新型微孔水凝胶的制备及其在软骨组织工程中的应用研究

发布时间：2018-03-22 20:39

本文选题：软骨细胞　切入点：微孔水凝胶　出处：《华南理工大学》2014年博士论文　论文类型：学位论文

【摘要】：软骨内部无血管，无神经，无免疫反应的独特特性导致其自愈能力极其有限，软骨修复是一大国际性难题。传统性修复技术尚不能完全满足临床需要，目前，软骨组织工程为软骨缺损修复与重建开辟了新的途径。如何保持长期有效的软骨组织修复效果是目前存在的重大挑战。细胞所处的微环境对其生长及分化有着重要的影响。因此，针对软骨修复材料的开发，研究材料提供的微环境对细胞行为及功能表达的影响具有重要意义。水凝胶以其良好的生物相容性，仿似ECM的3D微环境，合适的孔隙结构，且能提供短期的机械稳定性以及增加细胞附着表面积，附着力等特点从而能构成细胞生长和分化的良好微环境，，但目前传统的水凝胶的应用瓶颈便是它尚不能提供细胞足够的快速生长空间和适当的亲和位点。正因为如此，经过一段较长时间的体外培养，包埋在水凝胶边缘软骨细胞会突破水凝胶的阻碍，在边缘位置会分泌丰富的软骨组织基质蛋白，水凝胶外表面显示出真正的软骨组织特征，与水凝胶内部，细胞的保守生长形成鲜明对比。该现象被定义为“边缘发展edge flourish（EF）”。这种EF现象只是发生在传统水凝胶的边缘，缺乏可控性。受到EF的启发，我们通过在水凝胶内部构建微孔，从而人为地引入“凝胶微孔边缘现象”。这样，即使在水凝胶的内部，也能出现EF现象。本论文的研究目的是利用“微孔”这一结构的特殊EF效应来观察其对软骨细胞生长增殖以及细胞功能表达情况以研究材料的结构微环境与细胞的相互作用。本论文的研究工作主要开展了以下内容：本文首先采用II型胶原酶消化法将猪关节软骨细胞进行体外分离、培养和鉴定。不同代数的软骨细胞生长刚开始较为缓慢，之后进入快速增殖期和平台期，最后开始出现生长抑制。随着传代次数的增加，细胞形态由原代的近似椭圆形逐渐变为多角形或者纺锤形，原代软骨细胞的组织染色与天然软骨的组织染色相接近。II型胶原免疫组化染色表明其细胞质被染成棕黄色，细胞核基本不着色。证明该方法能获得纯度高，活性好的原代软骨细胞，成功建立了猪关节软骨细胞分离及培养体系。本文采用双乳液法成功制备出形状规整，尺寸可控的明胶微球，考察了搅拌速率对明胶微球粒径的影响，改变搅拌速率，制备并筛选出具有代表性的三种尺寸分别为小尺寸（80-120μm）、中尺寸（150-200μm）、大尺寸（250-300μm）的明胶微球。以明胶微球为致孔剂成功构建了具有以上三种不同尺寸大小结构的新型微孔水凝胶。研究了三种尺寸的“微孔”结构体系对软骨细胞生长的影响，微孔水凝胶比传统致密的海藻酸钠水凝胶具有更好的促细胞增殖作用。细胞会沿着明胶微球造成的“孔洞”结构边缘富集生长并最终完全填满整个“孔洞”。其中小孔水凝胶体系中软骨细胞的增殖效果最显著。且能显著表达软骨特征基因，微孔海藻酸钠水凝胶体系是通过激活MAPK/JNK信号通路来促进软骨细胞的增殖与生长，小孔尺寸结构的海藻酸钠水凝胶对保持软骨特征效果最优。其次，本文采用微孔水凝胶成功介导了去分化软骨细胞的再分化，微孔水凝胶具有使得去分化软骨细胞重新恢复并保持软骨细胞特征的能力，表现为软骨特征基因表达显著上调，能重新合成和分泌细胞外基质II型胶原和糖胺聚糖，基质分泌增强，且有大量软骨陷窝形成。且去分化的软骨细胞在微孔水凝胶中重新分化成软骨细胞的能力是优于传统致密海藻酸水凝胶。Westernblot结果进一步证明该再分化过程有p38和Erk1/2信号通路的参与。最后，考察了RGD修饰的微孔水凝胶对介导ATDC5细胞成软骨分化早期的影响。RGD的接枝率为1.4%-3.5%，RGD微孔水凝胶的内部仍呈多孔结构，孔隙连通性保持完整，且生物相容性提高。ATDC5细胞在RGD修饰的微孔水凝胶中体外培养4周后，成软骨相关基因均有大量上调表达。II型胶原和糖胺聚糖的蛋白表达也得到相应增强，会有序的形成类天然软骨陷窝结构，致密均匀，基质分泌明显。在生长因子TGF-β3的诱导下，RGD修饰的微孔水凝胶能显著增强ATDC5细胞的成软骨分化。
[Abstract]:No cartilage vessels, no nerve, no immune response leads to the unique characteristics of the self-healing ability is extremely limited, cartilage repair is a major international problem. The traditional repair technology is still not fully meet the clinical needs, at present, has opened a new way for the repair and reconstruction of cartilage tissue engineering cartilage defects. How to maintain the effect of cartilage tissue repair long term is a major challenge at present. The micro environment of the cells have important influence on their growth and differentiation. Therefore, for the development of cartilage repair materials, plays an important role in micro environment to provide research material on the expression and function of cell behavior.
Hydrogels with good biocompatibility, like ECM 3D micro environment, suitable pore structure, and can provide mechanical stability and increase the short-term cell adhesion surface, adhesion etc. thus constitute cell growth and differentiation of good micro environment, but the application bottleneck of traditional hydrogels is that it cannot provide the rapid growth of space enough cells and appropriate affinity sites. Because of this, after a long time of in vitro culture, entrapped in the hydrogel edge of cartilage cells will break through the obstacles in the edge position of the hydrogel, secrete abundant cartilage matrix protein, hydrogel surface showed the cartilage tissue features real, and within the hydrogel cell growth, the conservative stark contrast. This phenomenon is defined as "the edge of edge flourish development (EF)". This phenomenon only occurred in traditional EF hydrogels The edge of the lack of controllability. Inspired by EF, we construct microporous hydrogel in the inside, thereby artificially introduced "gel microporous edge phenomenon." in this way, even in the hydrogel interior, also can appear EF phenomenon. The purpose of this study is to observe the cartilage cell proliferation and cell function expression to the interaction structure of the material micro environment and cells. Using the special structure of the microporous EF effect "of the research work of this thesis mainly includes the following contents:
