Synergistic Effects of Bacterial Mimetic and Mechanical Stimulation in Dental Pulp Cell Inflammatory Signalling: A Laboratory Investigation
Orla M. Dunne, Lorcan P. McGarvey, Imad About, Fionnuala T. Lundy, Ikhlas El‐KarimABSTRACT
Objectives
Mechanical and/or microbial stimuli can prompt extracellular ATP release in many cell types, where it acts as an inflammatory mediator to amplify pain signals and inflammation. This study aimed to investigate the synergistic effects of microbial and/or mechanical stimulation on ATP and cytokine release from dental pulp cells (DPCs) and to determine if the inflammatory signalling identified was dependent on ATP acting via the purinergic receptor, P2X3.
Methods
DPCs were prepared from sound third molars using explant culture method. P2X3 protein expression and functionality in DPCs were investigated by immunostaining and calcium mobilisation assays using the P2X3 agonist αβmeATP and antagonist AF‐353. ATP or interleukin‐6 (IL‐6) release from DPCs treated with lipoteichoic acid (LTA) and fluid shear stress or stretch was measured using the ATPlite luciferase assay (PerkinElmer) or ELISA (R&D Systems), respectively. To assess the role of P2X3 activation in IL‐6 release, the P2X3 antagonist AF‐353 was included in ELISA experiments. Data were checked for normality and analysed by Mann–Whitney test for two‐group comparisons and Kruskal–Wallis or ANOVA test with Dunn's or Dunnett's multiple comparisons test where appropriate for multiple‐group comparisons.
Results
The DPCs expressed functional P2X3 receptors. The cells' response to αβmeATP was significantly inhibited by AF‐353 ( p < 0.0001). Increased ATP and IL‐6 release were observed following co‐stimulation with LTA and fluid shear stress ( p < 0.01, p < 0.05) or LTA and stretch ( p < 0.05). The addition of AF‐353 reduced IL‐6 release by 34% and 36% ( p < 0.05) in DPCs co‐stimulated with LTA and fluid shear stress or stretch, respectively.
Conclusion
This study demonstrates that the combined bacterial mimetic and mechanical stimulation of DPCs significantly enhances ATP and IL‐6 release. The inhibition of IL‐6 release by the P2X3 antagonist AF‐353 indicates the importance of P2X3 receptor activation in this process. These findings offer insights into the molecular mechanisms of dental pulp inflammation and potential therapeutic targets.