Effects of prolonged stimulation with heated tobacco products (Ploom TECH+ ) on gingival epithelial cells.

OBJECTIVE AND BACKGROUND: Heated tobacco products have recently become commercially available. These products, as well as combustible cigarettes, produce aerosols; the risk of various diseases associated with heated tobacco products may be the same or higher than that with combustible cigarettes. In this study, we examined the effect of Ploom TECH+ extract on gingival epithelial cells. METHODS: Tobacco leaves from Ploom TECH+ tobacco capsules and water were mixed and heated; the supernatant subsequently collected was the heated tobacco product (HTP; control: HTP not added). Normal human gingival epithelial progenitors were cultured alternately with or without HTP for a total of 1 month. Subsequently, RNA, DNA, and proteins were isolated from these samples and comprehensively analyzed using RNA sequencing (RNA-seq), reduced representation bisulfite sequencing (RRBS), and western blotting, respectively. RESULTS: RNA-seq revealed that 284 genes showed a twofold increase and 145 genes showed a twofold decrease in gene expression. A heat map showed genetic differences between the control and HTP groups. A principal component analysis plot showed a clear genetic distribution between the control and HTP. Gene Ontology (GO) analysis showed that genes related to seven GO terms, including cornification and keratinization, were induced by long-term HTP stimulation. By contrast, GO pathways with a significant decrease in component expression were not detected. RRBS revealed that CpG island methylation increased more than twofold in 158 genes and decreased to less than twofold in 171 genes. Methylation of these CpG islands was not correlated with changes in gene expression levels. HTP treatment increased S100A7 expression. CONCLUSION: Long-term HTP stimulation affected epithelial differentiation and keratinization of gingival epithelial cells. Thus, habitual use of Ploom TECH+ may be a risk factor for tobacco-related oral mucosal diseases.

The effect of Porphyromonas gingivalis on the gut microbiome of mice in relation to aging.

BACKGROUND AND OBJECTIVE: The translocation of oral bacteria, including Porphyromonas gingivalis, to the gut has been shown to alter gut microbiome. However, the effect of P. gingivalis on gut microbiome in relation to aging has not been demonstrated. We hypothesize that P. gingivalis has more detrimental effect on gut environment with increased age. The objective of this study is to investigate the effect of P. gingivalis on gut environment using aged mice. MATERIALS AND METHODS: C57BL/6J mice aged 4 weeks (young) or 76 weeks (old) were divided into four groups: control-young, control-old, P. gingivalis-administered young, and P. gingivalis-administered old. P. gingivalis was orally administered thrice weekly for 5 weeks. At 30 days after the last P. gingivalis administration, 16S rRNA sequencing was performed to study the gut microbiome. The mRNA and protein expression of intestinal junctional barrier molecules and the levels of the inflammatory cytokines IL-1ß and TNF-α in the serum were evaluated. RESULTS: Significant differences in the gut microbiomes between the groups, in terms of taxonomic abundance, bacterial diversity, and predicted metagenome function, were observed. A significant reduction in the alpha diversity and in the abundance of beneficial bacteria, such as Akkermansia and Clostridiaceae, in the P. gingivalis-administered old mice was observed. The mRNA and protein levels of Claudin-1 and Claudin-2 in the intestine were significantly elevated, while E-cadherin was significantly downregulated in the P. gingivalis-administered old mice, as were the serum levels of IL-1ß and TNF-α. CONCLUSION: The effect of P. gingivalis on the gut environment is more pronounced in old mice than in young mice.

An in vitro senescence model of gingival epithelial cell induced by hydrogen peroxide treatment.

Gingival tissue shows progressive changes with aging and an in vitro model of gingival tissue could be useful in understanding age-associated oral diseases. The present study aims to establish a hydrogen peroxide (H2O2) treatment model to induce aging in human gingival epithelial cells. In addition, fisetin, a flavonoid component studied for the anti-aging property is used to examine if it could reverse the induced senescence. Primary human gingival epithelial progenitor (HGEPp) cells were cultured and treated with different concentrations of H2O2. A cell vitality and morphology, senescence-associated beta-galactosidase (SA-ß-gal) staining, mRNA and protein expression analysis of known senescence markers p16, p21, and p53, and cell cycle assay were performed. The cells showed dose-dependent changes in vitality and morphology, SA-ß-gal staining, relative mRNA and protein expression, and cell cycle assay after H2O2 treatment. Based on these results, 400 µM H2O2 was considered as an optimal concentration to induce senescence. Treatment of senescence-induced cells with fisetin downregulated all the senescence markers used in this study. In conclusion, a senescence model of gingival epithelial cells induced by hydrogen peroxide treatment was established which could be employed to study age-related periodontal diseases.

