| 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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