Photoactivated disinfection procedure for denture stomatitis in diabetic rats

Preparation of fungal suspension

C. albicans SC5314 was provided by Shijiazhuang Hera Biotechnology Co., Ltd (Hebei, China). The C. albicans strain stored at -80 °C was inoculated onto CHROMagar^TM Candida chromogenic medium (Comagal Microbial Technology Co., Shanghai, China) and incubated at 37 °C for 24 h under a constant temperature incubator. After growth, a single colony was selected and re-inoculated onto a new Candida chromogenic medium and incubated at 37 °C for another 24 h. The activated single colony was then inoculated into 20 mL of yeast extract-peptone-dextrose medium broth medium (Kehua Jingwei Technology Co., Beijing, China) for amplification at 37 °C with shaking at 150 rpm overnight. The resulting fungal suspension was centrifuged at 4,000 rpm for 15 min in a high-speed centrifuge, and the supernatant was discarded. The fungal cells were rinsed in 10 mL of the PBS solution, the above mentioned centrifugation and rinsing steps were repeated thrice before discarding the supernatant to collect the fungal pellets. The pellets were then stored at 4 °C until further use. On the day of denture ligation, the spare fungal cells were diluted with PBS and counted on a blood cell count plate to 1 × 10⁹ CFU/mL for inoculation into the rat palate.

Experimental animals and establishment of diabetes model in rats (Deeds et al., 2011; King, 2012)

Based on the animal attrition rate calculation of approximately 10% in the pre-experiment, 40 male SD rats (age: 2 months, weight: 300–350 g; Beijing Huafukang Biological Technology Co., Ltd., Beijing, China) (animal quality certificate number: 110322220101519632) were acclimatized at Bethune International Peace Hospital Animal Laboratory for 1 week; the environment was maintained at a constant temperature and humidity, and a paste diet (prepared by mixing ground standard pellet rat food with warm water) and water was provided to them ad libitum. This study was approved by the Ethics Committee of the Hospital of Stomatology, Hebei Medical University (Approval No: [2020]016).

A total of 40 rats were fasted for 12 h and then injected intraperitoneally with 60 mg/kg of 1% streptozotocin to induce diabetes. On the day of streptozotocin injection, the rats were fed with 5% sterile glucose water to prevent hypoglycemia, and normal sterile water was provided on the following day. After 72 h, the rats with a fasting blood glucose level of >16.7 mmol/L were considered to have successfully developed diabetes and were included in the experiment, while one rat that did not meet this criterion was excluded. Finally, 39 rats were found to have met the criterion and were analyzed in this study.

Denture fabrication (Yano et al., 2016)

The alginate impression material (Haijiya Medical Equipment Co., Beijing, China) was evenly spread on a tongue depressor and then placed in the rat mouth to obtain an impression of the palate (Fig. 1A). A corresponding gypsum (Hanhe Medical Equipment Co., Linyi, China) model was developed according to the impression (Fig. 1B). The denture was then prepared using light-cured acrylic resin (Huge Medical Equipment Co., Shanghai, China) on the gypsum model to approximately 3-mm thickness and used to cover the entire hard palate area (Fig. 1C). After polishing, it was placed in distilled water at 37 °C for 48 h to release any residual monomers. The prepared denture was then immersed in sterile distilled water and microwaved at 650 W for 3 min for sterilization.

Denture seeding (Lie Tobouti et al., 2016)

On the day of denture ligation, the prepared fungal suspension was diluted in PBS and counted using a hemocytometer to a concentration of 1 × 10⁷ CFU/mL in RPMI-1640 medium for denture seeding. To allow C. albicans to adhere to the denture tissue surface, the denture was placed in a 6-well tissue culture plate, and each well contained 2 mL of 1 × 10⁷ CFU/mL of C. albicans suspension in RPMI-1640 medium. The 6-well plate was placed in 37 °C water bath oscillation incubator and shaken at 75 rpm for 90 min. The denture was then gently immersed in 2 mL of PBS to remove any non-adherent fungal cells.