Gut flora alterations due to lipopolysaccharide derived from Porphyromonas gingivalis.

Gut dysbiosis induces 'leaky gut,' a condition associated with diabetes, NASH, and various auto-immune diseases. Porphyromonas gingivalis is a periodontopathic bacterium which causes periodontal tissue breakdown, and often enters the systemic blood flow. Oral administration of P. gingivalis induced gut dysbiosis in mice model, but no systemic administration of P. gingivalis has been reported thus far. In the present study, we investigated the effect of P. gingivalis-derived lipopolysaccharide (Pg-LPS) on the intestinal flora of our established mouse model. Eight-week-old C57BL/6J mice were intraperitoneally administered Pg-LPS. Three months later, DNA was extracted from stool, and RNA from the small and large intestines. After euthanizing the mice, pathological sections of the intestinal tract were prepared and stained with hematoxylin and eosin (H&E). Tumor necrosis factor alpha (TNF-α), interleukin (IL)-1ß, and IL-6 expression levels were evaluated using quantitative PCR. 16S rRNA gene PCR amplicon analysis data were acquired using NGS. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. Furthermore, alterations in microbial function were performed by PICRUSt2. No significant inflammatory changes were observed in the H&E. No significant differences in the mRNA levels of IL-1ß, IL-6, and TNF-α were observed between the groups. Pg-LPS administration decreased the abundance of Allobacterium in the gut. A predictive metagenomic analysis by PICRUSt2 and STAMP showed that 47 pathways increased and 17 pathways decreased after Pg-LPS administration. Systemic application of periodontal pathogens may cause changes in the intestinal flora which may affect the physiological functions of the intestinal tract.

Aberrant expression of DUSP4 is a specific phenomenon in betel quid-related oral cancer.

Oral cancer due to betel quid chewing habit is very common in South Asian countries. We attempted to detect the presence of a novel gene in epithelial cells stimulated with arecoline, a main component of betel quid. Human gingival epithelial progenitors were cultured and treated with a 3-day alternating regimen with/without 50 µg/ml arecoline for 1 month. DNA microarray and methylation arrays were analyzed to identify the candidate genes. Immunohistochemical staining was performed in the tissue samples. Genome-wide analyses, quantitative reverse transcription PCR and quantitative methylation-specific PCR revealed DUSP4 as the most significant and promising gene. The methylation levels of DUSP4 were significantly higher in the betel quid-related oral squamous cell carcinoma (OSCC) than those in the non-related OSCC and controls (Mann-Whitney U test, p < 0.05). The number of DUSP4 immunopositive cells in betel quid-related OSCC was significantly higher than those from the non-chewing patients and the controls (p < 0.05). Hypermethylation of DUSP4 may be considered as a specific event in betel quid-related oral cancer.

Pharmacotherapy in relieving the symptoms of burning mouth syndrome: A 1-year follow-up study.

OBJECTIVE: Burning mouth syndrome (BMS) is a chronic intraoral burning sensation with no identifiable causes. In this study, we aim to demonstrate the effectiveness of treatment strategy using ethyl loflazepate monotherapy or in combination with milnacipran or amitriptyline. METHOD: A hospital-based, retrospective study was conducted in 86 patients. The patients were divided into remission group and non-remission group. The remission group comprised patients who were satisfied with their pain relief within a year of treatment initiation and did not require any follow-up treatment. The treatment was considered effective if the patient got remission within 1 year or was able to reduce the visual analogue scale (VAS) score to <20, in the absence of remission. RESULTS: The treatment strategy was effective in 76.7% of the patients. Significant reductions (p < .05) in VAS scores from 73.5 ± 14.2 at first visit to 14.7 ± 8.7 at last visit in the remission group, and from 79.7 ± 14.3 at first visit to 33.4 ± 23.7 after 1 year of treatment in the non-remission group were noted. CONCLUSION: The treatment strategy using ethyl loflazepate monotherapy or in combination with milnacipran or amitriptyline can be very effective in reducing pain in BMS patients.

Chronic stress-induced elevation of IL-1ß in the saliva and submandibular glands of mice.

