| Objective: The aim was to prepare and characterize the injectable a FGF/Sulfonated chitosan oligosaccharide/thermo-sensitive hydrogel, and also establish its quality standard system to determine the contents of a FGF. Furthermore, the models of in vitro hypoxia-induced PC12 nerve cell injury and rat sciatic nerve injury were simultaneously tested to investigate the repair effects on the injured nerve. Moreover, acute toxicity test and biocompatibility test were preliminary conducted on its safety evaluation. These results would provide an experimental basis for the application of a FGF/Sulfonated chitosan oligosaccharide /thermo-sensitive hydrogel.Methods:(1) Chlorine sulfonic acid was used as sulfonated reagent to modify the precise fixed-point of chitosan oligosaccharide.(2) Poloxamer was selected as temperature sensitive matrix to prepare a FGF/Sulfonated chitosan oligosaccharide/thermo-sensitive hydrogel. Scanning electron microscope was employed to investigate its homogenous microstructure.(3) A double antibody sandwich enzyme-linked immunosorbent assay(ELISA) was developed to determine the content of a FGF. Besides, a 3T3 cell MTT method was established to test the bioactivity of a FGF in thermo-sensitive hydrogel.(4) A hypoxia injury model of PC12 nerve cells was developed and induced by Na2S2O4. And then, the protection of the thermo-sensitive a FGF hydrogel was tested.(5) The paw withdrawal threshold to mechanical stimulation and thermal withdrawal latency of rats with injured sciatic nerve were measured to study the repair function on the transected sciatic nerve.(6) A series of preliminary assessment, including mice acute toxicity test, blood biocompatibility test, mice tissue compatibility test by subcutaneous injection, were all conducted to examine the safety of the thermo-sensitive a FGF hydrogel.Results:(1) Sulfonated chitosan oligosaccharide was successfully prepared and its structure was confirmed by infrared spectrum, high resolution mass spectrometry, and nuclear magnetic resonance data.(2) Poloxamer was compatibly used as thermo-sensitive material to prepare the injectable a FGF/Sulfonated chitosan oligosaccharide/thermo-sensitive hydrogel.(3) A rapid, sensitive, stable and reliable ELISA method was established to analyze the content of a FGF in samples. The sensitivity could reach to 12 pg/m L and the bioactivity was tested by MTT method.(4) The prepared thermo-sensitive a FGF hydrogel had excellent stability during three months.(5) The viability of PC12 cells increased, and the LDH level of PC12 cells decreased after treated with a FGF solution and sulfonated chitosan oligosaccharide, respectively.(6) In the rat sciatic nerve injury experiment, after treated with different concentration of thermo-sensitive a FGF hydrogel, the paw withdrawal threshold to mechanical stimulation and thermal withdrawal latency obviously reduced, which indicated that the drug could repair the rats injured sciatic nerve.(7) During acute toxicity test, the general behavior and growth of mice were all normal, no abnormal behavior and deaths were observed. In the biocompatibility tests, the general behaviors of mice in treatment group were all normal. The phenomenon of irritability, anorexia, weight loss and so on did not appear. The surface of skin mounds in injection site was well-balanced and did not appear redness and ulcers.Conclusion: In this study, the precise fixed-point modification of chitosan oligosaccharide was performed based on the reported reference. And also, a quality evaluation standard based on ELISA method was developed to determine the content of prepared injectable a FGF/Sulfonated chitosan oligosaccharide/thermo-sensitive hydrogel. Furthermore, the in vivo and in vitro pharmacodynamic results indicated that a FGF/Sulfonated chitosan oligosaccharide /thermo-sensitive hydrogel could protect the injured PC12 cells induced by Na2S2O4 and repair the sciatic nerve injury of rats. Preliminary safety evaluation exhibited that a FGF/Sulfonated chitosan oligosaccharide /thermo-sensitive hydrogel was safe, and also it did not induce hemolytic reaction and had good biocompatibility. |
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