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Dimethyl fumarate (DMF), originally proposed to treat multiple sclerosis, is considered to have a spectrum of anti-inflammatory effects that effectively control periodontitis, mainly when applied with a hydrogel delivery system. Chemokine expression by gingival fibroblasts is a significant driver of periodontitis; thus, hydrogel-based strategies to deliver DMF, which in turn dampen chemokine expression, are of potential clinical relevance. To test this approach, we have established a bioassay where chemokine expression is induced by exposing gingival fibroblast to IL1β and TNFα, or with saliva. We show herein that DMF effectively reduced the expression of CXCL8, CXCL1, CXCL2, and CCL2—and lowered the phosphorylation of ERK and JNK—without affecting cell viability. This observation was confirmed by immunoassays with CXCL8. Consistently, the forced chemokine expression in HSC2 oral squamous epithelial cells was greatly diminished by DMF. To implement our hydrogel-based delivery system, gingival fibroblasts were cocultured with gellan gum hydrogels enriched for DMF. In support of our strategy, DMF-enriched gellan gum hydrogels significantly reduced the forced chemokine expression in gingival fibroblasts. Our data suggest that DMF exerts its anti-inflammatory activity in periodontal cells when released from gellan gum hydrogels, suggesting a potential clinical relevance to control overshooting chemokine expression under chronic inflammatory conditions. [ABSTRACT FROM AUTHOR]

Oxidative stress and high levels of reactive oxygen species (ROS) are linked to various age-related diseases and chronic conditions, including damage to oral tissues. Dexamethasone (DEX), a widely used glucocorticoid in dentistry, can have side effects like increased ROS production and delayed wound healing. Resveratrol (RSV) is known for its antioxidant properties, but its limited bioavailability hinders its clinical use. This study investigated the potential of two RSV derivatives (1d and 1h) to address these limitations. The antioxidant abilities of 1d and 1h (5 μM) against DEX-induced oxidative stress (200 μM) were evaluated in human gingival fibroblasts (hGFs) and osteoblasts (hOBs). The effects of these compounds on cell viability, morphology, ROS levels, SOD activity, gene expression, and collagen production were evaluated. RSV derivatives, under DEX-induced oxidative stress condition, improved cell growth at 72 h (191.70 ± 10.92% for 1d+DEX and 184.80 ± 13.87% for 1h+DEX), morphology, and SOD activity (77.33 ± 3.35 OD for 1d+DEX; 76.87 ± 3.59 OD for 1h+DEX at 1 h), while reducing ROS levels (2417.33 ± 345.49 RFU for 1d+DEX and 1843.00 ± 98.53 RFU at 4 h), especially in hOBs. The co-treatment of RSV or derivatives with DEX restored the expression of genes that were downregulated by DEX, such as HO-1 (1.76 ± 0.05 for 1d+DEX and 1.79 ± 0.01 for 1h+DEX), CAT (0.97 ± 0.06 for 1d+DEX and 0.99 ± 0.03 for 1h+DEX), NRF2 (1.62 ± 0.04 for 1d+DEX and 1.91 ± 0.05 for 1h+DEX), SOD1 (1.63 ± 0.15 for 1d+DEX and 1.69 ± 0.04 for 1h+DEX). In addition, 1d and 1h preserved collagen production (111.79 ± 1.56 for 1d+DEX and 122.27 ± 1.56 for 1h+DEX). In conclusion, this study suggests that the RSV derivatives 1d and 1h hold promise as potential antioxidant agents to counteract DEX-induced oxidative stress. These findings contribute to the development of novel therapeutic strategies for managing oxidative stress-related oral conditions. [ABSTRACT FROM AUTHOR]