Psychological stress is involved in the development of various oral diseases. Alterations in the levels of cytokines in the saliva of patients with stress-related oral diseases have been reported. However, the inconsistencies in the results of these studies might be attributed to differences in the local and systemic factors in the oral cavities of the patients. We examined the effect of chronic stress on three major inflammatory cytokines Interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in the saliva and salivary glands of chronically stressed mice. Six-week-old C57BL/6 J mice were randomly divided into a control and a stress group. The mice in stress group were exposed to 4 h of stress daily for 10 days and subsequently saliva, as well as the submandibular glands, were collected from both groups. The expression levels of cytokines in the saliva were examined by enzyme-linked immunosorbent assay. The submandibular glands were subjected to histopathological and mRNA expression analyses. IL-1ß was significantly elevated in saliva of the chronic stressed mice. Furthermore, the mRNA expression levels of both IL-1ß and IL-6 were significantly elevated in the submandibular gland of chronic stressed mice. IL-1ß may be a potential salivary biomarker in response to chronic stress in mice.

Ingenuity pathway analysis of gingival epithelial cells stimulated with estradiol and progesterone.

OBJECTIVE: Periodontal disease is a risk factor for preterm delivery, and elevated female hormone levels during pregnancy promote hormone-dependent periodontopathogenic bacterial growth and gingivitis. Although the saliva of pregnant women contains female hormones at elevated levels, their effects on the gingiva are poorly understood. Therefore, in this study, we investigated the effects of estradiol and progesterone stimulation on gingival epithelial cells via ingenuity pathway analysis. METHODS: Human gingival epithelial progenitors were cultured in a CnT-Prime medium; 17ß-estradiol (E2) and progesterone (P4) were used as the reagents. Cells treated with dimethyl sulfoxide alone were used as the control group. Cells in the control and experimental groups were incubated for 12 h. RNA was extracted from the cultured cells, RNA-Seq was performed, and pathway analysis was conducted. RESULTS: Differentially expressed genes were detected for 699 (over 2-fold increase) and 348 (decrease) genes in group E2 and for 1448 (increase) and 924 (decrease) genes in group P4 compared with those in the control group (FDR <0.05, n = 4). The z-scores of the pathways suggest that E2 and P4 increased the activity of the wound healing signaling pathway. The activation of this pathway was higher in the E2 and P4 groups than that in the control group. CONCLUSIONS: The results of this study suggest that estradiol and progesterone may affect gingival homeostasis and wound healing.

Oral infection with Porphyromonas gingivalis augmented gingival epithelial barrier molecules alteration with aging.

OBJECTIVE: Disruption of the gingival epithelial barrier is often mediated by aging or the pathogen Porphyromonas gingivalis. This study examined the combined effects of aging and P. gingivalis exposure on gingival epithelial barrier molecules. METHODS: In vitro experiments involved treating young- and senescence-induced primary human gingival epithelial progenitor cells (HGEPp) with P. gingivalis lipopolysaccharide (LPS). Transepithelial electrical resistance (TER) and paracellular permeability were measured. In vivo, male C57BL/6J mice aged 10 (young) and 80 (old) weeks were divided into four groups: young, old, young with P. gingivalis (Pg-Young) inoculation, and old with P. gingivalis (Pg-Old) inoculation. P. gingivalis was inoculated orally thrice a week for 5 weeks. The mice were sacrificed 30 days after the last inoculation, and samples were collected for further procedures. The junctional molecules (Claudin-1, Claudin-2, E-cadherin, and Connexin) were analyzed for mRNA expression using qRT-PCR and protein production using western blotting and immunohistochemistry. The alveolar bone loss and inflammatory cytokine levels in gingival tissues were also assessed. RESULTS: LPS-treated senescent cells exhibited a pronounced reduction in TER, increased permeability to albumin protein, significant upregulation of Claudin-1 and Claudin-2, and significant downregulation of E-cadherin and Connexin. Furthermore, the Pg-Old group showed identical results with aging in addition to an increase in alveolar bone loss, significantly higher than that in the other groups. CONCLUSION: In conclusion, the host susceptibility to periodontal pathogens increases with age through changes in the gingival epithelial barrier molecules.

Systemic Administration of Lipopolysaccharide from Porphyromonas gingivalis Decreases Neprilysin Expression in the Mouse Hippocampus.

