In this article, properties of thermosensitive chitosan hydrogels prepared with the use of chitosan chloride with β-glycerophosphate disodium salt pentahydrate enriched with calcium glycerophosphate are presented and compared with chitosan hydrogels with β-glycerophosphate disodium salt pentahydrate. The study is focused on the determination of hydrogel structure and biological testing of hydrogels with human osteoblasts line Saos-2. The structure of gels was visualized by scanning electron microscopy and was investigated by infrared spectroscopy. The crystallinity of gel structure was determined by X-ray diffraction analysis and thermal effects were determined using differential scanning calorimetry thermograms.