Several in vitro studies utilizing 2-dimensional (2D) cell culture systems have linked 2-hydroxyethyl methacrylate (HEMA) with cytotoxic effects in oral mucosa and dental pulp cells. Although such studies are invaluable in dissecting the cellular and molecular effects of HEMA, there is a growing interest in the utilization of appropriate 3-dimensional (3D) models that mimic the structure of oral mucosa. Using a previously characterized 3D-organotypic co-culture model, this study aimed to investigate the cellular and molecular effects of HEMA on a 3D-co-culture model consisting of primary normal oral keratinocyte (NOK) grown directly on top of collagen I gel containing primary oral fibroblasts (NOF). The second aim was to examine the suitability of a 3D-co-culture system consisting of oral squamous cell carcinoma (OSCC) cells as a model system to investigate the biological effects of HEMA. We demonstrated that HEMA treatment led to reduced viability of NOK, NOF and OSCC-cell lines in 2D-culture. The keratinocytes in 3D-co-cultures of NOK and OSCC-cells reacted similarly with respect to cell proliferation and activation of autophagy flux, to HEMA treatment. Nevertheless, NOK was found to be more susceptible to apoptosis following HEMA treatment than OSCC in 3D-co-cultures. These results indicate that 3D-organotypic co-cultures of NOK might represent an appropriate model system for the investigation of the biological effects of HEMA and other dental biomaterials. Given the challenges in obtaining primary cultures of NOK and issues associated with their rapid differentiation in culture, the possible use of OSCC cells as an alternative to NOK for 3D models represents an area for future research. [ABSTRACT FROM AUTHOR]

Oxidative stress and high levels of reactive oxygen species (ROS) are linked to various age-related diseases and chronic conditions, including damage to oral tissues. Dexamethasone (DEX), a widely used glucocorticoid in dentistry, can have side effects like increased ROS production and delayed wound healing. Resveratrol (RSV) is known for its antioxidant properties, but its limited bioavailability hinders its clinical use. This study investigated the potential of two RSV derivatives (1d and 1h) to address these limitations. The antioxidant abilities of 1d and 1h (5 μM) against DEX-induced oxidative stress (200 μM) were evaluated in human gingival fibroblasts (hGFs) and osteoblasts (hOBs). The effects of these compounds on cell viability, morphology, ROS levels, SOD activity, gene expression, and collagen production were evaluated. RSV derivatives, under DEX-induced oxidative stress condition, improved cell growth at 72 h (191.70 ± 10.92% for 1d+DEX and 184.80 ± 13.87% for 1h+DEX), morphology, and SOD activity (77.33 ± 3.35 OD for 1d+DEX; 76.87 ± 3.59 OD for 1h+DEX at 1 h), while reducing ROS levels (2417.33 ± 345.49 RFU for 1d+DEX and 1843.00 ± 98.53 RFU at 4 h), especially in hOBs. The co-treatment of RSV or derivatives with DEX restored the expression of genes that were downregulated by DEX, such as HO-1 (1.76 ± 0.05 for 1d+DEX and 1.79 ± 0.01 for 1h+DEX), CAT (0.97 ± 0.06 for 1d+DEX and 0.99 ± 0.03 for 1h+DEX), NRF2 (1.62 ± 0.04 for 1d+DEX and 1.91 ± 0.05 for 1h+DEX), SOD1 (1.63 ± 0.15 for 1d+DEX and 1.69 ± 0.04 for 1h+DEX). In addition, 1d and 1h preserved collagen production (111.79 ± 1.56 for 1d+DEX and 122.27 ± 1.56 for 1h+DEX). In conclusion, this study suggests that the RSV derivatives 1d and 1h hold promise as potential antioxidant agents to counteract DEX-induced oxidative stress. These findings contribute to the development of novel therapeutic strategies for managing oxidative stress-related oral conditions.

AbstractSeveral in vitro studies utilizing 2-dimensional (2D) cell culture systems have linked 2-hydroxyethyl methacrylate (HEMA) with cytotoxic effects in oral mucosa and dental pulp cells. Although such studies are invaluable in dissecting the cellular and molecular effects of HEMA, there is a growing interest in the utilization of appropriate 3-dimensional (3D) models that mimic the structure of oral mucosa. Using a previously characterized 3D-organotypic co-culture model, this study aimed to investigate the cellular and molecular effects of HEMA on a 3D-co-culture model consisting of primary normal oral keratinocyte (NOK) grown directly on top of collagen I gel containing primary oral fibroblasts (NOF). The second aim was to examine the suitability of a 3D-co-culture system consisting of oral squamous cell carcinoma (OSCC) cells as a model system to investigate the biological effects of HEMA. We demonstrated that HEMA treatment led to reduced viability of NOK, NOF and OSCC-cell lines in 2D-culture. The keratinocytes in 3D-co-cultures of NOK and OSCC-cells reacted similarly with respect to cell proliferation and activation of autophagy flux, to HEMA treatment. Nevertheless, NOK was found to be more susceptible to apoptosis following HEMA treatment than OSCC in 3D-co-cultures. These results indicate that 3D-organotypic co-cultures of NOK might represent an appropriate model system for the investigation of the biological effects of HEMA and other dental biomaterials. Given the challenges in obtaining primary cultures of NOK and issues associated with their rapid differentiation in culture, the possible use of OSCC cells as an alternative to NOK for 3D models represents an area for future research.