BACKGROUND/AIM: Alzheimer's disease is the most common type of neurodegenerative disorder in elderly individuals worldwide. Increasing evidence suggests that periodontal diseases are involved in the pathogenesis of Alzheimer's disease, and an association between periodontitis and amyloid-ß deposition in elderly individuals has been demonstrated. The aim of the present study was to examine the effects of systemic administration of Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS) on neprilysin expression in the hippocampus of adult and senescence-accelerated mice. MATERIALS AND METHODS: PG-LPS diluted in saline was intraperitoneally administered to male C57BL/6J and senescence-accelerated mouse prone 8 (SAMP8) mice at a dose of 5 mg/kg every 3 days for 3 months. Both C57BL/6J and SAMP8 mice administered saline without PG-LPS comprised the control group. The mRNA expression levels of neprilysin and interleukin (IL)-10 were evaluated using the quantitative reverse transcriptase-polymerase chain reaction. The protein levels of neprilysin were assessed using western blotting. Sections of the brain tissues were immunohistochemically stained. RESULTS: The serum IL-10 concentration significantly increased in both mouse strains after stimulation with PG-LPS. Neprilysin expression at both mRNA and protein levels was significantly lower in the SAMP8 PG-LPS group than those in the SAMP8 control group; however, they did not differ in PG-LPS-treated or non-treated C57BL/6J mice. Additionally, the immunofluorescence intensity of neprilysin in the CA3 region of the hippocampus in PG-LPS-treated SAMP8 mice was significantly lower than that in control SAMP8 mice. CONCLUSION: Porphyromonas gingivalis may reduce the expression of neprilysin in elderly individuals and thus increase amyloid-ß deposition.

Analysis of the cells isolated from epithelial cell rests of Malassez through single-cell limiting dilution.

The epithelial cell rests of Malassez (ERM) are essential in preventing ankylosis between the alveolar bone and the tooth (dentoalveolar ankylosis). Despite extensive research, the mechanism by which ERM cells suppress ankylosis remains uncertain; perhaps its varied population is to reason. Therefore, in this study, eighteen unique clones of ERM (CRUDE) were isolated using the single-cell limiting dilution and designated as ERM 1-18. qRT-PCR, ELISA, and western blot analyses revealed that ERM-2 and -3 had the highest and lowest amelogenin expression, respectively. Mineralization of human periodontal ligament fibroblasts (HPDLF) was reduced in vitro co-culture with CRUDE ERM, ERM-2, and -3 cells, but recovered when an anti-amelogenin antibody was introduced. Transplanted rat molars grown in ERM-2 cell supernatants produced substantially less bone than those cultured in other cell supernatants; inhibition was rescued when an anti-amelogenin antibody was added to the supernatants. Anti-Osterix antibody staining was used to confirm the development of new bones. In addition, next-generation sequencing (NGS) data were analysed to discover genes related to the distinct roles of CRUDE ERM, ERM-2, and ERM-3. According to this study, amelogenin produced by ERM cells helps to prevent dentoalveolar ankylosis and maintain periodontal ligament (PDL) space, depending on their clonal diversity.

Induction of Periodontal Ligament-like Cells by Coculture of Dental Pulp Cells, Dedifferentiated Cells Generated from Epithelial Cell Rests of Malassez, and Umbilical Vein Endothelial Cells.

INTRODUCTION: Apart from the epithelial cell rests of Malassez (ERMs), dental pulp (DP) contains the same types of mesenchymal cells as the periodontal ligament (PDL). ERMs may affect the characteristics of the mesenchymal cells in the PDL. The aim of this study was to examine whether DP cells cultured with ERMs and human umbilical vein endothelial cells (HUVECs) could transform into PDL-like cells. METHODS: Progenitor-dedifferentiated into stem-like cells (Pro-DSLCs) were produced by the induction of ERMs with 5-Azacytidine and valproic acid. DP cells were cultured in mesenchymal stem cell medium for 1 week under the following conditions: DP cells alone (controls); PDL cells alone; coculture of DP cells and ERMs (DP + ERM) or Pro-DSLCs (DP + Pro-DSLC); and coculture of DP cells, HUVECs, and ERMs (DP + ERM + HUVEC) or Pro-DSLCs (DP + Pro-DSLC + HUVEC). Quantitative real-time reverse transcription polymerase chain reaction, quantitative methylation-specific polymerase chain reaction, and flow cytometry were performed. RESULTS: The expression levels of PDL-related markers Msx1, Msx2, Ncam1, Postn, and S100a4 and mesenchymal stem cell-positive markers Cd29, Cd90, and Cd105 were significantly higher in the PDL cells and DP + Pro-DSLC + HUVEC cultures than in the controls (P < .05). The DNA methylation levels of Msx1 and Cd29 in the PDL cells and the DP + Pro-DSLC + HUVEC culture were significantly lower than in the controls (P < .01). We found a significant increase in the number of cells stained with MSX1 (P < .05) and CD29 (P < .01) in the DP + Pro-DSLC + HUVEC culture than in the controls. CONCLUSIONS: Coculture of DP cells with Pro-DSLCs and HUVECs induced their transformation into PDL-like cells. This method may prove to be useful for periodontal regeneration via tissue engineering.

