论文标题:透明质酸化学交联修饰及其衍生物的体外降解研究
Study on Chemical Cross-linking Modification of Hyaluronan and the in Vitro Degradation of Its Derivatives
论文作者 胡帼颖
论文导师 顾汉卿,论文学位 硕士,论文专业 生物医学工程
论文单位 天津医科大学,点击次数 4,论文页数 102页File Size5570k
2004-05-01论文免费下载 http://paper.dic123.com/lunwen_106364522/ 透明质酸;糖胺聚糖;粘度法测量分子量;交联;二乙烯基砜;双环氧化物;戊二醛;透明质酸四丁基铵盐;体外降解
hyaluronic acid (hyaluronan); glycosaminoglycans; testing molecular weight by viscosity method; cross-linking; vinyl sulfone; diepoxy compound; glutaraldehyde; tetrabutylammonium salt of hyaluronan; in vitro degradation
天然细胞外基质(ECM)中广泛存在的天然高分子具有良好的生物相容性和生物降解性，有望成为代表组织工程与再生医学优点的支架材料。在这些天然高分子材料中，透明质酸(HA)作为滑液和ECM中一种丰富的糖胺聚糖(GAG)成分，扮演着多种生理角色，其制备的医疗用品也具有很多独特的优点。然而，由于天然HA生物机械性能缺乏，必须经交联、酯化等化学修饰作用提高其化学和机械性能，才能作为生物材料广泛使用。本研究旨在探明HA羟基交联及其衍生物的体外降解规律，并为今后进一步开发HA交联及酯化衍生物提供科学基础和实验依据。 1．粘度法测量HA分子量(Mr_(HA))： 采用粘度法测量Mr_(HA)，使用多点法测得本研究使用的HA样品Mr_(HA)=2.547±0.135 MDa，并将多点法与一点法的测量值进行配对t检验，得到使用一点法测量Mr_(HA)及交联衍生物分子量(Mr)的最适HA溶液浓度为1.25×10~(-1)g／L，可用于测量后续实验中交联HA流体的Mr。 2．HA羟基交联衍生物的制备： 使用二乙烯基砜(DVS)在碱性介质中以不同HA／DVS配比、不同温度及不同NaOH溶液浓度交联HA，分别得到性能变化很大的HA-DVS衍生物，包括HA-DVS凝胶、软凝胶和流体。使用1，3-双环氧丁二烯、1，2，7，8-双环氧辛二烯和双环氧二聚环戊二烯三种双环氧化合物(DEC)为交联剂，在碱性介质中改变碱的浓度以及DEC的种类，得到HA-DEC流体；并采用戊二醛(GTA)为交联剂，在酸性介质中使用丙酮作催化剂，改变丙酮的比例及酸性介质的浓度，得到HA-GTA流体。 通过测量HA-DVS凝胶、软凝胶的浸液粘度及三种交联剂制得交联HA流体的Mr，发现DVS、DEC及GTA这三种双功能分子均能使HA发生羟基交联，并得到以下规律：1) 交联剂加量增大，可缩短交联反应时间，增大HA的交联度，提高交联HA衍生物机械强度；2) 反应温度升高，可缩短交联反应时间，但在相当大的范围内不改变HA的交联度，温度过低或过天津医科大学硕士学位论文摘要高会使HA交联衍生物机械强度降低;3)碱性介质对DvS和DEC交联HA的催化作用和对HA的降解作用相互竞争，酸性介质对GTA交联HA的催化作用和对HA的降解作用相互竞争，介质浓度过高或过低均会使交联度降低，介质参与交联反应;4)丙酮可催化GTA对HA的交联;5)DVS的交联效果明显优于DEC和GTA，本研究采用的三种DEC和GTA的交联能力差异不大。 3.HA及其经基交联衍生物的降解: 将 HA依1 .25 xlo一19/L溶解于不同浓度酸、碱性介质中，发现1h内HA降解迅速，Mr。显著降低，但延长降解时间不导致MrHA的显著下降。HA在蒸馏水中24h即可溶解，溶解48h后MrHA未显著下降，可认为蒸馏水对HA的降解作用很弱。对HA一DVS凝胶密封保存，数月后部分凝胶形态改变为软凝胶或流体，将软凝胶或流体在蒸馏水中浸泡48h，测量流体溶解后的M:，发现凝胶在贮存条件下发生降解，但流体的Mr仍明显大于Mr。。HA一DEC及HA一GTA流体在一定交联反应时间后在蒸馏水中溶解48h，测其Mr均比HrRA显著增大，但随交联反应时间延长，在不同浓度反应介质中，流体的Mr或下降或波动，说明反应过程中交联和降解同时发生。 4.狱四丁基按盐[(e.H，)‘N一HA]的制备: 为进一步开发狱的交联及醋化衍生物，本课题探索了一种以HA和四丁基氯化钱[(c，H，)。Ncll为原料，利用很普及的磺酸基阳离子树脂进行二次离子交换，再将洗脱液冻干得到(C.Hg)。N一HA的方法，并比较了固、液态两种不同的(C。H，).Ncl的制备效果及固态(c’H，)。Ncl在离子交换柱中及在烧杯中制备(C’H，)‘N一HA的制备效果。红外光谱检测发现三种方法均可制得(C‘H，)‘N一HA。
Hyaluronan (HA), an abundant glycosaminoglycan (GAG) component of synovial fluid and extracellular matrices (ECM), has wonderful biocompatibility and biodegradability, plays many important physiological roles, and its medical products possesses many distinctive merits. However, in order to be widely used, purified HA needs chemical modification, such as cross-linking and esterification, to improve its chemical and biomechanical properties. This study aims at exploring the regularity of hydroxyl cross-linking modification of HA and that of degradation of its derivatives, as well as providing experimental support for the future study of cross-liking and esterification of HA.1. Testing molecular weight of HA (Mrha) by viscosity methodMrha tested by Multi-point Method is 2.547 +0.135 MDa, and is compared with Mrha tested by Single-point Method by paired t-test. The appropriate concentration of HA solution for Single-point Method is 1.25 x 