Assessment of the regenerative ability of two GSK3 inhibitors (Tideglusib & CHIR99021) on the Wnt/beta catenin pathway activation, the proliferative capacity, and the odontogenic differentiation of the human dental pulp stem cells (hDPSCs)

Resumen

Statement of problem: New vital pulp therapy concept, using GSK 3 inhibitors (Tideglusib) incorporated in resorbable scaffold, is being introduced nowadays into literature promising superior results than the present pulp caping materials especially in inducing full dentin regeneration and the induction of tooth regeneration. Using this newly introduced drug is still under investigation for its safe in-vivo use and the confirmation of the expected outcomes. Aims of the study: The current study aims at assessing the regenerative the two proposed GSK3 inhibitors drugs namely (Tideglusib & CHIR99021) on activation of Wnt/beta catenin pathway regarding the proliferative capacity odontogenic differentiation potential of human dental pulp stem cells. Materials and Methods: Study design set for this study is in-vitro study. Human Dental Pulp Stem Cells (hDPSC's) was obtained from extracted sound anterior teeth for periodontal and aesthetic problems, after receiving their informed consents and was isolated and cultivated according to Gronthos et al., 2000 technique. Flow cytometric analysis was used to confirm the mesenchymal stem cells properties of the hDPSCs. Wnt Induction Assay was achieved by placing hDPSCs celis in 24-well plates and after 24 hrs incubation (37 oc, 5% C02/95% air, 100% humidity) using standard culture medium, selected drugs with the appropriate dosage, were used in triplicates to treat the cells for 24 h and Trizcl were used for cells lyses and extraction of RNA and real time qRT-PCR was achieved. Later, proliferation assessment via Cell Counting Kit-8 (CCK-8) was done followed by analysis of surface marker expression of ANNEXIN V by flow cytometry. The Effect of GSK3 inhibitors on the stemness properties of human dental pulp stem cells was also analysed by qRT-PCR, Finally, the effect on the odontogenic differentiation of human dental pulp stem cells was assessed by Alizarin red Staining and qRT- PCR for odontogenic markers. Results: Within the cytotoxicity assessment of different drug concentrations over the HDPSCs, no statistical significance was registered however, CHIR99021 at 5 n!vl dose and Tideglusib at 100 nM concentrations resulted in the highest cell viability. To assess the activation of Wnt pathway, AXIN2 expression was quantified using RT-PCR. Tideglusib group and CHIR99021 were seen to express the AXIN2 gene 7.462 and 1.414 times higher than the control respectively and Tideglusib group expressed the AXIN2 gene in statistically significant less cycle than the CHIR99021. Within the analysis of both drugs effect over the stemness of the hDPSCs, both drugs expressed the three quantified genes more than the control group and Tideglusib group expressed the NANOG gene, statistical significance less cycles in comparison to the CHIR99021 while for the OCT4 gene, CHIR99021 expressed the gene in statistically significant less cycles in comparison to the control. Regarding proliferation and cell viability, no statistical significance was registered between the effect of the three groups over cell viabilities and proliferation. However, it was noted that both drugs at the chosen concentration increased cellular proliferation in comparison to the control group which was also confirmed with the low level of expression of ANNEXIN V within the tideglusib group 6.62% and CHIR99021 1.34 %. Finally odontogenic differentiation ability was confirmed with both drugs with Alizarin red staining showing Intense Orangered nodules on the 14th day and by the high expression of odontogenic differentiation genes (DSPP, DMPI, RUNX, (OPN) and ALP) in comparison to the control group. However, for the cycle threshold, no statistical significance was registered between the groups within each gene's expression. Conclusion: Both drugs were seen to be safe to use with proximity to the hDPSCs. Both drugs had positive effect over the proliferation and cell viability of the stem cells and over the stemness ability of those celIs. Also, authors can conclude that thway activation and the both drugs showed promising results with the Wnt pa odontogenic differentiation.