The use of electronic cigarettes (e‐cigarettes) has increased rapidly in the United States, especially among high school students. e‐Cigarettes contain some recognized carcinogens and may induce DNA damage in oral cells. The aim of this review is to summarize studies reporting DNA adducts or other types of DNA damage in oral cells in vitro or in vivo upon exposure to e‐cigarette vapor and to evaluate the possible connections between e‐cigarette exposure and oral cancer. Three databases including PubMed, Scopus, and EMBASE and gray literature were searched for articles published up to April 24, 2022. After screening 321 articles, we extracted 27 for further investigation. Based on the inclusion criteria, 22 articles were eligible for this review. The in vitro studies demonstrate that e‐cigarette liquid or vapor can induce DNA damage, oxidative stress, DNA double‐stranded breaks, apoptosis, cytotoxicity, and genotoxicity in different types of oral cells. The clinical studies showed that e‐cigarette users have significantly higher levels of N′‐nitrosonornicotine, acrolein DNA adducts, metanuclear anomalies, gene regulation, and lactate dehydrogenase enzyme expression and significantly lower levels of apurinic/apyrimidinic sites than non‐users. Comparison of micronuclei levels between e‐cigarette users and non‐users gave inconsistent results. e‐Cigarettes are implicated in DNA damage to oral cells, but publications to date present limited evidence. Future studies with larger sample sizes are required to investigate the long‐term consequences of e‐cigarette use. [ABSTRACT FROM AUTHOR]

Objectives: To study whether damaged epithelial cells and gingival fibroblast could affect the expression of inflammatory cytokines in healthy cells. Materials and methods: Cell suspensions were submitted to different treatments to obtain the lysates: no treatment (supernatant control), sonication, and freeze/thawing. All treatments were centrifuged, and the supernatants of the lysates were used for experimentation. Cell viability assays, RT-qPCR of IL1, IL6 and IL8, IL6 immunoassay, and immunofluorescence of NF-kB p65 were applied to verify the inflammatory crosstalk of damaged cells over healthy plated cells. Furthermore, titanium discs and collagen membranes were treated with lysates and checked for IL8 expression by RT-qPCR. Results: Lysates obtained upon sonication or freeze/thawing of oral squamous carcinoma cell lines provoked a robust increase in the expression of IL1, IL6, and IL8 by gingival fibroblasts, which was confirmed by IL6 immunoassays. Lysates obtained from the gingival fibroblasts failed to increase the expression of inflammatory cytokines in oral squamous carcinoma cells. Additionally, oral squamous carcinoma cell lysates caused the activation of the NF-kB signalling cascade in gingival fibroblasts as indicated by the phosphorylation and nuclear translocation of p65. Finally, oral squamous carcinoma cell lysates adhered to the titanium and collagen membrane surfaces and increased IL8 expression by gingival fibroblasts growing in these materials. Conclusions: Injured oral epithelial cells can release factors that incite gingival fibroblasts to become pro-inflammatory. Clinical relevance: Injuries affecting the oral mucosa generate epithelial fragments that may reach the underlying connective tissue and provoke inflammation. These injuries are routinely caused by mastication, sonication for teeth cleaning, teeth preparation, prostheses maladaptation, and implant drilling. [ABSTRACT FROM AUTHOR]