Denture ligation

The experimental rats were anesthetized via intraperitoneal injection of 0.6% (40 mg/kg) sodium pentobarbital. Two stainless steel ligatures (5-cm length, 0.2-mm diameter) were threaded between the first and second molars on either side of the maxilla and through the holes on both sides of the denture. The excess wire at the end was cut, and the tip was covered with a self-curing resin to protect the oral soft tissues of the rat. A fungal suspension (density 1 × 10⁹ CFU/mL) was applied to the rat’s palate after denture ligation (Moraes et al., 2022). Preceding modeling experiments showed that a stable DS model in diabetic rats could be established after 3 weeks of ligation and denture seeding.

PAD treatment methods

A total of 36 rats were selected as the molded rats and assigned to four groups by using a random number table method (n = 9 per group). For the PAD-1 group, 1 mg/mL of the TBO solution (Denfotex, Edinburgh, UK) was applied to the palate and denture tissue surface with a small brush, incubated for 1 min, and then irradiated with 750-mW output power for 1 min (PAD^TM Plus instrument, Denfotex, UK; model: DX9001). The light source was systematically maneuvered to ensure that the entire palate and denture surface were irradiated. This treatment was conducted once a day for 1 day. For the PAD-2 group, the same treatment as for the PAD-1 group was administered, but with an interval of 1 day for irradiation, totaling three times within 5 days. The NYS group was treated with 100,000 IU of nystatin suspension applied to the palate and denture once a day for five consecutive days, while the infection group was not treated.

Efficacy observation and euthanasia

The rats in each group were anesthetized via intraperitoneal injection of 0.6% sodium pentobarbital at 40 mg/kg at 1 day and 7 days after the end of the treatment. The samples underwent two assessments: first, scoring for the extent of palatal inflammation, and second, measuring the fungal burden on both the palate and the denture tissue surface. Three rats from each group were randomly executed 1 day after the end of treatment (death by inhalation anesthesia with excessive isoflurane), and all remaining rats were executed 7 days after the end of treatment. The palate tissues were collected for histopathological examination and immunohistochemistry to detect the changes of interleukin-17 (IL-17) (Boorsen Biotechnology Co., Beijing, China) and tumor necrosis factor (TNF-α) (Bowan Biotechnology Co., Shanghai, China). Since preceding experiments have proven that the stable period of the model occurred at 3–6 weeks after denture seeding, the infection group was evaluated at the same time points as the PAD-1 and PAD-2 groups after treatment (at 1 day and 7 days, respectively), with the average data of the two-time points when compared to eliminate any bias caused by the time difference. The effectiveness of PAD therapy for diabetic rat DS was evaluated based on the results obtained from these evaluations.

Palate mucosal inflammation score. Newton’s method was applied to visually evaluate the palatal tissues, and the scores were assigned based on the severity of inflammation (Johnson et al., 2012). 0: no inflammation; 1: punctate erythema; 2: diffuse erythema and edema; 3: diffuse erythema/edema and papillary hyperplasia.

Palatal and denture tissue surface fungal burden measurement. The palate and denture of each group of rats were swabbed with sterile cotton for 1 min. The cotton swab was then placed into a centrifuge tube containing 1 mL of saline and shaken for 1 min. A 10-μL aliquot of each dilution was spread onto BIGGY agar culture medium and cultured at 25 °C for 48 h. The fungal colony count was measured in CFU/mL and analyzed statistically by taking the logarithm of the CFU number.

Statistical analysis

Data analysis was performed using SPSS25.0 software (IBM Corp., Armonk, NY, USA). The measurement data was tested for normality and homogeneity of variance tests. If normality and homogeneity of variance were met, two-factor ANOVA was used and group comparisons were performed using Student t-tests. Otherwise, the Friedman rank-sum test was applied and group comparisons were performed using the Kruskal-Wallis test. As the grade data (the palate mucosal inflammation score of the rats) used a constituent ratio, it was described and analyzed with the Friedman rank-sum test. P < 0.05 was considered to indicate statistical significance.

