Pilot study

To simulate the dryness associated with dental treatment or oral surgery, intraoral vacuum was applied for 5 min, followed by a saliva ejector for 5 min in seven healthy volunteers. The oral cavity was exposed to dryness for a total of 10 min. Trehalose (Hayashibara Biochemical Laboratories, Inc., Okayama, Japan) and distilled water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) were used to produce a 10% trehalose solution, and a control solution was prepared from distilled water. The pH values of both solutions were adjusted to 7.4 with 200 mM sodium bicarbonate solution. These solutions (0.4 ml each) were sprayed into the oral cavity before applying the oral vacuum, and their effects were compared. The intraoral vacuum system was the aspirator used to remove saliva from patients’ mouths, which dries the oral mucosa. The mucosa of the anterior dorsum of the tongue was assessed before drying, and 5 min and 10 min after applying the oral vacuum.

The two variables assessed were changes in the surface area of the fungiform papillae and changes in the water content of the mucosal surface of the tongue. A flow chart of this study is shown in Fig. 1 A .

Effects of trehalose on oral dryness under the drying process. (A) Flow chart of the simulated drying process using vacuum in a pilot study. (B) Fungiform papilla area ratio. Data are expressed as means (bars) ± SDs (error bars) for 7 patients * p < 0.01, ^# p < 0.05. (C) Moisture content score for the drying time course. Data are expressed as means (bars) ± SDs (error bars) for 7 patients * p < 0.01, ^# p < 0.05.

The authors measured the surface area of the fungiform papillae before and after the oral vacuum was applied. Two standardized indicators were placed on the mucosa of the anterior dorsum of the tongue and the surface area of the fungiform papillae was calculated using the homothetic ratio for indicators on the recorded still image ( Fig. 1 B). The average number of the fungiform papillae was 10.96 (range 5–24). The surface area of the fungiform papillae was calculated using the following formula as the area of an ellipse: area = πab /4 ( a : major axis, b : minor axis). The water content of the mucosal surface on the tongue was measured before and after applying the oral vacuum using a moisture measuring system (Mucos ^® , Life Co., Ltd., Saitama, Japan).

Clinical study

Ten patients (4 males and 6 females) with dental caries, ranging in age from 20 to 67 years (mean, 49 years), received root canal treatment. They did not have serious conditions. To assess the clinical efficacy and acceptability of trehalose solution, an oral spray (0.4 ml of 10% trehalose solution) was applied before root canal treatment, and intraoral vacuum performed for about 10 min. After treatment, the authors evaluated the discomfort and pain due to dryness using a self-rated, visual analog scale (VAS) as subjective assessment, and the atrophic changes on the surface of the tongue using a three-point scale as an objective assessment. They obtained the patients’ written informed consent for root canal treatments. A flow chart of this study is shown in Fig. 2 A .

Effect of trehalose on oral dryness during root canal treatment. (A) Flow chart of drying process during root canal treatment in a clinical study. (B) Post-stimulatory levels of dryness and pain intensity after drying, measured by VAS. Data are expressed as means (bars) ± SDs (error bars) in 10 patients * p < 0.01, ^# p < 0.05. (C) Objective assessment of the oral mucosa: dryness of the tongue and erythrogenic tongue. Data are expressed as means (bars) ± SDs (error bars) in 10 patients * p < 0.01. (D) Appearance of the tongue before and after treatment.

The VAS used for assessing dryness and pain intensity consisted of a 100 mm horizontal line, with ‘no dryness or pain’ and ‘maximum dryness or pain’ at the extreme ends, and the words ‘slight, moderate, severe’ distributed along the length of the line. ‘Maximum dryness or pain’ was defined as the worst dryness or pain ever experienced.

Atrophic changes on the surface of the tongue were scored on a scale of 0–2 (0 indicating minimal change involving <25% areas, 1 indicating partial change involving <50% areas, 2 indicating marked change involving >50% areas with mucosal dryness and erythrogenic changes after treatment).

This study was approved by the institutional review board of the University of Tokyo (No. 1740).

Cell culture

Human oral cancer cell line Ca9-22 cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). Ca9-22 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and 1% (v/v) penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO). Ca9-22 cells (1 × 10 ⁵ ) were seeded in a 96-well collagen 1-coated plate (AGC Techno Glass Co., Ltd, Ciba, Japan).

Measurement of cell viability

After the medium was aspirated completely, the cells were washed with phosphate buffered saline (PBS), and incubated for 15 min in 100 μl of 7.5% trehalose/PBS or in PBS alone as a control. These solutions were aspirated completely, and the cells were left to dry up for 0, 40 or 50 min at room temperature and morphological changes observed under the microscope. Cells were washed with PBS and incubated for 30 min with LIVE/DEAD reagent (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes, Eugene, OR) in PBS. Live and dead cells were visualized using fluorescence microscopy (BZ-8000, Keyence Co., Osaka, Japan) following filters for FITC (535/50) and Dapi (460/50) (emission maximum/full width at half maximum in nm, respectively) and quantified using a fluorescence plate reader (Fluoroscan Ascent FL, Labsystem, Helsinki, Finland) following filters for Live cells (485/527) and Dead cells (544/650) (excitation/emission in nm, respectively).

Statistical analyses

Statistical analysis was performed using a paired t -test. Data are shown as means ± SD p < 0.05 or p < 0.01 were considered significantly different.