材料表面拓扑形貌的细胞响应以及PLGA组织工程多孔支架的制备与软骨组织修复研究
发布时间：2019-05-16 17:48
【摘要】：软骨缺损修复是再生医学领域非常有挑战性的一个课题。组织工程学的出现,将可能使人们从传统的器官移植和植入这种以伤补伤的方法进入到一个器官制造的新时代,其为软骨缺损修复提供了新的思路和方法。软骨组织的损伤或者缺损往往会伴随软骨下骨的改变,近年来,采用双层支架来模拟关节软骨和软骨下骨各自内在结构和生理功能以实现软骨和软骨下骨的同时修复成为一个新思路,但关于双层支架的很多基本问题还有待于进一步探索。 未来生物材料的设计需要对于材料的细胞响应有深入的认识。近几十年来的相关研究已经大大加深了人们对于“生物材料”涵义的理解,同时也提供了更多探索的方向。材料的诸多因素可以影响细胞行为,其中,表面形貌的效应普遍存在,并且由于形貌改变可不涉及化学成份的改变,故相应的材料改性容易最终获得批准应用。 本博士论文以聚乳酸—羟基乙酸共聚物(PLGA)这种有应用价值的可降解高分子为基质材料,开展了生物医用材料方面的研究。论文首先从二维平面探索微米拓扑形貌的细胞响应行为并得出基本规律,继而将其应用于三维多孔支架中,最后再用组织工程双层支架进行关节软骨和软骨下骨同时修复的动物实验。该研究有助于理解材料物理因素对于细胞的作用,为生物材料设计提供指导。 本论文的主要创新性工作包括以下几方面： 1.制备了一系列不同尺寸的微米阵列,研究其细胞响应行为,并发现适合骨髓基质干细胞黏附的尺寸。本论文在二维PLGA膜表面制备了一系列不同高度(深度)的微柱(微坑)阵列,然后研究骨髓基质干细胞在这些拓扑形貌表面的生长情况。根据实验结果,我们发现1μm高度(或深度)的微柱(或微坑)阵列相对于其它高度(或深度)的阵列更利于细胞的生长。进一步我们比较了1μm微坑和1μm微柱,发现1μm微柱比微坑和光滑膜更适合骨髓基质干细胞的黏附。 2.利用材料表面微柱阵列实现了对于细胞核严重自我变形的调控。本论文工作过程中,意外发现新生大鼠骨髓基质干细胞细胞核在特定高度的PLGA微柱阵列表面能发生严重自我变形；细胞核变形并非是由细胞核的重力作用引起的,而可能是由细胞铺展时的内应力导致；尽管骨髓基质干细胞发生了如此严重的细胞核变形,其仍具备增殖和成骨分化的能力。本文通过设计不同微柱阵列,成功控制了细胞核的形状,在国际上首次实现了哑铃形、方形、十字交叉等异常细胞核形状。此外进一步研究了另外五种细胞(Hela、PC12、NIH3T3、MC3T3、 Chondrocyte),发现细胞在微柱阵列表面的细胞核变形现象具有普适性；同时其响应程度具有细胞类型依赖性。 3.通过设计新型致孔剂实现了三维PLGA多孔支架内部孔结构以及孔壁拓扑形貌的控制。基于粒子浸出技术,首次通过对石蜡球致孔剂表面物理修饰的办法调控了组织工程支架内表面微米拓扑形貌。在石蜡球这种致孔剂粒子表面通过氯化钠粒子的撞击制备出微米尺寸的凹坑,然后再把这些微米拓扑形貌转移到三维多孔支架孔表面,从而获得微米凸起结构。三维多孔支架中的这些微米凸起能促进新生Sprague Dawley (SD)大鼠骨髓基质干细胞的黏附,同时并不影响干细胞的增殖和分化。此外,还首次发明“糖粘盐”这种新型致孔剂,使得制备得到的组织工程支架具有规则的球形孔形状以及良好的连通性,并且孔表面具有微米拓扑形貌。制备得到的组织工程多孔支架利于SD大鼠骨髓基质干细胞的生长。这些研究成果为组织工程和再生医学中的材料设计提供新的视野和技术。 4.设计了PLGA双层多孔支架,并通过合作研究,探讨了支架物理参量对于体内关节软骨修复效果的影响。在动物实验之前,本论文按照国家标准“医疗器械生物学评价”的相关要求设计了PLGA支架材料体内外的生物相容性实验,并研究了其在新西兰大白兔体内的降解行为。通过实验,发现PLGA支架材料细胞毒性小,血液相容性好,且不会在骨/软骨缺损处引起明显的炎症反应,同时具有合适的降解速率。另外,制备了双层支架以修复新西兰大白兔骨/软骨缺损,并首次实现对双层支架中各层最优孔隙率、孔径探索。设计并制备好双层支架后,通过合作研究,将PLGA双层支架植入新西兰大白兔骨/软骨缺损处(直径4mm,深度5mm),以检验各组支架对其修复效果。术后12周,从大体观、HE染色、甲苯胺蓝染色、免疫组化染色、组织学评分和相关基因的表达这些结果中我们得出：软骨段支架孔隙率为92%,骨段支架孔隙率为77%的双层支架及软骨段孔径为100-200μm,骨段孔径为300-450μm的双层支架能实现比其它孔隙率或孔径组合更好的修复效果。 除了上述工作以外,在“复旦大学博士生短期国际访学资助计划”资助下赴美国密歇根大学进行为期4个月短期交流期间,我还合成了两组分的聚酸酐,并研究了其表面溶蚀性能、制备了药物释放装置。
[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
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08;R683
本文编号：2478454
[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
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08;R683
【参考文献】
相关期刊论文 前2条
1 景殿英;张俊川;吴林波;丁建东;;聚乳酸多孔支架的变温降解研究[J];高分子材料科学与工程;2005年06期
2 ;Distribution of bone marrow stem cells in large porous polyester scaffolds[J];Chinese Science Bulletin;2009年17期
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