AIM: To investigate the ability of dead odontoblasts to initiate NLRP3 inflammasome-dependant sterile inflammation and to explore the effect on dental pulp cells (DPCs) migration, proliferation and odontogenic differentiation.
METHODS: Odontoblast like cells were subjected to freezing-thawing cycles to produce odontoblast necrotic cell lysate (ONCL). DPCs were treated with ONCL to assess proliferation and migration. THP-1 differentiated macrophages stimulated with ONCL and live cell imaging and western blotting were used to assess NLRP3 inflammasome activation. Cytokines were measured with multiplex arrays and ELISA. qPCR, alkaline phosphatase and Alizarin red assays were used to assess odontogenic differentiation of DPCs. Data were analysed using the t-test or ANOVA followed by a Bonferroni post hoc -test with the level of significance set at p≤0.05.
RESULTS: ONCL induced migration and proliferation of DPCs. Treatment of THP-1 macrophages with ONCL resulted in the release of the inflammatory cytokines IL-1β, IL-6, IL-8, TNFα, IFN-γ, CCL2 and angiogenic growth factors, angiogenin and angiopoietin. This inflammatory response was associated with activation of NFκB, p38MAPK and NLRP3 inflammasome. To confirm that ONCL induced inflammatory response is NLRP3 inflammasome-dependent, treatment with a caspase-1 inhibitor and a specific NLRP3 inhibitor significantly reduced IL-1β release in THP-1 macrophages (p=0.01and 0.001). Inflammasome activation product, IL-1β induced odontogenic differentiation of DPCS as evident by the increase in odontogenic genes expression DMP-1, RUNX-2, DSPP and SPP, alkaline phosphatase activity and mineralisation.
CONCLUSION: Dead odontoblasts induced NLRP3 inflammasome-dependent sterile inflammation and activated the migration, proliferation and differentiation of DPCs.