【作者】 张柏青；

【导师】 张仲文；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 硕士

【摘要】 目的:天然胶原蛋白是结缔组织的主要成分之一,具有良好的生物相容性,但胶原存在加工性差、缺乏柔韧性、抗拉力差等缺点,单独用做修复软骨缺损的载体材料难以达到要求。软骨细胞外基质(ECM)富含蛋白聚糖、糖胺聚糖、Ⅱ型胶原蛋白及其它成分。关节软骨依靠大量的自分泌因子维持软骨细胞的表型和正常基质的代谢。生长因子可调节软骨细胞增殖、分化及代谢功能,并参与了关节软骨的生理及病理过程。本课题拟用牛真皮为原料,制备脱细胞真皮基质胶原膜(ADM),并在此基础上进一步应用硫酸软骨素(CS)、透明质酸钠(HA)、Ⅱ型胶原(Col-Ⅱ)、转化生长因子-β(TGF-β)和胰岛素样生长因子-Ⅰ(IGF-Ⅰ)等修饰ADM,以便最大程度上模拟软骨细胞的生长环境,促进软骨细胞更好的增长,更好的用于软骨缺损的治疗。方法:采用积水潭医院创伤骨科研究所提供的技术方法制备ADM。消毒包装后备用。取新生新西兰胎兔4只,行软骨细胞分离,原代培养,传代培养。绘制生长曲线,行HE及双醋酸荧光素(FDA)-碘化丙啶(PI)荧光染色。分为空白组、CS组、HA组、Col-Ⅱ组、TGF-β组、IGF-I组、混合组。配制0.5%(m/v)的CS高糖DMEM溶液;1.0%(m/v)的HA高糖DMEM溶液;0.5%(m/v)Col-Ⅱ高糖DMEM溶液;1ng/ml的TGF-β高糖DMEM溶液;10ng/ml的IGF-I高糖DMEM溶液;将上述五种修饰材料配成0.5%的CS+1.0%的HA+0.5%Col-Ⅱ+1ng/ml的TGF-β+10ng/ml的IGF-I高糖DMEM混合溶液。共6组溶液与ADM的复合。消毒包装后备用。软骨细胞体外培养至第3代,制成浓度为5×106/ml的软骨细胞悬液。与备用的7组ADM复合培养。于1、3、7、14、21天行扫描电镜观察,包埋切片,HE染色,S100免疫组化染色。免疫组化结果行阳性细胞计数。于1、3、7天时,取复合软骨细胞的ADM,行RT-PCR。BanScan4.3凝胶图像分析软件,分析所得结果。制作兔膝关节软骨缺损模型,分为3组,混合修复组植入物为多种材料修饰ADM复合软骨细胞,单纯胶原膜组植入物为未修饰的ADM复合软骨细胞,空白对照组不复合任何材料。分别于第1、2、3月取材行大体观察、HE染色、Col-Ⅱ免疫组化染色,并用Wakitani法进行组织学评分。结果:制备所得的ADM外观呈白色,具有弹性。电镜下可见呈多孔疏松结构,孔隙率较佳,适合软骨细胞生长。关节软骨经Ⅱ型胶原酶消化2～4小时后离心去上清液,加入培养液进行原代细胞培养,并能通过传代增殖获得大量软骨细胞。HE染色示细胞形态较好,FDA-PI荧光染色示细胞活性率达98%以上。电镜结果显示各组修饰效果:混合组>Col-Ⅱ>TGF-β>CS>HA及IGF-Ⅰ。HE及免疫组化结果示:混合修饰组细胞形态最佳,阳性率最高。显微镜下对阳性细胞数进行计数,统计结果表明:修饰效果同电镜结果,混合组效果最佳。RT-PCR结果示,第1天时,Col-Ⅱ组,TGF-β组及混合组扩增出的条带明显。其余各组未见明显的扩增条带。第3,7天Col-Ⅱ组及混合组扩增出的条带明显。BanScan4.3凝胶图像分析软件分析结果显示混合组Col-ⅡmRNA的表达最高。大体观察及HE染色显示:第1、2、3月时混合修复组修复为透明软骨样修复,单纯材料组和空白对照组为纤维软骨及纤维性修复。Col-Ⅱ免疫组化结果显示:混合修复组阳性表达>单纯胶原膜组强>空白对照组。Wakitani组织学评分结果显示:混合修复组修复结果明显优于单纯材料组及空白对照组。结论:ADM具有良好的生物相容性;适宜的生物降解性;较好的细胞黏附表面活性;良好的可塑性;良好的三维多孔结构。混合修饰的ADM与软骨细胞有较好的相容性。混合修饰的ADM最适宜作为修复软骨缺损的载体。其修复关节软骨缺损的效果较佳。更多还原