Necrotic cell damage occurs as a consequence of invasive dental procedures. Loss of membrane integrity being the hallmark of necrotic cells leads to the release of cytoplasmic and membranous components. Macrophages are predestined to respond to lysates originating from necrotic cells. Here, we implement necrotic lysates from human gingival fibroblasts, HSC2, and TR146 oral epithelial cell lines, and RAW264.7 macrophage cell lines to be tested for their potential to modulate the inflammatory response of macrophages. To this aim, necrotic cell lysates were prepared by sonication or freezing/thawing of the respective cell suspension. Necrotic cell lysates were tested for their potential to modulate the lipopolysaccharide (LPS)-induced expression of inflammatory cytokines using RAW264.7 macrophages as a bioassay. We show here that all necrotic cell lysates, independent of the origin and the preparation way, reduced the expression of IL1 and IL6 in LPS-induced RAW264.7 macrophages, most obviously shown for TR146 cells. This finding was supported in a bioassay when macrophages were exposed to poly (I:C) HMW, an agonist of TLR-3. Consistently, all necrotic lysates from gingival fibroblasts, HSC2, TR146, and RAW264.7 cells reduced the nuclear translocation of p65 in LPS-exposed macrophages. This screening approach supports the overall concept that necrotic cell lysates can modulate the inflammatory capacity of macrophages. [ABSTRACT FROM AUTHOR]

Rutin is a bioflavonoid found mostly in medicinal plants, but also in food, and other products. Throughout the years, rutin has been proven to exhibit important pharmacological properties, including antioxidant, anti-inflammatory, and anti-cancer activities. Due to its low oral solubility, the flavonoid is preferable to be included in nanoformulas. The main aim of the present study was to assess the cytotoxic potential of rutin and rutin formulations on one healthy cell line (primary gingival keratinocytes - PGK) and two tumoral oral cell lines (tongue carcinoma - SCC-4 and pharynx carcinoma - Detroit 562). The in vitro assay employed was MTT test which revealed that rutin has a cytotoxic effect on cancer cells, without affecting the healthy ones, and its inclusion in liposomes increases its anticancer potential, especially at 10 and 25µM. The results warrant further detailed studies and in-depth anti-cancer mechanistic research. [ABSTRACT FROM AUTHOR]

Periodontitis patients suffering concomitantly from rheumatoid arthritis (RA) often present with less inflamed periodontal tissues due to the ongoing anti-rheumatic therapy. This in vitro study was aimed to analyze whether anti-inflammatory drugs used in the therapy of RA can modulate the release of IL-8 and IL-1β by professional and non-professional immune cells stimulated with microorganisms. Periodontal ligament (PDL) fibroblasts, monocytic MONO-MAC-6-cells, and gingival keratinocytes were exposed to ibuprofen, prednisolone, and methotrexate with and without lysates of Fusobacterium nucleatum or Candida albicans. Supernatants were obtained and the levels of interleukin(IL)-8 and IL-1β (only MONO-MAC-6) were quantified. The addition of F. nucleatum lysate resulted in the strongest release of proinflammatory cytokines by PDL fibroblast and MONO-MAC-6 cells, while the modification by the tested anti-rheumatic drugs was only minor. After stimulation of the MONO-MAC-cells with F. nucleatum, prednisolone increased the release of IL-8, whereas methotrexate decreased the level. Anti-inflammatory drugs increased the adherence of C. albicans to epithelial cells. In patients with RA, the reduction of the microbial load in subgingival biofilm (biofilm removal) is of major importance; however, the intake of inflammatory drugs may interfere with the inflammatory response.