Psychological Backgrounds of Medically Compromised Patients and Its Implication in Dentistry: A Narrative Review.

The number of medically compromised dental patients is increasing every year with the increase in the super-aged population. Many of these patients have underlying psychiatric problems and diseases, which need to be recognized by dental professionals for better treatment outcomes. The aim of this narrative review article is to summarize the psychological and psychiatric backgrounds of medically compromised patients who are frequently visited and taken care of by dentists using findings from recent systematic reviews and meta-analyses. Anxiety and symptoms of depression, post-traumatic stress disorders, panic disorders, poor cognitive functions, and poor quality of life were some of the common psychological backgrounds in medically compromised patients. Additionally, the consequences of these psychological problems and the considerations that need to be taken by the dentist while treating these patients have been discussed. Dental professionals should be aware of and recognize the different psychological backgrounds of medically compromised dental patients in order to provide appropriate dental treatment and to prevent oral conditions from worsening.

Alteration of oral flora in betel quid chewers in Sri Lanka.

BACKGROUND: Betel quid chewing is known as a crucial risk factor for oral diseases such as periodontal diseases, oral cancer, and precancerous lesions in Southeast Asian countries. Although abnormal oral bacterial flora may be linked to betel quid related-oral diseases such as oral cancer, precancerous lesions, and periodontal diseases, little information is available on alterations of their oral flora thus far. To identify these alterations, we analyzed the oral flora in betel quid chewers (BQC) and non-chewers (NC) in Sri Lanka. METHODS: Samples obtained from buccal swabs of BQC and NC were analyzed with a next generation sequencer. Data were processed and analyzed using the QIIME software package. Mann-Whitney U test and Permutational multivariate analysis of variance were used for statistical analyses. P values < 0.05 were considered to be statistically significant. RESULTS: In BQC, the proportion of periodontal pathogens including Actinomyces, Tannerella, and Prevotella was higher than that in NC (P < 0.05), while the proportion of cariogenic pathogens including Streptococcus, Lautropia, and Actinobacillus was lower than that in NC (P < 0.05). A statistically significant difference in Shannon index and PD Whole tree was observed between BQC and NC (P < 0.05). PCoA analysis detected different clusters in BQC and NC (P < 0.05). CONCLUSION: The results suggested that betel quid chewing significantly altered oral flora. Adequate oral health care may help prevent BQC from developing bacterial pathogen-related oral diseases.

Bee Pollen Diet Alters the Bacterial Flora and Antimicrobial Peptides in the Oral Cavities of Mice.

BACKGROUND: Bee pollen (BP) has a broad range of beneficial effects on health. The aim of this study was to examine the effect of BP on the oral environment, including the microbiome and antimicrobial peptides. METHODS: C57BL/6J mice were randomly divided into two groups: control and BP. The BP group was fed with a 5% BP diet for 1 month. Swabs from the oral and buccal mucosa and samples of the intestinal stool were collected. Genomic DNA was extracted and the microbiome was analyzed via 16S rRNA sequencing. RESULTS: BP inhibited the growth of P. gingivalis at a concentration of >2.5%. The metagenomic study showed that the abundance of genus Lactococcus was significantly elevated in the oral and intestinal microbiomes of the BP group when compared to those of the control group. Significant alterations in alpha and beta diversity were observed between the oral microbiomes of the two groups. The mRNA levels of beta-defensin-2 and -3 were significantly upregulated in the buccal mucosa of the BP group. CONCLUSION: A BP diet may have a beneficial effect on oral and systemic health by modulating the bacterial flora and antimicrobial peptides of the oral cavity. Further investigations are needed to clarify how a BP diet affects overall human health.