关节软骨细胞外基质源性微粒的制备及生物相容性评估

发布时间：2018-06-11 16:38

本文选题：组织工程 + 软骨细胞外基质　；参考：《中国人民解放军医学院》2014年硕士论文

【摘要】：研究背景：关节软骨无血液、淋巴供应，因此关节软骨的再生修复能力相当有限。目前在临床上应用的外科手术治疗方法在后期均存在一定问题，如纤维软骨形成、修复边缘区域整合性差等。近年来组织工程技术作为新的治疗手段正逐步兴起，为软骨损伤的治疗带来了希望。软骨组织工程三大关键问题，即种子细胞、生物支架、细胞因子微环境。自体软骨细胞是组织工程的首选种子细胞，临床应用最为广泛。但随着而来的难题是如何在体外短时间内获得足够数量、高质量的种子细胞，即扩增问题。微载体培养技术因其具有扩增种子细胞并维持细胞表型的特点而广泛用于动物细胞的大规模培养。微载体作为种子细胞的扩增载体，细胞可在微载体表面快速增殖，以满足后期检测分析或应用种子细胞的需求，特别是近年来以组织细胞外基质为原料制备的微载体。目前在皮肤、脂肪组织再生领域，组织细胞外基质源性微载体已获得良好结果，但在软骨组织再生领域未见相关报道。目的：本研究以天然软骨组织为基础，通过湿法粉碎、分筛过滤等处理后制备软骨细胞外基质源性微粒，并对其与软骨细胞复合后的生物相容性进行评估。研究内容主要包括：以天然软骨组织为材料制备关节软骨细胞外基质源性微粒，并观察其主要成分；观察其体外软骨细胞相容性；探究关节软骨细胞外基质源性微粒对兔软骨细胞表型的影响。方法：将猪关节软骨进行粉碎处理，通过分筛过滤得到直径220-300μm的软骨微粒，经脱细胞处理后获得天然去细胞关节软骨细胞外基质源性微粒，对此软骨微粒进行组织学成分鉴定、DNA残留量检测、GAG含量和总胶原含量检测。体外将软骨细胞与关节软骨细胞外基质源性微粒共培养，通过Dead/Live细胞活性染色和MTT，观察软骨细胞的生长、增殖情况。以普通平面静态培养和Cytodex-3微载体为对照，在三维微重力培养条件下，通过Rt-PCCR观察关节软骨细胞外基质源性微粒对软骨细胞表型的影响，，通过流式细胞仪，检测软骨细胞周期变化。结果：扫描电镜显示关节软骨细胞外基质源性微粒表面有纳米级别绒毛附着；组织学染色显示关节软骨细胞外基质源性微粒无细胞残留，蕃红花“O”、甲苯胺蓝染色、II型胶原免疫组化染色阳性；总胶原、GAG定量检测结果显示该软骨微粒保留了软骨组织的大部分细胞外基质成分。Dead/Live细胞活性染色显示关节软骨源性微粒表面的软骨细胞成绿色，细胞形态呈短梭形或多角形；细胞数量逐渐增多，未见红色细胞存在；MTT结果显示关节软骨细胞外基质源性微粒组的OD值呈持续上升趋势。Rt-PCR结果显示关节软骨细胞外基质源性微粒能够促进软骨细胞Collagen-II、SOX-9、Aggrecan基因表达水平，抑制Collagen-II基因表达水平；细胞周期检测结果显示关节软骨细胞外基质源性微粒使多数细胞处于S、G2-M期。结论：以天然软骨组织为材料制备的关节软骨细胞外基质源性微粒，脱细胞处理彻底，软骨微粒呈类圆形结构，表面附有绒毛状微丝结构，保留了软骨组织的大部分细胞外基质成分；无细胞毒性，生物相容性良好，能够促进细胞增殖，是软骨组织工程种子细胞的优良扩增载体；刺激细胞生长、增殖的能力优于Cytodex-3微载体；可有效维持软骨细胞的特有表型其效果与优于Cytodex-3。
[Abstract]:Background: articular cartilage has no blood and lymph supply, so the ability of regeneration and repair of articular cartilage is very limited. There are some problems in the clinical application of surgical treatment at the present time, such as fibrous cartilage formation, and poor integration of edge areas. In recent years, tissue engineering technology is being taken as a new treatment method. Bu Xingqi, which has brought hope for the treatment of cartilage injury. The three key problems of cartilage tissue engineering are seed cells, biological scaffolds, and cytokine microenvironments. Autologous chondrocytes are the preferred seed cells for tissue engineering, which are the most widely used in clinical practice. However, the problem is how to obtain sufficient quantity and high quality in short time in vitro. Microcarrier culture is widely used in the large-scale culture of animal cells because it has the characteristics of amplifying seed cells and maintaining the phenotype of the cells. As a carrier of seed cells, microcarriers can rapidly proliferate on the surface of microcarriers to meet the needs of later detection analysis or application of seed cells. In recent years, especially in the tissue of tissue extracellular matrix as a microcarrier, good results have been obtained in the field of skin, adipose tissue regeneration, tissue extracellular matrix derived microcarriers, but no related reports have been reported in the field of cartilage tissue regeneration.
