Iterative Hyperosmotic- and Thermal-Stimuli Modify TRPV1 Activation in Odontoblast Like-Cells

Objectives: To evaluate the changes in TRPV1 function after hyperosmotic and thermal repeated stimuli in OLC
Methods: Human odontoblast-like cells (OLC) were differentiated from dental pulp stem cells using TGF-β1. Hyperosmotic stimuli were performed with mannitol, xylitol and sucrose solutions (370, 500, 1000 mOsm/L) and thermal stimuli using heated or cooled medium (45°C or 15°C). Capsaicin (CAP, 50, 100, 200 μM) was used as TRPV1 activation control. Each stimulus was carried out for 30 min, followed by a recovery period of 8 h, then repeated two more times (three cycles), and cytotoxicity was evaluated by resazurin metabolic assay. Other cells at the end of reiterative stimulus were loaded with the calcium fluorescent probe Fluo-4 AM (2 μM) and challenged with CAP 100 μM, and fluorescence measured and normalized with fluorescence signal of unstimulated cells control (310 mOsm/L)
Results: Mannitol and xyitol at 370 or 500 mOsm/L did not induce cytotoxicity, however 1000 mOsm/L solutions significantly reduced cell viability (65.35%±0.58 and 31.4%±5.97 reduction, respectively). In the case of sucrose, all three concentrations induced more than 50% cytotoxicity of the treated OLC. Repeated stimuli with 45°C or 15°C did not affect the cell survival percentage (96% and 99% respectively). Regarding [Ca2+]i levels, repeated stimuli with mannitol and xylitol significantly decreased CAP-induced activation (p < 0.05), suggesting a desensitization process. Conversely, thermal 45°C or 15°C stimuli sensitize TRPV1, yielding an increment in fluorescence ratios F/F0 of 3.07±0.18 and 3.2±0.16, regarding the F/F0 ratio of 2.29±0.47 in OLCs treated with a single acute CAP stimulus
Conclusions: Iterative challenge with stimuli associated with dental pain (hyperosmotic solutions or thermal challenges) modifies the activation of TRPV1 channels, suggesting that this channel is important in the transduction of these stimuli in the dental pulp. The modulation of TRPV1 can be considered a potential strategy in the management of dental pain.