| Chitosan is a unique polysaccharide derived from chitin by partial deacetylation with alkali. Chitin and chitosan are recommended as suitable functional materials, for their excellent biological properties such as non-toxicity, biodegradation, immunological, antibacterial and wound-healing activities. Chitosan thermosensitive hydrogels prepared with different methods are of great interest in drug delivery, cell encapsulation, tissue engineering and so on. Chitosan/glycerophosphate (CS/GP) hydrogel, a novel thermosensitive hydrogel, is an important system due to its sol-gel transition at body temperature. The system remained to be solution at physiological pH (7.0) and room temperature, but changed into a gel upon heating at physiological temperature 37°C. CS/GP hydrogel has recently been proposed as a suitable vehicle for the extravascular parenteral administration of drugs, tissue engineering applications, cell culture, nerve regeneration and so on.In this work, quaternary ammonium salt of chitosan (H T C C) is synthesized in the heterogeneous system and its parameters such as water solubility, pH-dependence, viscosity, and degree of quaternization DQ are tested. An in situ injectable thermosensitive hydrogel constitutes of CS, H T C C withα,β-glycerophosphate (α,β-GP) was designed and synthesized without any chemical stimulation for its formulation. The influence of organic and inorganic acids on chitosan/glycerophosphate (CS/GP) hydrogel has been investigated by dissolving chitosan in different acids. In addition, CS/GP hydrogel films were prepared and evaluated.The main contents and conclusions are as follows:HTCC was prepared by reacting CS with glycidyltrimethylammonium chloride (GTMAC), had good water solubility, high stability, and weak dependence in pH. It can be preserved for a long time with good stability, while its quality is not so stable in HAc solution.Added HTCC into CS/GP solution, the sol-gel transformation time can be shortened. When the proportion of CS: HTCC=5: 1, gelation time declined to 3 min. But continue to increase the content of H T C C, CS- H T C C /GP failed in gelation. Under the low temperature conditions, CS- H T C C /GP solutions were transparent flowing liquid, but when the temperature became higher than 25°C, the CS- H T C C /GP solution can transform into ivory-white hydrogel. Under the 25°C condition, CS-HTCC/GP solution takes a long time (20 min) to gel. When the temperature increased to 40°C and above, the gelation became rapid. When the temperature in the range of normal body temperature (35-37°C), it needs 3-5 min to gel. High glycerophosphate concentration can shorten the gelation time. SEM results showed that, CS/GP hydrogel and CS-HTCC/GP hydrogels are porous structure in the interior of hydrogel, HTCC made the aperture larger.Chitosan was soluble in all monovalent acids of wide concentration(0.05-0.15 mol/L) but a part of multivalent acids. Chitosan/glycerophosphate solutions prepared with monovalent acids all could transform into hydrogel within 2–5 min at 37°C, while the multivalent acid solutions failed to gel. The factors that promote gelation including: higher chitosan and GP concentration, low acid concentration. All the nine hydrogels prepared with monovalent acids were injectable viscous liquid below body temperature, and they would transform into soft and transparent non-flowing gel at 37°C. The SEM micrographs clearly illustrated the dependence of hydrogel morphology on the type of acids. Network-like structures and lots of ramified configuration were formed in CS/GP hydrogels and the pores were connective each other. The viscosity of the CS/GP system depended on the acid which dissolved chitosan. Hydrogels prepared with carboxylic acids showed higher viscosity than inorganic acids. The viability of MEFs and Hela cells after incubation with the extracts of CS/GP hydrogels was determined with the MTT assay. The results showed that the CS/GP hydrogels prepared in this paper were biocompatible with low cell cytotoxicity except that prepared with chloroacetic.Compared with pure chitosan films, the CS/GP hydrogel films prepared by solvent evaporation method were ivory-white in appearance, more flexible, still integrity after swelling. And the pure chitosan films were curly and friable after swelling. Scanning electron microscope (S E M) microphotographs of the surfaces and cross-section of different films showed that the surfaces of pure chitosan films were relatively flat, homogeneous, and smooth. The cross-section of the samples was dense, non-porous.However, the surface of chitosan/GP hydrogel films were roughness, and the cross-section showed porous network microstructure. The chitosan/GP hydrogel films showed higher hydrophilicity than the films prepared with pure chitosan. Compared with the pure chitosan films, CS/GP hydrogel films exhibited lower tensile strength, the elongation property of CS films is lower than that of CS/GP hydrogel films. The CS/GP hydrogel films adsorbed more proteins than the chitosan films both for BSA and LYZ. From the results of MTT assay and cell morphology evaluation, it could be concluded that the CS/GP hydrogel films had good biocompatibility. The cells remained relatively flat and well spread in the light microscope. The cells attached and spread on the hydrogel film surfaces without apparent impairment of cell morphology. And the network of the dried hydrogel films could partly absorb free water in culture medium, thus allowing diffusion of oxygen, nutrients, and waste throughout the scaffold. In this way, the GP/CS hydrogel films are ideal for cell culture.
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