【作者】 张晓燕；

【导师】 韩冰；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2014， 硕士

【摘要】 运用组织工程修复缺损的软骨已成为关节软骨缺损修复与再生领域的研究热点，其中组织工程支架的选择是这一修复治疗中，我们最先遇到的难题。目前急需开发出物理性能良好，生物相容性良好，而且适合细胞生长和组织形成的支架。吉林大学化学院通过多年实验研究，运用静电纺丝技术，以生物相容性均优良的丝素蛋白（silk fibroin，SF）与左旋聚乳酸（poly L-lactic acid，PLLA）为原料，制备出了物理性能优良的SF/PLLA复合纳米级材料。本实验组通过动物皮下埋植实验、兔膝关节软骨细胞与支架材料体外复合培养实验、急性全身毒性实验、体外细胞毒性实验，来评价该材料的生物相容性及安全性，并以此来探讨其作为生物植入材料的可行性。本研究的具体内容如下:1、将材料植入小鼠背部，以3-0缝线作为对照，于术后1、2、3、4周分别取材，HE染色，拍照。结果：材料植入小鼠背部后，开始有少量成纤维细胞长入材料中，伴随出现少量的淋巴细胞及异物巨细胞，随后成纤维细胞数量增加，淋巴细胞及异物巨细胞无明显增多，材料慢慢降解，直至第4周时，材料出现明显的降解；材料周围组织炎症反应，第2周时最严重，然后逐渐减轻，其周围组织炎症反应与缝线周围组织一致或略轻；将细胞接种于支架上24h后，有细胞贴附于材料纤维上，48h后贴附于纤维上的细胞数量增多。2、成功分离、培养兔膝关节软骨细胞，将细胞接种至材料表面，在体外继续培养，倒置光学显微镜及电镜下观察细胞的黏附和生长情况。结果：将细胞接种于支架上24h后，有细胞贴附于材料纤维上，48h后贴附于纤维上的细胞数量增多。3、制备浸提液，并以生理盐水作为对照，注射入小鼠体内，观察72h内小鼠的一般情况及不良反应。结果：注射浸提液后，小鼠的全身状况与注射生理盐水后无差别。4、将关节软骨细胞分为阴性对照组（空白培养液）、实验组（含材料的培养液）及阳性对照组(含苯酚培养液)3个组，不同组材料浸提液与细胞共培养24和48h，MTT法检测材料对软骨细胞增殖的影响。结果：实验组24和48h细胞毒性均为Ⅰ级。除阳性对照组外，随着培养时间的延长，吸光度（Absorbance，A）值均有增加，同一时间点，实验组与阴性对照组比较差异无统计学意义（P>0.05），与阳性对照组比较A值明显升高(P<0.01）。综上所述，运用静电纺丝工艺，以丝素蛋白与左旋聚乳酸为原料制备的复合纳米材料，适合细胞生长和组织形成，且具有良好的生物相容性，属于安全植入性材料。为今后应用此支架，修复缺损的关节软骨，提供了一定的生物学实验基础。更多还原

【Abstract】 It has already become the focus studied to repair defects by means oftissue engineering in the domain of defects repair and regeneration.It is the firstdifficult problem for us to prepare ideal scaffolds. So far,it’s urgent to ownscaffolds with good physical and biological properties,which are suitable forcell growth. After years of experimental research, we prepare SF/PLLAnanocomposite with silk fibroin and poly L-lactic acid by static spinning inChemical College of Jilin University.In our experimental group,we evaluate the biological compatibility andsafety of the material by the experments of in vivo implantation test,observation after the rabbit knee joint cartilage cells seeded to the surface ofmaterials,acute systemic toxicity test,and cytotoxicity test.The main content of this research are as follows:1.The materials were implanted into the back of the mice,and3-0suturewas used as control.Tissues were collected at1,2,3,4weeks after operation,dyed by HE staining and then photos were taken.The tissue reactions inexperimental group and control group were observed.Result: There were littlefibroblasts and a little of lymphocytes and macrophage cells in the materialswhich were implanted into the back of the mice at the beginning.Then thenumber of the fibroblasts increased,but the number of the lymphocyte andmacrophage cells did not change obviously.The materials degraded slowly,andthe material degraded obviously at4weeks. The inflammation of tissue aroundthe material reduced gradually from2nd week. And the inflammation oftissue around the material was the same to the suture,sometimes was slighterthan the suture. After seeding24h,there were cells attaching to the fibers of thematerial.More and more cells attached to the fibers.2.The rabbit knee joint cartilage cells were separated and cultured, and then subculture cells were seeded to the surface of materials. After culturing invitro, the adhesion and growth of the cells were observed with inverted opticalmicroscope. Result: After seeding24h,there were cells attaching to the fibersof the material.More and more cells attached to the fibers.3. Physiological saline as control,the leaching solution was prepared andinjected into the mice,then the rates were observed for72h. Result:Afterinjection，he body status of the mice was the same to the control group.4．The bioactivities of the rabbit knee joint cartilage cells in negativecontrol group(DMEM culture media), experimental group(DMEM containmaterials) and positive control group(DMEM contain phenol solution)weredetermined by MTT Tassay after coculture for24and48h. Result: The reasultof MTT Tassay showed that the cytotoxicity in experimental groups were all onLevel Ⅰ at24and48h.Except for positive control group,the A values wereincreased in other groups with the extended response time.At the same time,there was no significant difference in cytotoxicity between experimental groupand negative control group(P>0.05)and the A values of experimental group washigh than positive control group(P<0.01).This showed that material has goodsafety.In conclusion,the materials prepared with silk fibroin and poly L-lacticacid by static spinning have good biocompatible,which are suitable for cellgrowth and belong to safe implant materials.The article provide experimentbasis for studying how to repair defective articular cartilage with the materials.更多还原