生物材料：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-β_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-β,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-β1(TGF-β1)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-β1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-β1及牛血清白蛋白(BSA),分别制备TGF-β1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35μm,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-β1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-β1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200-350μm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,x±s)；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0.3467±0.1075,0.4020±0.0796,0.4932±0.0838,各时间点与生理盐水组组间配对t检验P>0.05；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0.17±0.06；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-β1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-β1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-β1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-β1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,Ⅱ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7.67±0.47；3.83±0.75；1.00±0.89；0.83±0.75。

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Ⅱ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：4.50±1.12：10.83±0.37；13.67±0.47,有明显统计学差异(P<0.01)结论：壳聚糖复合支架在一定程度上可以修复非负重区关节软骨缺损,可促进细胞归巢,原诱导软骨细胞分化,修复软骨缺损英文摘要】PartⅠPreparation and evaluation of the characteristics of different deacetylated degree of chitosan scaffoldTo evaluate the effect of different deacetylated degree of chitosan scaffold, it was using the SEM to observe the morphology and the rate of porosity, evaluate the swelling of water absorption and degradation in vitro and in vivo test. The results showed that the different deacetylation of scaffold was highly porous and three-dimensional structure; with increasing of the deacetylated degree, theircorresponding porosity was 93.6%,90.0%、85.1%; the rate of swelling was 820%,803%,772%; On the fourth week in vitro, the degraded rates were 30.44%, 22.88%,17.10%; while, in vivo the corresponding rate were 57.48%,40.23%, 29.53%.The degraded rate of chitosan scaffold were negatively correlated to deacetylated degree, furthermore, it showed that the speed of degradation in vivo was faster than the level in vitro. By controlling deacetylated degree of chitosan (almost 80%). it would be a booming and suitable material for the reparation of cartilage defects.PartⅡThe preparation and detection of chitosan loaded with TGF-β1microspheres:Using emulsified cross-linking methods to prepare the chitosan loaded with transforming growth factor-β1(TGF-β1) microspheres, it detected the characteristics of swelling ratio, loading drug and TGF-β1.releasing amount of chitosan, and further to assesse the feasibility of using biodegradable chitosan scaffold as a carrier for controlled release of TGF-β1.Methods:to take the Sorbitol Oleate Benzene-80 as the emulsifier and sodium tripolyphosphate (TPP) as cross-linking agent, it used emulsified cross-linked methods to prepare the chitosan microspheres. It was embedded TGF-β1-releasing or bovine serum albumin (BSA) microspheres. The surface of the specimens of chitosan were detected by means of Scanning Tunneling Microscopy, the microsphere diameter measurement by using the laser particle distribution method; The microsphere swelling rate, drug loading amount, entrapment efficiency and sustained releasing rate by vitro assay.Results:it found that the microspheres diameters were concentrated centripetally and evenly distributed, with an average particle size of 35μm, smoothly spherical surface; the swelling rate in the acidic environment was the highest, which was up to 800%; the encapsulated efficiency of them was 88%. the TGF-β1 loading amout was 11ng/mg, the cumulative releasing amount 。