Palate mucosal inflammation score

One day after treatment, the infection group exhibited apparent symptoms of palate swelling and redness, and the inflammation score was primarily 2 (Fig. 2A). In the three treatment groups, there was less palate inflammation, with scores between 0 and 1 (Figs. 2B–2D). The infection group’s palate inflammation persisted and worsened on day 7, with inflammation scores between 2 and 3 (Fig. 2E). Partial redness and edema of the palate were also observed in the PAD-1 group, with scores between 1 and 2 (Fig. 2F). The PAD-2 and NYS groups, with scores between 0 and 1, did not exhibit any discernible redness or swelling on the palate (Figs. 2G and 2H). One day after treatment, pairwise comparisons revealed that inflammation scores in the infection group were considerably higher than those of the three treatment groups (P < 0.05). Seven days after treatment, the inflammatory score in the infection group was noticeably greater than that in the PAD-2 group 7d after treatment (P < 0.05). The inflammation score in the PAD-1 group was significantly higher at 7 days after treatment than at 1 day after treatment (P < 0.05). However, there was no statistically significant difference in inflammation scores between the infection, PAD-2, and NYS groups at either 1 day or 7 days after treatment (P > 0.05) (Fig. 3). The inflammatory scores showed no significant difference between the infection, PAD-1, and NYS groups (P > 0.05, Table 1). Notably, the PAD-2 group demonstrated a statistically significant reduction in palatal inflammation, suggesting that its efficacy is potentially greater than that of the NYS group (Table 2). These findings suggest that only PAD-2 treatment can effectively reduce inflammation in this context.

Fungal burden analysis

The fungal burden in the palate and dentures of the PAD-1 group was significantly higher at 7 days after treatment than at 1 day after treatment (P < 0.05). However, there were no significant differences in the palate and denture fungal load between the infection, PAD-2, and NYS groups at either 1 day or 7 days after treatment (P > 0.05; Fig. 4).

When comparing pairwise, the fungal load in the palate of the three treatment groups was significantly lower than that in the infection group 1day after treatment, with statistical significance (P < 0.05; Fig. 5A). There were no significant differences in the palate fungal load between the three treatment groups (P > 0.05). On day 7 after treatment, the fungal load in the palate of the infection and PAD-1 groups was significantly higher than that of the PAD-2 and NYS groups (Fig. 5B, P < 0.05). There was no statistically significant difference between the PAD-1 and infection groups, as well as between the PAD-2 and NYS groups (P > 0.05; Fig. 5B). These results suggest that, when used efficiently, PAD can kill C. albicans, achieving a level similar to that of NYS.

When comparing pairwise, the denture fungal burden in the infection group was significantly higher than that in the three treatment groups 1 day after treatment, with statistical significance (Figs. 4B and 6; P < 0.05). There were no significant differences in denture fungal colonization between the three treatment groups (P > 0.05). However, 7 days after treatment, the denture fungal load in the infection group was significantly higher than that in the PAD-2 and NYS groups (Fig. 4B; P < 0.05). Interestingly, the denture fungal burden in the PAD-1 group was also significantly higher between days 1 and 7 (Fig. 4B) than that in the PAD-2 group, with significant differences (Fig. 6B; P < 0.05). However, there were no significant differences in the denture fungal load between the PAD-1 and infection groups; the PAD-2 and NYS groups; and the PAD-1 and NYS groups (P > 0.05; Fig. 6B).

Palate tissue histology

As observed in the HE staining of the tissue in Fig. 7, one day after colonization, the infection group had notable thickening of the epithelial layer with papillary projections (Fig. 7A), while the epithelial layer structure appeared more homogenous in the three treatment groups (Figs. 7B–7D). In contrast, 7 days after treatment, the epithelial tissue in the infection group showed significant and abnormal proliferation, while partial proliferation was observed in the epithelium of the PAD-1 group (Figs. 7E and 7F). The epithelial structure appeared more normal in the PAD-2 and NYS groups, with no significant differences in epithelial structure among these treatment groups (Figs. 7G and 7H).

