生物材料论文：TGE-β_1缓释微球壳聚糖支架对兔关节软骨缺损修复的研究.doc

生物材料论文：TGE-_1缓释微球壳聚糖支架对兔关节软骨缺损修复的研究【中文摘要】第1章不同脱乙酰度壳聚糖支架制备及降解性能评价制备不同脱乙酰度壳聚糖三维支架并考察其体内、外降解性能,为壳聚糖作为软骨缺损修复支架材提供前期实验依据。方法：用脱乙酰度分别为65%、80%、95%壳聚糖,以相分离法制备三维支架,SEM观察其表面形态、孔径,以液体替代法检测孔隙率,吸水溶胀率；含溶菌酶107U/L的PBS溶液(pH7.4)37气浴振荡,测不同时间(1d,3d,7d,14d,21d,28d)支架降解率,植入SD大鼠竖棘肌内,分别检测2周、4周、6周、8周、10周、12周降解率及观察其降解支架与组织局部情况。结果：不同脱乙酰度支架均具高孔隙三维结构,随脱乙酰度增加孔隙率分别为93.6%、90.0%、85.1%,支架颜色由淡黄渐变白,溶胀率分别为820%、803%、772%,在体外溶菌酶的作用下逐步降解,至第28d时不同脱乙酰度支架分别降解为30.44%、22.88%、17.10%,且逐步降解第4周时降解率为57.48%,40.23%,29.53%。HE检测示与周围肌组织具有良好的相容性结论：支架具有良好的三维孔隙结构,能在体内外逐步降解,脱乙酰度越高降解越慢,相同时间点体内降解速率快于体外降解,脱乙酰度为80%的壳聚糖降解速率与正常软骨8周修复相一致。第2章缓释TGF-,壳聚糖微球的制备及性能检测运用乳化交联法制备壳聚糖微球,以包裹转化生长因子-1(TGF-1)并检测其溶胀率、载药量及缓释等性能,评估利用壳聚糖微球作为控释TGF-1载体的可行性。方法：以液体石蜡为乳化剂,三聚磷酸钠(TPP)为交联剂,采用乳化交联法制备壳聚糖微球。以包裹TGF-1及牛血清白蛋白(BSA),分别制备TGF-1壳聚糖微球与BSA壳聚糖微球。应用扫描电镜、激光颗粒分布测量仪检测微球形态,检测球溶胀率,ELISA夹心法测定微球载药量、包封率及体外药物缓释率等综合分析微球特性。结果：制备的微球粒径分布集中,平均粒径35m,球形良好,球体均匀表面光滑,在酸环境下溶胀率最高,达800%；具有较高的包封效率88%,载药量为11ng/mg,药物释放试验表明TGF-1及BSA均可以从微球中缓慢释放,第7天累积释药量达90%,63.3%；在溶菌酶降解作用下逐步降解,6周时降解为57%。结论：乳化交联法制备壳聚糖缓释微球方法简单易行,所得TGF-1壳聚糖微球具有良好的缓释性能,其作为软骨组织工程材料具有潜在的应用价值。第3章负载可降解缓释微球壳聚糖支架生物相容性实验研究制备负载缓释微球壳聚糖支架并对其生物相容性进行体内外评价,为壳聚糖作为一类有前途的动物软骨缺损修复支架材料提供实验依据。方法：以乳化交联法及相分离法制备负载缓释微球多孔壳聚糖支架材料。以溶血试验、急性毒性实验、皮内刺激实验、热源性实验、肌内植入实验,整体评价自制负载缓释微球壳聚糖支架的生物相容性。结果：材料孔径多为200-350m且相互贯通的三维立体多孔结构,各孔以板状分开,孔隙率为93.63%0.51%(n=6,xs)；支架溶血率为1.6%,镜下未见明显红细胞破坏；材料急性毒性评价程度为无毒,材料浸提液组小鼠24h,48h,72h体重变化分别为0.34670.1075,0.40200.0796,0.49320.0838,各时间点与生理盐水组组间配对t检验P0.05；皮内原发刺激记分及原发刺激指数(PII)均为0；热源性实验体温升高度为0.170.06；肌内植入实验大鼠均成活,全身良好、无感染,4周左右新生毛正常分布,8周大体观察支架周围血管明显增多,与周围肌组织整合良好,心肝肺肾等内脏均无特殊,1周、2周、4周、8周、12周随时间延长,淋巴细胞浸润逐渐减少,可见血管及纤维长入支架,包裹逐渐变薄,支架渐降解。结论：负载微球多孔壳聚糖支架具有优良的生物相容性,具有良好的三维孔隙结构及可降解性,有望成为一种良好的软骨修复材料。第4章制备负载TGF-1微球壳聚糖支架并考察其对兔关节软骨缺损修复研制负载缓释TGF-1微球壳聚糖支架,探讨其体内吸附自身髓腔中骨髓细胞及微环境中信号因子,诱导软骨缺损处原位成软骨细胞再生分化的效应性。方法：乳化交联法制备具有缓释TGF-1功能的壳聚糖微球,与液相分离法制得壳聚糖支架共混得复合支架,采用环境扫描电镜(SEM)观察支架及微球形态,激光颗粒分布测量仪检测微球直径分布,ELISA夹心法测微球TGF-1包封率,载药量以及缓释率,体外检测微球及支架4周降解率；选用兔作为实验动物造成双侧股骨滑车部全层软骨缺损,采用不同的材料构成四组,观察修复效果。于术后1月、3月取材,大体观察软骨修复状况并予Masuoka评分,固定组织行甲苯胺蓝染色,型胶原免疫组织化学及Wakitani评分综合评估组织修复质量。结果：四组植入物的兔膝关节均无关节腔感染、积液,Masuoka评分MS-TGFs, CS-TGF, CS, Empty组依次为7.670.47；3.830.75；1.000.89；0.830.75。组织1、3月取材TB染色示MS-TGFs修复最佳,填充面光滑平整,关节软骨排列整齐,细胞结构完整,连续；CS-TGF修复欠佳,表面平整性差,软骨量少；CS为大量纤维软骨组织填充；Empty组无修复,且周围软骨继发损坏,缺损直径约为5mm；第1月CD34、CD44双组化鉴定吸附细胞为干细胞的来源。MS-TGFs组CD34(-)CD44(+)细胞最多,其次为CS-TGF组。