Experimental Study On Cs/Hacc/Gp As Temperature-Sensitive Hydrogel

Aim To synthesize a quaternary ammonium salt of chitosan,2-Hydroxypmpyltrime thylammonium chloride chitosan, HACC. To fabricate a temperature-sensitive hydrogel which have injectability, easy to handle with and suitable to cell growth in it. To identify its characteristics and evaluate the potential of this hydrogel to be used for nerve tissue engineering. To determined the porosity, degradation rate, microscopic structure, biocompatibility of the cell scaffold and measure pH of degradation liquid. To observe growth and proliferation of BMSCs in the scaffold.Methods In this work, we modified Cs with cationic etherifying agent through grafting copolymerizetion method to obtain a new derivative of Cs, HACC. The generation of quaternary ammonium group was confirmed by FTIR and1H NMR. The hydrogel was fabricated by proper riato of CS/HACC/GP mixed solution prompted by temperature. The gelation time was decided by inverting the tube. The cross-section morphology of the scaffolds was examined by scanning electron microscopy, SEM. The scaffold trimmed into block were weighed accurately and immersed in PBS (PH=7.4) buffer under37℃. The PBS buffer was changed weekly. The scaffolds samples at different time points were washed and weighed accurately. The weight loss was calculated according to the weight loss formula and the weight loss curve was drawn. The PH of degradation liquid and change of its aspect and shape was measured at set time. The porosity was calculated according to the porosity formula by substitute method of anhydrous alcohol. The biological safety of the hydrogel was evaluated through the experiments including hemolysis test, short-term systemic toxicity test and cytotoxicity test (MTT test). Then MSCs were seeded into the hydrogel. Cell adhesion and morphology of MSCs were analysed by SEM. MSCs nurtureed in a growth flask was used for control.Result The successful synthesis of HACC was confirmed by the FTIR spectrum and’H NMR spectrum which implied the presence of group of quaternary ammonium. HACC showed good solubility and more electropositive in water solutions. The osmolality of the hydrogel was range of290-310mmol/kg. Gelation time of CS/HACC/GP hydrogel was around10minutes. Macroporous internal morphology with pore size ranging from50μm to100μm was observed in CS/HACC/GP scaffold. It had a high macro porosity of greater than85percent. The time of CS/HACC/GP scaffold degradation completely was12weeks in vitro. The weight loss of the scaffold increased with time according to the degradation test. No haemolysis or agglutination observed in vitro hemolysis test. The hemolysis rate was less than5percent. The weight increasing of the rats was not influenced by extracts of the scaffolds. It showed no cytotoxic effect to MSCs in MTT test. From the SEM observation, the proliferation and metabolism of MSCs cultured on the three-dimensional hydrogel cell culture grew well but slowly than that in the control group.Conclusion In this experiment, HACC was successfully synthesized. The hydrogel have high porosity and a moderate degradation rate. The good physical properties of CS/HACC/GP hydrogel made the composite scaffold suitable for a cell scaffold in nerve tissue engineering. The hydrogel have the huge potential to be used as an optional carrier of transplanted cells for nerve tissue engineering.