The presence of fungal colonization was analyzed with PAS staining of the mucosa. One day after treatment, PAS staining revealed that a number of C. albicans yeast cells adhered to the tissue surface in the infection group and that some hyphae invaded the superficial epithelium. The number of C. albicans in the three treatment groups was less evident compared to that in the infection group, and occasional C. albicans yeast cells were observed to adhere to the mucosal surface but without hyphae invasion (Figs. 8A–8D). There were no significant differences in fungal load among the treatment groups. Seven days after treatment, a large number of C. albicans yeast cells adhered to the mucosal surface in the infection group, with hyphae invading the superficial part of the epithelium (Fig. 8E). The PAD-1 group showed an intermediate number of C. albicans yeast cells adhered to the mucosal surface and partial hyphae invasion of the epithelium, but less than that observed in the infection group (Fig. 8F). Occasionally, a few C. albicans yeast cells were observed on the mucosal surface in the PAD-2 and NYS groups (Figs. 8G and 8H), supporting the previous findings.

The effect of PAD on IL-17 and TNF-α expressions in the palate mucosa of diabetic DS rats

IL-17 is a secretory protein that can be detected through immunohistochemistry as a brownish-yellow pattern in the cytoplasm and intercellular space of various layers of epithelial cells in normal tissues. One day after treatment, the infection group exhibited strong positive expression in various layers of epithelial cells, stroma, and vascular endothelial cells (Fig. 9A). The treatment groups were weakly positive in epithelial cells, stroma, and vascular endothelial cells, with lighter staining compared to the infection group (Figs. 9B–9D). After 7 days of treatment, the infection and PAD-1 groups showed strong positive expression in epithelial cells, stroma, and vascular endothelial cells (Figs. 9E and 9F). In comparison, the PAD-2 and NYS groups exhibited a weaker expression pattern in these cell types, with lighter staining than the infection and PAD-1 groups (Figs. 9G and 9H).

On day 1 after treatment, the average optical density value of the infection group was significantly higher than that of the treatment groups (Table 3, a super index P < 0.05). The average optical density value of the PAD-1 group was also slightly higher than that of the PAD-2 and NYS groups (P < 0.05). There was no significant difference in the optical density value between the PAD-2 and NYS groups (Table 3, b super index P > 0.05). Seven days after treatment, the average optical density values of the infection group and PAD-1 group were significantly higher than those of the PAD-2 and NYS groups (P < 0.05). There was no significant difference in the optical density value between the infection and PAD-1 groups, as well as between the PAD-2 and NYS groups (P > 0.05) (Table 3).

TNF-α can also be expressed in normal tissues, mainly in the cytoplasm of cells across the epithelial layer. It appears as yellow-stained particles in the cytoplasm via immunohistochemistry. A strong positive expression of TNF-α was observed in the epithelial and vascular endothelial cells in the infection group. One day after treatment, a weak positive expression of TNF-α was detected in the epithelial and vascular endothelial cells of the treatment groups, with lighter staining compared to the infection group. Seven days after treatment, a strong positive expression of TNF-α was observed in the surface and basal layers of the epithelium and vascular endothelial cells in the PAD-1 group, and a positive expression was observed in the granular and spinous layers. A weak positive expression of TNF-α was observed in the epithelial and vascular endothelial cells of the PAD-2 and NYS groups, with lighter staining than the infection and PAD-1 groups (Fig. 10).

When comparing pairwise, the average optical density value in the infection group was significantly higher than that in the treatment groups 1 day after treatment (P < 0.05). Conversely, there was no significant difference in the optical density value among the treatment groups (P > 0.05). Seven days after treatment, the average optical density values in the infection and PAD-1 groups were significantly higher than those in the PAD-2 and NYS groups (P < 0.05). There was no significant difference in the optical density values between the infection and PAD-1 groups, as well as between the PAD-2 and NYS groups (P > 0.05) (Table 4).