Objective: to prepare cartilage extracellular matrix derived particles by wet crushing, sifting and filtration, and evaluate the biocompatibility of cartilage cells with cartilage cells. The main contents are as follows: the preparation of extracellular matrix derived from articular cartilage by natural cartilage tissue, The main components were observed, the chondrocyte compatibility in vitro was observed, and the effects of extracellular matrix derived particulates of articular cartilage on the phenotype of rabbit chondrocytes were explored.
Methods: the cartilage of porcine articular cartilage was crushed and the cartilage particles with a diameter of 220-300 m were obtained by sifting and filtration. The extracellular matrix derived from the natural acellular articular cartilage was obtained by dehydration. The histological composition of the cartilage particles, the detection of DNA residue, the content of GAG and the total collagen content were detected. The cell and articular cartilage extracellular matrix derived microparticles were co cultured. The growth and proliferation of chondrocytes were observed by Dead/Live cell activity staining and MTT. The normal plane static culture and Cytodex-3 microcarrier were compared. Under the condition of three-dimensional microgravity culture, the extracellular matrix derived from articular cartilage was observed by Rt-PCCR. The effects of cell phenotype were detected by flow cytometry.
Results: the scanning electron microscope showed that the surface of the extracellular matrix derived from articular cartilage was attached to the surface of nanoscale villus; histology staining showed that the extracellular matrix of articular cartilage was no cell residue, crocus "O", toluidine blue staining, II collagen immunohistochemical staining positive; total collagen, GAG quantitative detection results showed the cartilage The microparticles retained most of the extracellular matrix components of the cartilage tissue.Dead/Live cell activity to show that the cartilage cells of the articular cartilage derived particles were green, the cell morphology was short spindle or polygonal, and the number of cells increased gradually, and no red cells were found. MTT results showed the extracellular matrix group of articular cartilage extracellular matrix. The.Rt-PCR results showed that the extracellular matrix derived from articular cartilage cells could promote the expression level of Collagen-II, SOX-9, Aggrecan gene and the expression level of Collagen-II gene, and the results of cell cycle detection showed that the extracellular matrix of articular cartilage made most of the cells in S, G2-M phase.
Conclusion: the extracellular matrix derived from articular cartilage was prepared by natural cartilage tissue. The removal of cells was complete, the cartilage particles were round structure, and the surface was attached with villous microfilament structure, which retained most of the extracellular matrix components of cartilage tissue, and had no cytotoxicity and good biocompatibility, which could promote cell proliferation. An excellent carrier of seed cells for cartilage tissue engineering; the ability to stimulate cell growth and proliferation is superior to Cytodex-3 microcarrier; it can effectively maintain the endemic phenotype of chondrocytes and is superior to Cytodex-3.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08

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