Thermosensitive Chitosan-Gelatin-Glycerol Phosphate Hydrogels as a Cell Carrier for Nucleus Pulposus Regeneration: An In Vitro Study

Injectable hydrogel is one of the great interests for tissue engineering and cell encapsulation. In the study, the gelatin molecules were added to the thermosensitive chitosan/beta-glycerol phosphate (C/GP) disodium salt hydrogels to form chitosan/gelatin/beta-glycerol phosphate (C/G/GP) disodium salt hydrogels which were applied as a cell carrier for nucleus pulposus (NP) regeneration. The gelation temperature, gelation time, and gel strength of the C/G/GP hydrogels were analyzed by the rheometer. NP cells were then harvested from the intervertebral discs of the adult New Zealand white rabbits and cultured in monolayer or in C/G/GP hydrogel, respectively. The cell viability, material-mediated cytotoxicity, cell proliferation, production of sulfated glycosaminoglycans, anabolic/catabolic gene expressions, and extracellular matrix-related gene expressions of the NP cells were demonstrated. The results show that the sol/gel transition temperature of the C/G/GP hydrogel was in the range of 31.1-33.8 degrees C at neutral pH value, the gelation time was shortened, and the gel strength also improved at body temperature when compared with the C/GP hydrogel. Among those, C/GP with 1% gelatin addition showed the most promising gelation time and gel strength and were utilized in the later experiments. From the results of cell activity, cytotoxicity, and cell proliferation assays, NP cells cultured in C/G/GP hydrogel had normal cell viability and cell proliferation that indicated the hydrogel was noncytotoxicity. The amounts of sulfated glycosaminoglycans of NP cells cultured in C/G/GP hydrogels were significantly higher than monolayer cultured. Considering the extracellular matrix-related gene expression, type II collagen and aggrecan of NP cells cultured in the hydrogels greatly increased than those in monolayer culture. On the contrary, the unfavorable gene expression, such as that of type I collagen, was decreased significantly. The results reveal that gelatin added into C/GP hydrogel significantly shortened the gelation time and improved the gel strength without influencing the biocompatibility. NP cells cultured in the C/G/GP hydrogel also displayed better gene expressions when compared with the monolayer culture. This study indicates that using chitosan/gelatin hydrogel for NP cell culture is feasible and may apply in minimal invasive intervertebral disc surgery in the future.