第3月甲苯胺蓝及型胶原免疫组化染色可见明显软骨细胞及胶原异染,组织修复质量Wakitani评分示：4.501.12：10.830.37；13.670.47,有明显统计学差异(P 0.05; the points of skin to the primary stimulation and the primary stimulation index (PII) are 0; in the heat experiment,the temperature rised by 0.170.06; rats from implantation in muscle test were all survived well without systemic infection, and appearance of newborn normal hair was in the 4 weeks; and in the 8 weeks Naked-eye observed that there was significantly increased peripheral vascular stents, which well integrated with the surrounding muscle tissue, while other internal organs such as heart, liver, lung and kidney were ordinary, furthermore, from the 1 week to 12 weeks, infiltration of lymphocytes was gradually decreased,blood vessels and fibrous tissues around the frame is visible, simultaneously, wrapped fibrous tissues were gradually thin, chitosan scaffolds were gradually degraded.Conclusion:chitosan scaffold loaded porous microspheres had excellent biocompatibility, good three-dimensional pore structure and biodegrade ability, all that induce it was expected to be a good repair material for the treatment of osteochondral defects. Part IVRepair of articular cartilage defects in rabbits using porous Chitosan Scaffold Containing Microspheres Loaded with Transforming Growth Factor-1:To investigate the effect of MS-TGFs homing bone marrow cells and the signal factor from the marrow microenvironment in vivo, inducing bone marrow cells differentiated and regenerated into functioned cells in the cartilage defects location.Methods:To prepare chitosan loaded with the sustained release of transforming growth factor-1 microspheres and chitosan composite scaffold by cross-linking emulsion, liquid separation methods, respectively. The specimens were observed using a scanning electron microscopy SEM for the surface of microspheres and chitosan after being gold-coated with a sputter coater; the microsphere diameter by Measurement of laser particle distribution, ELISA sandwich method to measure the entrapment efficiency, drug loading and release rate of TGF-1 microsphere and the degradation of microsphere and scaffold in the fourth week; the full-thickness articular artilage defect deep to subchondral bone of 4.2mm diameter and 7 mm depth was created by drilling, then to implant ate different materials TGF-1 microspheres/ chitosan scaffold (MS-TGFs), TGF-1/chitosan scaffold (CS-TFG), pure chitosan scaffold (CS), spacious blank was constitution of the fourth group, compared to observe the recovery effect. In the first month and third month after surgery, samples were harvested, respectively. The repair