The Effect Of PDTC On The Odontogenic/osteogenic Differentiation Of Human Dental Pulp Stem Cells In Vitro/Viro

Dental caries, classified by WHO, is one of the three contemporarynoncommunicable diseases which harm to human health. Dental pulp tissue is often infected or inflamed because of the bacteria in dental caries. In the circumstances, the human dental pulp stem cells(h DPSCs), which could differentiate into odontoblast cells, osteoblast and nerve cells, will migrate to the injured portion and differentiate into odontoblast-like cells(OBLCs) in order to avoid further damage of pulp tissues.Researchers have showed that, NF-κB signaling pathway plays an important role in the regulation of differentiation of stem cells. Such as under the effect of TNF and IL-17, IKK-NF-κB signaling pathway was activated and promoted the degradation of beta-catenin in order to promote the osteogenic differentiation of bone marrow mesenchymal stem cells[1].In terms of h DPSCs, our previous studies have proved that LPS can induce the expression of IL-8 in h DPSCs through TLR4/My D88/NF-κB signaling pathways. PDTC as the NF-κB signaling pathway inhibitor, can regulate the differentiation of stem cells when inhibiting the NF-κB pathway[2].The pyrrolidine dithiocarbamate(PDTC) is a kind of stable compound which always be used as the NF-κB inhibitor. The researchers have demonstrated that, the PDTC play a vital role in the differentiation of stem cells. After the stimulation of LPS on the rat bone marrow mesenchymal stem cells in vitro, anti-inflammatory factor IL-10 and TGF-beta 3 were increased by RT-PCR detection. And the NF-κB was involved in this regulation. PDTC could inhibit the expression of anti-inflammatory factors, suggesting that PDTC was involved in the regulation of inflammatory immune cells[3]. PDTC could inhibit NF-κB pathway, which could down regulate Smad signaling pathway, and promote the bone marrow mesenchymal stem cells osteogenic differentiation of patients with systemic lupus erythematosus[4].While PDTC restrain the mesenchymal stem cells, derived from adipose, osteogenic differentiation by inhibiting NF-κB signaling pathway[5]. However, there is no report about the effect of the PDTC on the odontoblastic/osteoblastic differentiation of h DPSCs. In this study, we discuss the effect of PDTC on the odontogenic/osteogenic differentiation of the h DPSCs in vitro/viro in order to explore the function of the NF-κB inhibitor PDTC in the differentiation of h DPSCs, which also have profound significance in illuminating the molecular mechanism of the differentiation of the h DPSCs and to provide new ideas for the future clinical application of treatment of vital pulp conservation project. What we have done are as follows: 1. Isolation, culture and identification of h DPSCsBy using enzymatic digestion of the pulp tissue, the primary h DPSCs have been successfully cultured. Then limiting dilution method was used to get monoclonal cells, and expand culture to obtain relative homogeneous h DPSCs. For the purpose of detecting the biological characteristics of h DPSCs, the flow cytometry was used to make an phenotype analysis. The osteogenic and adipogenic differentiation characteristics of h DPSCs were confirmed by osteogenic and adipogenic induction for two and three weeks respectively. The experiment results showed that the h DPSCs lines were successfully established. 2. Effects of PDTC on the proliferation of h DPSCs.MTT assay was used to detect the effect of different concentrations(2-200 μmol/L) of PDTC on h DPSCs at different time points. The result showed 200 μmol/L PDTC significantly inhibit the cell proliferation and had toxic effect on h DPSCs, while the 2 μmol/L, 20 μmol/L PDTC inhibit the cell proliferation but have no harm effect on the h DPSCs compared with the control group. And there is no significant difference between the 2 μmol/L and 20 μmol/L PDTC. 3. Effects of PDTC on the differentiation of h DPSCs in 2D and 3D cultureenvironment in vitro.First of all, h DPSCs were incubated in mineralizing medium with different concentrations of PDTC in 2D culture environment for two weeks. Alizarin red staining and quantitative results showed that, compared with the control group, the quantity of mineralized nodules of 20 μmol/L PDTC was significantly higher than the control group, suggesting that PDTC may promote the odontogenic/osteogenic differentiation of h DPSCs.And then, h DPSCs were incubated in mineralizing medium with different concentrations of PDTC in 3D culture environment for two weeks.The effect of the complex on the proliferation of h DPSCs were determined by MTT. The results showed that the material was non-toxic and PDTC inhibit the proliferation activity of h DPSCs. And then, after 2 weeks mineralization induction, the alizarin red staining and quantitative results, compared with the control group, showed that PDTC stimulation group promoted the mineralization of h DPSC and 20 μmol/L PDTC has a stronger effect.4. In vivo subcutaneous implantation to explore the effect of PDTC on thedifferentiation of h DPSCsIn this experiment the pretreatment with 20 μmol/L PDTC induced mineralization of h DPSCs were seeded on 3D scaffold-nanofibros-gelatin and retrieved after 6 weeks of subcutaneous implantation in the nude mice. The specimens were fixed, dehydrated, embedded and sliced. The staining results showed that 20 μmol/L PDTC promoted the h DPSCs odontogenic/osteogenic differentiation by HE, Masson and Von Kossa stain the same as the analysis results of the X ray and Micro-CT. 5. Mechanism of the effect of PDTC on the differentiation of h DPSCsIn order to explain the mechanism of PDTC regulated odontogenic/osteogenic differentiation of h DPSCs, alizarin red staining and Western blot were used to investigate. h DPSCs were incubated in mineralizing medium containing 20 μmol/L PDTC with different inhibitors for two weeks. The results showed that the expression of p-p65 were increased unless stimulated by PDTC. ERK pathway inhibitor(U0126) observably decreased the formation of mineralized nodules of h DPSCs induced by PDTC. In addition, MAPK(JNK, p38) inhibitors(SP600125, SB203580) did not affect PDTC regulated odontogenic/osteogenic differentiation of h DPSCs.In brife, through in vitro/ vivo experiments confirming that certain concentration of PDTC have an effect on the proliferation and odontogenic/osteogenic differentiation ability of h DPSCs. The further study found that NF-κB signaling pathway functioned as a negative regulator, while the ERK signaling pathway had a positive regulatory function in odontogenic/osteogenic differentiation of PDTC regulating of h DPSCs, which has made a solid foundation for future study of the molecular mechanism of human dental pulp injury and repair.