Opalescence boost

Cells were implanted on the dentin side of the disc. After attachment, the enamel-dentin disc was overturned in the medium to expose the enamel surface above the liquid level with a 1 mm gap; the enamel surface was surrounded by a light cured resin barriers (Opalescence PF; Ultradent Products, South Jordan, Utah [UT], US) to prevent the bleach from spilling into the medium (Fig. S1). After that, approximately 0.5 mL of thickened bleaching gel was daubed on the enamel surface at 37 °C. We tested two concentrations of bleaching gel, 40% H₂O₂ and 15% carbamide peroxide (Opalescence Boost and Opalescence PF, respectively; Ultradent Products, South Jordan, Utah [UT], US)^{6 (link)}. Five groups were created: G1, untreated DPSCs; G2, 15% bleaching gel for 90 min; G3, 15% bleaching gel and 1 μM HC030031 (TRPA1 inhibitor, ApexBio; ApexBio, Houston, Texas [TX], US) for 90 min; G4, 40% bleaching gel for three 15-min periods in a row; and G5, 40% bleaching gel and 1 μM HC030031 for three 15-min periods in a row. As a small molecule drug that inhibits TRPA1, HC030031 can be used as a tool to study the role of TRPA1 channels in pain^{23 (link)}. In the inhibition group, HC030031 was dissolved in the culture medium to directly inhibit TRPA1.

Three different hydrogen peroxide-based treatments were tested (n = 5 for each treatment, see Table 1). A prototype of an in-situ liquid applicator, consisting of flexible aquaria tubing (2 cm in diameter) cut in half lengthwise to create an open chamber and coated and secured with silicone glue, was created to deliver a direct application of 30% hydrogen peroxide to BBD lesions. A one-way valve was used to allow the injection of hydrogen peroxide from a 60 ml blunt tipped plastic syringe into the chamber and, as the hydrogen peroxide entered, a second valve allowed seawater to escape. The prototype was filled with approximately 30 ml of hydrogen peroxide and held directly on the lesion for approximately one minute (S1A Fig). Additionally, a tooth whitening gel, Opalescence™ Boost™, containing 40% hydrogen peroxide (Opalescence™ Teeth Whitening Systems, Ultradent, South Jordan, UT, USA) was applied directly to the lesion and covered with marine epoxy (All-Fix Epoxy, Lafayette Hill, PA, USA) to help secure the gel to the coral (S1B Fig). In another treatment, the whitening gel was mixed with a delivery vehicle, Base2B (Ocean Alchemists LLC, Tampa FL, USA), which was applied directly to the lesion and anchored approximately every 5 cm with marine epoxy to hold the ointment in place (S1C Fig).