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The purpose of the present study was to provide insights into the interactive effects of β-glycerophosphate (β-GP) molecules on thermosensitive chitosan-based hydrogels, which experience sol–gel transformation around the physiological temperature. This study also investigated the influence of β-GP as a physical cross-linker and pH modifier on chitosan-based hydrogels by Fourier transform infrared spectroscopy with the aim of achieving a better understanding of the effect of β-GP action mechanism. For this purpose, some solutions with 2% (w/v) chitosan were mixed with different amounts of β-GP in the range of 0–20% (w/v) concentrations. It was shown that by further addition of β-GP, the anionic phosphate units of β-GP in combination with the cationic amine units of chitosan could provide electrostatic cross-linking for making chitosan-based hydrogels. All the β-GP–chitosan solutions remained at the physiological pH after complete mixing. In addition, the viability of 3T3 fibroblast cells determined by MTT assay and scanning suggested that the samples containing 12% β-GP would show optimum biological characteristics. This study demonstrated that the synthesized chitosan–β-GP is a biocompatible thermosensitive system, which can be potentially used for tissue engineering applications in different areas, such as cartilage injections, and that injectability and appropriate gelation behavior are important factors.

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