10-1g/L, which can be applied to test Mr of cross-linked HA fluid in our successive experiments.2. Preparation of hydroxyl cross-linked HA derivativesIn alkaline media (NaOH solution), DVS is used as cross-linking agent to prepare HA-DVS gels, soft gels and fluid with different ratio of HA/DVS, temperature, and concentration of NaOH solution. Also in alkaline media, 3 types of diepoxy compound (DEC), 1,3 butadiene diepoxide (C4H6O2), 1,2,7,8-diepoxyoctane (C8H14O2) and dicyclopentadiene dioxide (C10H12O2), are used seperately as cross-linking agent, to prepare HA-DEC fluid with different concentration of NaOH solution. In acidic media (HC1 solution), glutaraldehyde (GTA) is used as cross-linking agent and acetone is used as catalyst, to prepare HA-GTA fluid with different concentration of acetone andthat of HCl solution.By testing the viscosity of steeps of cross-linked HA gels and soft gels, as well as Mr of cross-linked HA fluid, we confirm that DVS, DEC and GTA have cross-linked hydroxyls of HA.1) Reducing the ratio of HA/DVS can shorten the time of gelating, improve cross-linking degree and enhance mechanical stability of HA derivatives.2) Improving cross-linking temperature can reduce time of gelating, but influence cross-linking degree little within a wide range of temperature; high and low temperature may weaken the mechanical stability of HA derivatives.3) Both alkaline media in cross-linking HA by DVS or DEC and acidic media in cross-linking HA by GTA compete with degradation caused by the media. Such competition may reduce cross-linking degree when they are excessively high or low. The media take part in cross-linking.4) Acetone catalyzes the reaction of cross-linking HA by GTA.5) The cross-linking effect of DVS is much better than those of DEC and GTA, which have little difference from each other.3. Degradation of HA and its hydroxyl cross-linking derivatives Dissolving in different concentration of acidic and alkaline media as 1.25 x 10-1g/L, HA can be degraded within 1h, and MrHa changes little after 1h. HA can be solved within 24h in distillated water, but little reduction of MrHA after 24h demonstrates the little degradation ability of distillated water to HA. Sealed up and preserved for several months, some of HA-DVS gels have degraded into soft gels or fluids, but Mr of the fluids is significantly higher than MrHA HA-DEC and HA-GTA fluids dissolved in distillated water in 48h after a certain period of cross-linking, their Mr has significantly improved compared with MrHA With the time of cross-linking prolonging, Mr of thefluids reduces or fluctuates in different concentration of reaction media, demonstrating the coincidence of cross-linking and degradation.4. Preparation of tetrabutylammonium salt of HA [(C4H9)4N-HA] In order to further developing cross-linked and esterified HA, we have prepared (C4H9)4N-HA by (C4H9)4NCl and sulfonic positive ion-exchange resin in three methods and compared their effect. FT-IR spectra shows that all the three methods can successfully prepare
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