the biodegradable polymer with the application value of poly (lactic acid)-glycolic acid copolymer (PLGA) is used as the matrix material, and the biological medical material is carried out. In this paper, we first study the cell response behavior of the micro-topological morphology from the two-dimensional plane and get the basic law, then apply it in the three-dimensional porous scaffold, and finally use the tissue engineering double-layer support for the simultaneous repair of the articular cartilage and the subchondral bone. The study can help to understand the effect of the physical factors of the material on the cells, and to provide for the design of the biological materials. For guidance. The main innovative work of this paper includes In the following aspects:1. A series of microarrays of different sizes were prepared to study the response behavior of the cells and to find a suitable bone marrow stromal stem In this paper, a series of microcolumn (micropits) arrays with different height (depth) were prepared on the surface of the two-dimensional PLGA membrane, and then the morphology of the bone marrow stromal cells was studied. The growth of the surface. Based on the results of the experiment, we found that the array of microcolumns (or micropits) of a height (or depth) of 1. m The growth of the cells was facilitated. Further, we compared the microcells of 1. m u.m and the microcolumn of 1. m u.m, and found that the microcolumn of 1. m The adhesion of the stem cells.2. The array of microcolumn of the surface of the material is used to realize the cell nucleus. In the course of the work, the nuclei of the bone marrow stromal stem cells of the newly-born rats were found to be self-deformed on the surface of a specific height of the surface of the PLGA microcolumn array, and the deformation of the nucleus was not caused by the gravity of the nucleus, possibly by the cells. The internal stress at the time of spreading is caused; although the bone marrow stromal stem cells have so severe nuclear deformation, it still has an increase in Through the design of different microcolumn arrays, the shape of the nucleus is successfully controlled, and the dumbbell shape, the square and the cross are realized for the first time in the world. In addition, five other cells (Hela, PC12, NIH3T3, MC3T3, and Chondrcyte) were further studied. in that invention, a novel pore-forming agent is design to realize the internal hole structure of the three-dimensional PLGA porous support, Based on the particle leaching technology, the tissue engineering is regulated and controlled for the first time by means of physical modification on the surface of the paraffin-like pore-forming agent. the micron-sized pits are prepared by the impact of the sodium chloride particles on the surface of the pore-forming agent particle of the paraffin ball, and then the micro-topological morphology is transferred to the surface of the three-dimensional porous support hole, These microprojections in the three-dimensional porous scaffold can promote the adhesion of the bone marrow stromal stem cells of the neonatal Sprague Dawley (SD) rat, while not In addition, the novel pore-forming agent is also invented for the first time, so that the prepared tissue engineering scaffold has regular spherical pore shape and good connectivity, and the surface of the hole is provided with a micron topological appearance, and the prepared porous scaffold of the tissue engineering is beneficial to the large SD The growth of murine bone marrow stromal stem cells, which is a material for tissue engineering and regenerative medicine The design provides a new field of vision and technology.4. The PLGA double-layer porous scaffold is designed, and the physical parameters of the stent are discussed through the cooperation study. The effect of the repair of the articular cartilage in vivo was studied. The biocompatibility of the PLGA stent was designed according to the relevant requirements of the national standard "Biological evaluation of medical devices" before the animal experiment. The results showed that the cell toxicity of the PLGA scaffold was small, the blood compatibility was good, and no obvious inflammation was caused at the bone/ cartilage defect. In addition, a double-layer stent was prepared to repair the New Zealand white rabbit bone/ cartilage defect and for the first time a double-layer stent was achieved After the double-layer stent was designed and prepared, the PLGA double-layer stent was implanted in the defect of the bone/ cartilage of the New Zealand white rabbit (4 mm in diameter and 5 mm in depth). The results showed that the porosity of the cartilage segment was 92%, the porosity of the bone segment was 77%, and the cartilage of the two-layer scaffold and the cartilage was 77%. The diameter of the segment is 100-200 & mu; m, and the two-layer support with the pore size of 300-450 & mu; m can achieve the specific pore diameter of 100-200 & mu; m In addition to the above-mentioned work, during a four-month short-term exchange in "The Program of the Short-term International Fund for the Doctoral Students of Fudan University"-funded university, I also synthesized the two-component polyacids and studied their table
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R318.08;R683

【參考文獻】

相關(guān)期刊論文 前2條

1 景殿英;張俊川;吳林波;丁建東;;聚乳酸多孔支架的變溫降解研究[J];高分子材料科學(xué)與工程;2005年06期

2 ;Distribution of bone marrow stem cells in large porous polyester scaffolds[J];Chinese Science Bulletin;2009年17期

，

本文編號：2478454