Chronic arsenic exposure can lead to various health issues, including cancer. Concerns have been mounting about the enhancement of arsenic toxicity through co-exposure to various prevalent lifestyle habits. Smokeless tobacco products are commonly consumed in South Asian countries, where their use frequently co-occurs with exposure to arsenic from contaminated groundwater. To decipher the in vitro molecular and cellular responses to arsenic and/or smokeless tobacco, we performed temporal multi-omics analysis of the transcriptome and DNA methylome remodelling in exposed hTERT-immortalized human normal oral keratinocytes (NOK), as well as arsenic and/or smokeless tobacco genotoxicity and mutagenicity investigations in NOK cells and in human p53 knock-in murine embryonic fibroblasts (Hupki MEF). RNAseq results from acute exposures to arsenic alone and in combination with smokeless tobacco extract revealed upregulation of genes with roles in cell cycle changes, apoptosis and inflammation responses. This was in keeping with global DNA hypomethylation affecting genes involved in the same processes in response to chronic treatment in NOK cells. At the phenotypic level, we observed a dose-dependent decrease in NOK cell viability, induction of DNA damage, cell cycle changes and increased apoptosis, with the most pronounced effects observed under arsenic and SLT co-exposure conditions. Live-cell imaging experiments indicated that the DNA damage likely resulted from induction of apoptosis, an observation validated by a lack of exome-wide mutagenesis in response to chronic exposure to arsenic and/or smokeless tobacco. In sum, our integrative omics study provides novel insights into the acute and chronic responses to arsenic and smokeless tobacco (co-)exposure, with both types of responses converging on several key mechanisms associated with cancer hallmark processes. The generated rich catalogue of molecular programs in oral cells regulated by arsenic and smokeless tobacco (co-)exposure may provide bases for future development of biomarkers for use in molecular epidemiology studies of exposed populations at risk of developing oral cancer., Competing Interests: DECLARATION OF COMPETING INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Epidemiology studies link cigarillos and shisha tobacco (delivered through a hookah waterpipe) to increased risk for cardiopulmonary diseases. Here we performed a comparative chemical constituent analysis between 3 cigarettes, 3 cigarillos, and 8 shisha tobacco products. The potency for genotoxicity and oxidative stress of each product's generated total particulate matter (TPM) was also assessed using immortalized oral, lung, and cardiac cell lines to represent target tissues. Levels of the carcinogenic carbonyl formaldehyde were 32- to 95-fold greater, while acrolein was similar across the shisha aerosols generated by charcoal heating compared to cigarettes and cigarillos. Electric-mediated aerosol generation dramatically increased acrolein to levels exceeding those in cigarettes and cigarillos by up to 43-fold. Equivalent cytotoxic-mediated cell death and dose response for genotoxicity through induction of mutagenicity and DNA strand breaks was seen between cigarettes and cigarillos, while minimal to no effect was observed with shisha tobacco products. In contrast, increased potency of TPM from cigarillos compared to cigarettes for inducing oxidative stress via reactive oxygen radicals and lipid peroxidation across cell lines was evident, while positivity was seen for shisha tobacco products albeit at much lower levels. Together, these studies provide new insight into the potential harmful effects of cigarillos for causing tobacco-associated diseases. The high level of carbonyls in shisha products, that in turn is impacted by the heating mechanism, reside largely in the gas phase which will distribute throughout the respiratory tract and systemic circulation to likely increase genotoxic stress. [ABSTRACT FROM AUTHOR]

Telomere length, measured from blood cells, is commonly used as the standard telomere length of the whole body. The relationship between blood telomere length and oral telomere length is still unclear, especially among neonates. In this study, we measured blood telomere length as well as cheek cells telomere length to find out the overall telomere synchronization in neonates. Children aged 1 month or younger were included in this study. Blood and cheek cells were collected by heel stick method and nylon brush accordingly. Telomere length was measured by Southern blotting. A strong telomere length correlation (0.77, p<0.001) existed between oral and blood cells which showing telomere length synchronization among different tissue types. Mean telomere length and length variability were significantly higher (t=3.73, p=0.0004) in oral cells. Longer telomere among oral cells can be justified by progenitor cell pool theory. The cause of high telomere length variability in the oral source is not clear although the presence of leukocytes among cheek cells cannot be excluded. Therefore, for telomere length measurement purpose blood should be given preference over the oral source. [ABSTRACT FROM AUTHOR]

Astringency and bitterness are organoleptic properties widely linked to tannin compounds. Due to their significance to food chemistry, the food industry, and to human nutrition and health, these tannins’ taste properties have been a line of worldwide research. In recent years, significant advances have been made in understanding the molecular perception of astringency pointing to the contribution of different oral key players. Regarding bitterness, several polyphenols have been identified has new agonists of these receptors. This review summarizes the last data about the knowledge of these taste properties perceived by tannins. Ultimately, tannins’ astringency and bitterness are hand-in-hand taste properties, and future studies should be adapted to understand how the proper perception of one taste could affect the perception of the other one.