in general was graded according to the criteria reported previously as Masuoka score. The harvested tissue were fixed by toluidine blue, respectively, and then assessed by expression of COL II, and comprehensive assessment of the quality of tissue repair by Wakitani score.Results:The microspheres diameters were concentrated centripetally and evenly distributed, with an average particle size of 35m, smoothly spherical surface, The encapsulated efficiency of them was 88%. The TGF-1 loading drug was llng/mg, in the first 7 days, the cumulative release amount from the microspheres was about 63.3%.The degradation of post-implantation 4 weeks was 48.5%. Four groups have no joint cavity infection, effusion; the Masuoka score Of MS-TGFs, CS-TFG, CS, free group were 7.670.47; 3.830.75; 1.000.89; 0.830.75, respectively. Harvested tissue from the first and third month, TB staining in the MS-TGFs group showed the best repair, with smooth surface, neat rows in articular cartilage, the integrity and continuous in cell structural; CS-TFG was basically repaired, but the formation is poor, less cartilage; CS was filled with fibrous cartilage; free group without repair, the defect diameter was about 5mm; the first month tissues by CD34, CD44 double staining identified the homing cells from stem cells. The CD34 (-) CD44 (+) cells in the MS-TGFs group were the largest, following CS-TFG group. The results of toluidine blue Wakitani score 4.501.12; 10.830.37; 13.670.47 (p 0.01).Conclusion:The salt-leached chitosan scaffolds can be used for various tissue-engineering applications that cartilage defects can be repaired by homing cells.【关键词】生物材料 壳聚糖 脱乙酰度 体内降解 转化生长因子 微球 缓释 生物相容性 组织工程 软骨组织工程 TGF-1 缓释率 原位再生【英文关键词】Biological materials Chitosan Deacetylation Degradation transforming growth factor microspheres chitosan sustained releasing Microspheres chitosan stent biocompatibility biological materials Article cartilage engineering microsphere transforming growth factor1 sustained release regeneration in situ【目录】TGE-_1缓释微球壳聚糖支架对兔关节软骨缺损修复的研究摘要3-6ABSTRACT6-10前言14-17参考文献15-17第1章 不同脱乙酰度壳聚糖支架制备及降解性能评价17-26材料与方法17-211.1 实验材料17-181.1.1 主要试剂171.1.2 主要设备17-181.2 实验方法18-211.2.1 壳聚糖支架的制备181.2.2 支架形态、孔隙率、溶胀率测定18-191.2.3 体外降解实验191.2.4 体内降解试验19-201.2.5 制备石蜡切片201.2.6 苏木精-伊红染色20-21结果21-222.1 扫描电镜形貌、孔隙率、溶胀率212.2 体内、外降解21-22讨论22-23结论23参考文献23-26第2章 缓释TGF-1壳聚糖微球的制备及性能检测26-33材料与方法26-291.1 实验材料26-271.1.1 主要试剂261.1.2 主要仪器26-271.2 实验方法27-291.2.1 空白微球制备、外观形态、粒径分布271.2.2 TGF-1及BSA微球制备、包封率与载药量