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It has been reported that chitosan scaffolds, due to their physicochemical properties, stimulate cell proliferation in different tissues of the human body. This study aimed to determine the physicochemical, mechanical, and biological properties of chitosan scaffolds crosslinked with ammonium hydroxide, with different pH values, to better understand cell behavior depending on the pH of the biomaterial. Scaffolds were either neutralized with sodium hydroxide solution, washed with distilled water until reaching a neutral pH, or kept at alkaline pH. Physicochemical characterization included scanning electron microscopy (SEM), elemental composition (EDX), Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, thermogravimetric analysis (TGA), and mechanical testing. In vitro cytotoxicity was assessed via dental-pulp stem cells’ (DPSCs’) biocompatibility. The results revealed that the neutralized scaffolds exhibited better cell proliferation and morphology. It was concluded that the chitosan scaffolds’ high pH (due to residual ammonium hydroxide) decreases DPSCs’ cell viability.