Background: The bacterial biofilm at the gingival margin induces a host immune reaction. In this local inflammation epithelial cells defend the host against bacterial challenge. Porphyromonas gingivalis (P. gingivalis), a keystone pathogen, infects epithelial cells. The aim of this study was to investigate the activation of signaling cascades in primary epithelial cells and oral cancer cell lines by a profiler PCR array. Results: After infection with P. gingivalis membranes the RNA of 16 to 33 of 84 key genes involved in the antibacterial immune response was up-regulated, amongst them were IKBKB (NF-κB signaling pathway), IRF5 (TLR signaling) and JUN, MAP2K4, MAPK14 and MAPK8 (MAPK pathway) in SCC-25 cells and IKBKB, IRF5, JUN, MAP2K4, MAPK14 and MAPK8 in PHGK. Statistically significant up-regulation of IKBKB (4.7 ×), MAP2K4 (4.6 ×), MAPK14 (4.2 ×) and IRF5 (9.8 ×) (p < 0.01) was demonstrated in SCC-25 cells and IKBKB (3.1 ×), MAP2K4 (4.0 ×) MAPK 14 (3.0 ×) (p < 0.05), IRF5 (3.0 ×) and JUN (7. 7 ×) (p < 0.01) were up-regulated in PHGK. Conclusions: P. gingivalis membrane up-regulates the expression of genes involved in downstream TLR, NFκB andMAPK signaling pathways involved in the pro-inflammatory immune response in primary and malignant oral epithelial cells. [ABSTRACT FROM AUTHOR]

Objectives: This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs.Methods: The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 105cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using acytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett’s test and additionalt-tests.Results: The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005).Conclusions: OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells. [ABSTRACT FROM AUTHOR]

Objectives: Bisphosphonates-related osteonecrosis of the jaw (BRONJ) is a common problem in patients undergoing long-term administration of highly potent nitrogen-containing bisphosphonates (N-BPs). This pathology occurs via bone and soft tissue mechanism. Zoledronic acid (ZA) is the most potent intravenous N-BP used to prevent bone loss in patients with bone dysfunction. The objective of this in vitro study was to evaluate the role of different ZA concentrations on the cells from human oral cavity, as well as the potential of plasma rich in growth factors (PRGF) to overcome the negative effects of this BP. Material and methods: Primary human gingival fibroblasts and primary human alveolar osteoblasts were used. Cell proliferation was evaluated by means of a fluorescence-based method. A colorimetric assay to detect DNA fragmentation undergoing apoptosis was used to determine cell death, and the expression of both NF-κB and pNF-κB were quantified by Western blot analysis. Results: ZA had a cytotoxic effect on both human gingival fibroblasts and human alveolar osteoblasts. This BP inhibits cell proliferation, stimulates apoptosis, and induces inflammation. However, the addition of PRGF suppresses all these negative effects of the ZA. Conclusions: PRGF shows a cytoprotective role against the negative effects of ZA on primary oral cells. Clinical relevance: At present, there is no definitive treatment for bisphosphonates-related osteonecrosis of the jaw (BRONJ), being mainly palliatives. Our results revealed that PRGF has a cytoprotective role in cells exposed to zoledronic acid, thus providing a reliable adjunctive therapy for the treatment of BRONJ pathology. [ABSTRACT FROM AUTHOR]

This review article summarizes the recent progress in dental applications of natural products. Catechin gel showed selective antimicrobial activity, whereas the alkaline extract of various plant species rich in lignin carbohydrate complex (LCC) showed much higher antiviral activity than lower molecular weight polyphenols. Mouthwash with the alkaline extract of a plant classified as OTC effectively reduced halitosis. Unexpectedly, many polyphenolic compounds purified from the natural kingdom showed much lower tumor-specificity against human oral squamous cell lines as compared with antitumor agents, although they showed apoptosis-inducing activity. The alkaline extract of bamboo leaf, which exerted various common biological activities with LCC, showed osteogenic activity by stimulating differentiation toward osteoblasts while inhibiting differentiation toward osteoclasts. LCC enhanced the dectin-2 mRNA expression in macrophages, whereas glucan showed anti-osteoblastic action via dectin-1. These data suggest that natural products exert their biological activity by interacting with these molecules.