【Abstract】 Objective: Natural collagen protein was a major component of connective tissue and it had good biocompatibility. But there had some disadvantages about the collagen, such as poor of flexibility, poor anti-tensile force. There’s some difficults in meeting the needs of using it as a separate carrier material for the repair of cartilage defects. Cartilage matrix is rich in proteoglycan, glycosaminoglycan, type-Ⅱcollagen and other components. Articular cartilage relyed on a large number of autocrine factors in the maintenance of chondrocytes and normal phenotype matrix metabolism. Growth factors can regulate the proliferation, differentiation and metabolism of chondrocytes and participate in the physiological and pathological processes of articular cartilage. The objective of this experiment was using cattle dermis for the preparation of acellular dermal matrix collagen membrane (ADM). On this basis, further application of chondroitin sulfate (CS), sodium hyaluronate (HA), collagen typeⅡ(Col-Ⅱ), transforming growth factor -β(TGF-β) and insulin-like growth factor-Ⅰ(IGF-Ⅰ) were used for the modification of ADM. Simulating the greatest degree of chondrocytes growth environment, in order to promote the growth of chondrocytes.Methods: Using the Jishuitan Hospital Trauma Orthopedic Institute technical methods for the preparation of ADM. Disinfection and packaging.4 New Zealand fetal rabbit were sacrificed and the chondrocytes were gathered, then cultured and passed. Growth curve, HE and fluoresceindiacetate (FDA)-propidium iodide (PI) fluorescence staining were carried out.There’s blank group, the CS group, HA group, Col-Ⅱgroup, TGF-βgroup, IGF-I group and mixed group. Prepare 0.5%(m/v)CS high glucose DMEM solution;1.0%(m/v)HA high glucose DMEM solution;0.5%(m/v)Col-Ⅱhigh glucose DMEM solution;1ng/ml TGF-βhigh glucose DMEM solution;10ng/ml IGF-I high glucose DMEM solution;Mixing the above five modified materials as 0.5%CS+1.0% HA+0.5%Col-Ⅱ+1ng/ml TGF-β+10ng/ml IGF-I high glucose DMEM mixture solution. Combine the total of six groups of solution with ADM.Chondrocytes were cultured in vitro to the third passage. Concentration of the chondrocytes suspension was 5×106/ml, and then mixed the chondrocytes suspension with the ADM. Scanning electron microscopy, embedding, HE staining, S100 immunohistochemical staining were carried out in 1, 3, 7, 14, 21days. After that, the positive cell count of immunohistochemical staining was also done. In 1, 3, 7 days, ADM were taken out and then analyzed with RT-PCR. BanScan4.3 gel image analysis software, analyzed the results.Made the model of articular cartilge defects of rabbits, divided in into three groups. A mixed group repair implants was a variety of composite materials modification ADM with chondrocytes. Pure collagen membrane group was the ADM for non-modified composite chondrocytes. The blank group did not composed with any material. Rbbits were sacrificed in 1, 2, 3 month. Then, the general observation, HE staining, Col-Ⅱimmunohistochemical staining and Wakitani for histologic score were analyzed.Results: ADM appears white and flexible structure. Electron microscope showed the porous structure of it. The porosity was suitable for the implantation and growth of chondrocytes.The acquired articular cartilage was digested by type-Ⅱcollagenase for 2 to 4 hours, and then removed the supernatant after centrifugation. Adding culture medium to the chondrocytes, and then could get more chondrocytes through the transferring in vitro. HE staining showed that cell morphology better, the FDA-PI fluorescence staining showed that cell survival rate was over 98%.Electron microscope showed the modification effect of all groups: Mixed bank> Col-Ⅱ> TGF-β>CS>HA and IGF-Ⅰ. HE and immunohistochemical staining showed the mixed bank had the best effect and the highest positive rate. Under the microscope, the numbers of positive cells were counted. The results show that: modification effects were similar to the result showed by the electron microscopy. Mixed bank had the best effect. The results of RT-PCR showed that on the first day, Col-Ⅱbank,TGF-βbank and mixed bank appeared obviously strap. The other banks could not see obviously strap. On the third and seventh days, Col-Ⅱbank and mixed bank had obviously strap. BanScan4.3 suggested that the Col-ⅡmRNA expression of the mixed bank was the highest.On the 1, 2, 3 month, general observation and HE staining showed that, the repaired organization of the mixed group was likely hyaline cartilage repair. Pure collagen membrane group and the blank group was the fiber restoration. The positive rate of Col-Ⅱimmunohistochemical staining showed that the mixed group>Simple materials group>control group. The result of Wakitani histologic score suggested that the mixed group had a better effect of restoration for the defects.Conclusion: ADM had better biocompatibility, suitable biodegradation, better cell adhesion surface activity, better plasticity and better three-dimensional porous structure.Mixed modification ADM had a better compatibility with chondrocytes.Mixed modification ADM appears as the most suitable scaffold for repairing of cartilage defects.更多还原