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/β-glycerol phosphate (C/GP) disodium salt hydrogels to form chitosan/gelatin/β-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°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.

Coculture of human nucleus pulposus (NP) cells and mesenchymal stem cells (MSCs) using a noncontact system. To investigate the interaction between NP cells and MSCs through paracrine stimulation. Cell-based therapies have a potential role in the treatment of intervertebral disc degeneration. Upregulating the viability of NP cells and differentiating MSCs into NP-like cells are potential alternatives to achieve viable cells. Culture plates and inserts were used to coculture MSCs and NP cells without direct contact or exchange of cellular components. Cellular proliferation and RNA expression of selected genes were then evaluated after coculture. Coculturing slightly promoted the proliferation of MSCs, and expression of collagen I and Fas-associated death domain protein significantly decreased. MSCs, which initially expressed no collagen II, started to show collagen II expression after coculturing; the expression level was highest when the cells were cultured with a higher number of NP cells. On the converse, proliferation of NP cells significantly rose even after cocultured with a few MSCs. Increasing the number of cocultured MSCs did not further enhance proliferation of NP cells. Expression of aggrecan in the NP cells significantly increased when the cells were cultured with a higher number of MSCs. The results showed a possible mechanism of interaction between MSCs and NP cells mediated by secreted factors. The most significant effect on NP cells was enhancement of cellular proliferation when they were cocultured with even a small number of MSCs. To differentiate MSCs into NP-like cells with heightened collagen II expression, MSCs must be in an environment containing numerous NP cells.