Objective To investigate the effect of the roselle calyx extract (RCE) ( Hibiscus sabdariffa L.) on the in vitro viability and biofilm formation ability of oral pathogenic bacteria. Methods RCE was prepared by soaking roselle calyx powder with ethyl alcohol for 24 h at room temperature. After centrifugation, the extract was lyophilized. Then, the extract was dissolved in phosphate-buffered saline, the pH was adjusted, and the extract was aseptically filtered. We used Streptococcus mutans , Streptococcus sanguinis , Lactobacillus casei , Actinomyces naeslundii , Aggregatibacter actinomycetemcomitans , Fusobacterium nucleatum , Porphyromonas gingivalis and Prevotella intermedia in this study. The antibacterial activity of the RCE was determined by treating the cells of these bacteria with the extract for 10 or 20 min at room temperature. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration was determined using the microdilution method, and the effect of the RCE on the ability to form biofilm was determined using a polystyrene micro plate assay. In addition, we used the WST-1 assay to determine the cytotoxicity of the RCE on HGF, Ca9-22 and KB cells. Results The RCE had antibacterial activity against oral bacteria used in this study. In particular, most significant antibacterial activity was observed against Fusobacterium nucleatum , Prevotella intermedia and Porphyromonas gingivalis . The MIC and minimum bactericidal concentration were 7.2 mg/mL–28.8 mg/mL and 14.4 to >57.6 mg/mL. The RCE had an inhibitory effect on biofilm formation at the MIC and sub-MIC levels. In addition, the RCE had low cytotoxic effects on HGF, Ca9-22 and KB cells. Conclusions Thus, our results indicate that the RCE may be used for preventing oral diseases. [ABSTRACT FROM AUTHOR]

Aim The aim of this study was to investigate the smoking habit influence on DNA methylation status in the promoters of the cancer related-genes MLH1 , hTERT and TP53 in oral epithelial cells of healthy subjects. Materials and methods DNA methylation analysis was performed using methylation-sensitive restriction enzymes (MSRE) in oral epithelial cells from non-smokers, smokers and ex-smokers. Results The investigated CpG dinucleotides located at Hha I and Hpa II sites in the MLH1 gene promoter were observed to be fully methylated in the majority of DNA samples from the smoker group and statistical differences were found between non-smokers and smokers and between smokers and ex-smokers ( p < 0.05). The same was observed in the hTERT gene promoter at Hha I sites ( p < 0.05) and for Hpa II sites the unmethylated condition was more frequent in smokers in comparison to non-smokers ( p < 0.05). For TP53 , no differences were found among groups ( p > 0.05), with the fully methylated condition found to be a common event in healthy oral epithelial cells. Conclusion We conclude that smoking may induce changes in DNA methylation status in cancer-related genes of oral epithelial cells and that the cessation of smoking is capable of reversing this process. Based on our data, we suggest that DNA methylation status of the hTERT and MLH1 gene promoters are promising markers for screening a set of smoking-related alterations in oral cells. [ABSTRACT FROM AUTHOR]

In the context of cell-based oral hard tissue regeneration, especially assumed plasticity of oral host tissue cells in response to human mesenchymal stem cells (hMSCs), is poorly understood. To investigate this area, we assess osteogenic features in various oral cell types during hMSC coculture, including human alveolar osteoblasts (hOAs), periodontal ligament cells (hPDLs) and gingival fibroblasts (hGFs). Interactive hMSC coculture globally enhanced the transcription of osteogenic genes, in all oral cell types under study, as revealed by qRT-PCR and did not affect oral cell proliferation compared with controls in a transwell coculture system as evaluated by 5-bromo-2′-deoxyuridine proliferation assay. 3D gel-derived hMSC cocultures exhibited an abundance of bone-related key molecules in oral cells, which followed the ranking hOAs > hGFs > hPDLs. Compared to matched controls, this hierarchy also applied for the presence of higher amounts of extracellular matrix deposits and mineralization nodules in interactive hMSC coculture. Our results show for the first time that in the context of prospective periodontal tissue regeneration strategies, hMSCs influence oral cells by gradually shaping their plasticity, particularly features associated with an osteogenic phenotype. These novel findings contribute another piece to the conceptual hMSC action puzzle and valuably support the notion that hMSCs trigger osteogenesis in the oral cell context. [ABSTRACT FROM AUTHOR]

