| Hyaluronan(HA) is a linear macromolecule polysaccharide naturally existing in the body of humanbeings and animals, but in the presence of sodium salt- sodium hyaluronan(SH) in vitro. HA prossesses favorable viscoelasticity, biocompatibility and hygroscopicity, which widely used in cosmetics, foodstuff and medical fields, such as ophthalmology, orthopaedics and general surgery, and now the research and application as materials for tissue engineering gradually arouse people’s interest. Nevertheless, the natural HA is easily degraded in vivo and has a short retention time, which seriously affect the extension of application range. In order to enlarge its degradation period and enhance its mechanical properties, remodeling HA to its derivatives are traditional methods. The derivatives have increased molecular weight, enhanced viscoelasticity after remodeling, however it is hard for injection and clinical application. What is more, most materials used to prepare the derivatives are chemical reagents which have certain toxicity and can bring no telling harm to patients. Polyethylene glycol(PEG) which has widely used in medical fields, and genipin which coming from plant are elected as crosslinking agents in this research to prepare crosslinked sodium hyaluronan(CSH) and the preparation of granulated PEG-CSH, the enzyme-resistant degradation properties of the gels and its biological safety were studied.The effect of ionic concentration of phosphate buffer solution, reaction time, reaction temperature on PEG-CSH gels were investigated by single factor experiments. And the results of single factor experiments were screened and optimized by orthogonal test. The results of optimum preparation conditions of PEG-CSH gel were: the ionic concentration of phosphate buffer solution was 0.1 mol·L-1, reaction time was 10 h, reaction temperature was 60℃, and the enzyme-resistant degradation of the PEG-CSH gel was 88.71%.Taking the expansion rate as the index, the concentration of genipin, the molecular weight of SH, reaction time and temperature were investigated, and the results were optimized by orthogonal test. Finally, the optimum preparation conditions of genipin-CSH gel were: the concentration of genipin was 0.2%, the molecular weight of SH was 2.60×106 Da,the reaction time and temperature were 48 h, 60℃, and the expansion rate of genipin-CSH gel was 6.53. Granulated PEG-CSH gel was obtained after a series of technology and the enzyme-resistant degradation property of the gel was studied by carbazole colorimetric method. The optimum conditions of preparation technology were: the amount of 95% ethanol was 6.5 times of gel, drying the precipitation for 5.0 h at 50.0℃. The enzyme-resistant degradation ratio of the granulated PEG-CSH gel was 80.53%.According to the Medical biological evaluation standard(GB/T16886), the biological safety of PEG-CSH and granulated PEG-CSH gels were evaluated by animals skin sensitization test and intracutaneous stimulation test. Daubing the new type of gels and same amount of normal saline on the skin of mice to evaluate the effect of gels on animal sensitization condition, and injecting the new type of gels and same amount of normal saline under the skin of mice to evaluate the effects of gels on animal intracutaneous stimulation. The effect of daubing or injecting new gels on the animals’ growth were investigated by the change of feed intake and weight increment of the mice on the experiment stage. Observing the systemic and local reaction, behavioral responses of mice to evaluate the effect of daubing or injecting new gels on the reaction of mice. The results of animal testing were the new gels had no sensitization or stimulation effect.In this research, the optimized preparation technology and condition of new types of crosslinked sodium hyaluronan and granulated PEG-CSH gels lay a foundation for further research on the preparation of new types crosslinked SH and granulated gels, and provide scientific and experimental basis for the research of SH derivatives for biomaterials. |
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