Firstly, by type II collagenase digestion of porcine articular cartilage cells were isolated, cultured and identified. Chondrocyte growth beginning algebra is relatively slow, and after entering the stage platform period of rapid proliferation, finally began to appear. The growth inhibition with increasing passage number, cell morphology by primary ellipse gradually becomes polygonal or fusiform, tissue staining and natural cartilage chondrocytes by tissue staining is similar to type.II collagen immunohistochemical staining showed that the cytoplasm stained brown, nucleus basic color. The results show that the method can obtain high purity, primary chondrocytes activity, successfully established the isolation and culture system of pig the articular cartilage cells.
Double emulsion was successfully prepared by the regular shape, gelatin microspheres with controllable size, the effects of stirring rate on gelatin microspheres, changing the stirring rate, the preparation and selection of the three dimensions of the representative are of small size (80-120 m), size (150-200 m). Large size (250-300 m) gelatin microspheres. The gelatin microspheres as porogen successfully constructed a novel microporous hydrogel has more than three kinds of different size structure. On three dimensions of "micro" structure of cartilage cell growth, cell proliferation of microporous hydrogel is better than sodium alginate hydrogel the traditional dense cells. Along the gelatin microspheres caused by the "hole" edge enrichment and growth and eventually completely fill the "hole". The cartilage cells of small pore water in gel system and the proliferation of the most significant effect. It can significantly express cartilage characteristic genes. Microporous alginate hydrogel system promotes the proliferation and growth of chondrocytes by activating MAPK/JNK signaling pathway. Alginate hydrogel with small pore size has the best effect on maintaining cartilage characteristics.
Secondly, this paper uses the successful differentiation of microporous hydrogels mediated dedifferentiation of chondrocytes, the microporous hydrogel has the ability to restore and maintain the chondrocyte differentiation characteristics of chondrocytes, expressed in cartilage feature genes were up-regulated to synthesis and secretion of extracellular matrix type II collagen and glycosaminoglycan, matrix secretion increased and there is a lot of chondrocytes and cartilage lacuna formation. The differentiation in the porous hydrogel to chondrogenic differentiation ability is better than the traditional dense alginate hydrogel.Westernblot results further show that the differentiation process of p38 and Erk1/2 signaling pathways.
Finally, the study of RGD modified microporous hydrogel mediated effects of cartilage in early differentiation of the graft ratio of.RGD 1.4%-3.5% ATDC5 cells, RGD porous hydrogel was still inside the porous structure, pore connectivity remain intact, and improve the biocompatibility of.ATDC5 cells cultured in vitro in microporous hydrogels modified with RGD in 4 weeks, a there are a lot of cartilage related genes up-regulated the expression of type.II collagen and glycosaminoglycan protein has also been a corresponding increase, will be ordered form of natural cartilage lacuna structure, compact and uniform, matrix secretion was induced by growth factor TGF- beta 3, RGD modified porous hydrogel can significantly enhance the chondrogenic differentiation of ATDC5 cells.

【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.17

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