Recently, the modification of the initial structure of biopolymers, mainly chitosan, has been gaining importance with a view to obtain functional forms with increased practicality and specific properties enabling their use in tissue engineering. Therefore, in this article, the properties (structural and biological) of thermosensitive hydrogels obtained from chitosan lactate/chloride and two types of crosslinking agents (β-glycerol phosphate disodium salt pentahydrate and uridine 5′-monophosphate disodium salt) are discussed. The aim of the research is to identify changes in the structure of the biomaterials during conditioning in water. Structural investigations were carried out by FTIR spectroscopy. The crystallinity of gels was determined by X-ray diffraction analysis. The biocompatibility (evaluation of cytotoxicity and genotoxicity) of chitosan hydrogels was investigated by contact with human colon adenocarcinoma cell line for 48 h. The cytotoxicity was verified based on the colorimetric resazurin assay, and the genotoxicity was checked by the comet assay (percentage of DNA in the comet tail). The conducted research showed that the analyzed types of chitosan hydrogels are non-cytotoxic and non-genotoxic materials. The good biocompatibility of chitosan hydrogels surfaces makes them interesting scaffolds with clinical potential in tissue regeneration engineering.

Thermosensitive injectable hydrogels based on chitosan neutralized with sodium beta-glycerophosphate (Na-β-GP) have been studied as biomaterials for drug delivery and tissue regeneration. Magnesium (Mg) has been reported to stimulate adhesion and proliferation of bone forming cells. With the aim of improving the suitability of the aforementioned chitosan hydrogels as materials for bone regeneration, Mg was incorporated by partial substitution of Na-β-GP with magnesium glycerophosphate (Mg-GP). Chitosan/Na-β-GP and chitosan/Na-β-GP/Mg-GP hydrogels were also loaded with the enzyme alkaline phosphatase (ALP) which induces hydrogel mineralization. Hydrogels were characterized physicochemically with respect to mineralizability and gelation kinetics, and biologically with respect to cytocompatibility and cell adhesion. Substitution of Na-β-GP with Mg-GP did not negatively influence mineralizability. Cell biological testing showed that both chitosan/Na-β-GP and chitosan/Na-β-GP/Mg-GP hydrogels were cytocompatible towards MG63 osteoblast-like cells. Hence, chitosan/Na-β-GP/Mg-GP hydrogels can be used as an alternative to chitosan/Na-β-GP hydrogels for bone regeneration applications. However the incorporation of Mg in the hydrogels during hydrogel formation did not bring any appreciable physicochemical or biological benefit.

Chitosan colloidal systems, created by dispersing in aqueous solutions of hydrochloric acid, with and without the addition of disodium β-glycerophosphate (β-NaGP), were prepared for the investigation of forming mechanisms of chitosan hydrogels. Three types of chitosan were used in varying molecular weights. The impacts of the charge and shape of the macromolecules on the phase transition process were assessed. The chitosan system without the addition of β-NaGP was characterized by stiff and entangled molecules, in contrast to the chitosan system with the addition of β-NaGP, wherein the molecules adopt a more flexible and disentangled form. Differences in molecules shapes were confirmed using the Zeta potential and thixotropy experiments. The chitosan system without β-NaGP revealed a rapid nature of phase transition—consistent with diffusion-limited aggregation (DLA). The chitosan system with β-NaGP revealed a two-step nature of phase transition, wherein the first step was consistent with reaction-limited aggregation (RLA), while the second step complied with diffusion-limited aggregation (DLA).

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.