Effects Of IFN-γ On The Proliferation,Migration And Odonto/Osteogenic Differentiation Of Human Dental Pulp Stem Cells And The Signal Transduction Pathways Involved

Dental pulp is the only soft tissue located in the dentin pulp cavity. Pulpitis is a typical inflammatory disease of the pulpal connective tissue, whenever dentine and pulp are affected by caries, operative procedures or trauma. The inflammatory cytokines and chemokines confluence in the injuried site accompanied by the activation of macrophages, dendritic cells and T cells. Hahn et al have indicated that there are more lymphocytes in the reversibly inflamed, or irreversibly inflamed pulpal tissue when compared to the normal pulp tissue. Others have proposed that the expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-2, IFN-γ and IL-8 were evaluated.IFN-γ is a predominant cytokine in immune responses, with both pro-inflammatory and immunoregulatory properties. A higher prevalence of IFN-γ is obtained in shallow caries when compared to IL-4 or IL-10, while in deep caries IFN-γ is present too. IFN-γ significantly up-regulated CXCL10 and IL-6 production in the human dental pulp cells stimulated with ligands for PRRs. IFN-γ improves impaired dentinogenic functions of irreversible pulpitis-derived human dental pulp stem cells. IFN-γ-treated DPSCs could even promote human mesenchymal stem cells migration in vitro. All the above evidences remind us that IFN-γ may play an indispensable role in the pulp tissue repair.DPSCs is the sole mesenchymal stem cells in the dental pulp tissue with high proliferative and differentiation potential. In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp. DPSCs has been confirmed by their ability to differentiate into neural ectodermal cells and adipocytes, odontoblasts, osteoblasts, chondrocytes, and myoblast cells. DPSCs could form mineralized nodules after being cultured with osteogenesis induced liquid for 14 days in vitro and develop dentin-like tissue in vivo.During the early stage of pulpitis or in the reversible pulpitis, DPSCs proliferate, migrate and differentiate into odontoblast, finally, form tertiary dentin and contribute to host defence. The proliferation, migration and differentiation ability of DPSCs are all indispensable for the protective response.Our present study aims to investigate the effects of IFN-γ on the proliferation, migration and odonto/osteogenic differentiation of DPSCs both in vitro and in vivo, and the signal transduction pathways involved, which may provide cues for the pulp tissue repair.The major achievements as follows:1. Isolation, culture and identification of human dental pulp stem cellsPulp tissue was gained from freshly extracted third molars from adults aging from 18-22 years old with informed consent. Tissue block and enzymatic digestion method was used to obtain primary dental pulp cells. Then we use limiting dilution method to select monoclonal cell, then being expended in vitro. The surface molecular markers were determined by flow cytometry. Afterwards, the cells were exposed to adipogenic and osteogenic induction. The surface markers were consistent with previous study and the cells successfully formed mineralized nodule and lipid droplet in vitro. The results showed that we obtained relatively purified DPSCs.2. IFN-γ promotes the proliferation of human dental pulp stem cellsThe MTT and EdU incorporation assay were applied to test the proliferation ability of DPSCs treated with IFN-γ of different concentrations. The results showed that lower concentrations of IFN-γ significantly stimulated the proliferation of DPSCs. RT-PCR was performed by determination of cell proliferation-associated markers, e.g., PCNA, Cyclin B1, Cyclin D1 and P21. The RT-PCR result showed that the proteins promoting cell cycle, PCNA, Cyclin B1 and Cyclin D1 were up-regulated, while P21 negative-regulating cell cycle progression was down-regulated. Namely, IFN-γ promotes the proliferation of human dental pulp stem cells in vitro.3. IFN-γ enhances the migration of human dental pulp stem cellsThe Transwell and wound healing assay were used to test the motility of DPSCs. DPSCs were treated with IFN-γ of different concentrations. Both results showed that IFN-γ significantly increased the cell number that migrate vigorously. Then we conclude that IFN-γ enhances the migration of DPSCs.4. IFN-γ inhibits the odonto/osteogenic differentiation of h DPSCs through both NF-κB and MAPK signal transduction pathwaysThe alizarin red staining and western blot assay were introduced to evaluate the odonto/osteogenic differentiation ability of human DPSCs. Both results indicated that IFN-γ inhibits the odonto/osteogenic differentiation of human DPSCs, probably, trough both MAPK/P38 and NF-κB signal transduction pathways.The transplantation of DPSCs/ NF-gelatin scaffolds construct were designed to investigated the odonto/osteogenic differentiation ability of human DPSCs in vivo. Micro-CT and histological analysis results showed that IFN-γ inhibited the odonto/osteogenic differentiation of human DPSCs, and the inhibitors of MAPK/P38 or NF-κB signal transduction pathway reversed the inhibitory effect.In conclusion, we investigated the effects of IFN-γ on the proliferation, migration and odonto/osteogenic differentiation ability of DPSCs both in vitro and in vivo, and the signal transduction pathways involved.