Astringency is a highly complex sensation which involves multiple mechanisms occurring simultaneously, such as the interaction between flavan-3-ols and salivary proteins (SP). Moreover, astringency development can be affected by the presence of polysaccharides such as mannoproteins (MP). The aim of this work was to evaluate the molecular mechanisms whereby MP could modulate the astringency elicited by tannins, using a cell-based model of the oral epithelium (TR146 cells), and the effect of salivary proteins on these interactions. The binding of flavan-3-ols to oral cells was evaluated by DMACA assay, while the content of unbound flavan-3-ols after the interactions was assessed by means of HPLC-DAD-MS. Results obtained confirm the existence of cell-tannin interactions, that can be partially inhibited by the presence of SP and/or MP. The most significant decrease was obtained in the system containing MPF (38.16%). Both mannoproteins assayed seem to have modulating effect on flavan-3-ol-SP interactions, acting by two different mechanisms: MPF would lead to the formation of SP/MPF/flavan-3-ols ternary soluble aggregates, while MPL seems to prevent flavan-3-ol-saliva interaction by a competitive mechanism, i.e., MPL would reduce cell-tannin interactions, similar to SP. This study suggests that mannoproteins with different compositional characteristics could exhibit preferential interaction with distinct flavan-3-ol families.

Backround: The human oral cells have different physiological properties and different origins in developmental stages. Mechanical, physiological, and chemical stress can cause damage and irritation during clinical treatment in various oral tissues. Purpose: The effects of DNA damage response and gene silencing of checkpoint kinases (Chk1/2) is not unclear in oral primary and cancer cells. Method: Treatment with doxorubicin involving DNA damage and gene silencing of Chk1/2 by shRNA constructs was performed in pulp, periodontal ligament, gingival tissues (HGF), and mouth epithelial carcinoma cells (KB). Results: The KB cells were more sensitive to genotoxic stress response than oral primary cells. Endogenous levels of Chk1/2 in KB cell were higher than in pulp cells. When doxorubicin was administered, Chk2 activation was induced in KB cell, but not in pulp cells. However, viability in KB cells did not decrease by the suppression of the checkpoint proteins, whereas primary cells were defective in gene silencing. When doxorubicin treatment and gene silencing were combined, both primary cells and KB cell were defective. Moreover, in case of KB cell, cell death was increased and activation of Chk2 was increased in doxorubicin dose-dependent. Conclusion: These data indicate that not only stress response mechanism may be different in oral primary and cancer cells but also Chk1/2 proteins may have essential roles in oral primary cells. Based on these data, checkpoint proteins may be crucial drug targets for oral cancer therapy. [ABSTRACT FROM AUTHOR]

• Gallotannins and flavonols bind in different ways to oral constituents. • Salivary proteins were crucial contributors to gallotannin-driven interactions. • Interactions involving flavonols appeared to be oral cell-dependent. • Salivary proteins–flavonols interactions were negligible. • Astringency sub-qualities can be associated to different types of interactions. Astringency is a tactile sensation of puckering, tightening and dryness in the oral cavity, commonly induced by polyphenols. In this study, the interaction of two phenolic compound mixtures, one rich in gallotannins and the other in flavonols, with two oral models (tongue (HSC3) or buccal mucosa (TR146) was evaluated. Results provided evidence that gallotannins and flavonols seem to bind in a different way to the different oral constituents and models used. Gallotannins seems to bind more to the tongue than to the buccal mucosa cell line, but this difference is overcome by the presence of salivary proteins. Conversely, for the flavonol mixture, the presence of salivary proteins seems to restrain the interaction with oral cell lines. Structure–binding activity relationships were evidenced within each mixture: for gallotannins, interactions seem to increase along with the galloylation degree while for flavonol it was observed that increasing numbers of glucose residues decreased the binding activity. [ABSTRACT FROM AUTHOR]

The effect of thimerosal on cytosolic free Ca2+ concentrations ([Ca2+]i) in human oral cancer cells (OC2) is unclear. This study explored whether thimerosal changed basal [Ca2+]i levels in suspended OC2 cells using fura-2. Thimerosal at concentrations between 1and 50 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Thimerosal-induced Ca2+ influx was not blocked by L-type Ca2+ entry inhibitors and protein kinase C modulators (phorbol 12-myristate 13-acetate [PMA] and GF109203X). In Ca2+-free medium, 50 μM thimerosal failed to induce a [Ca2+]i rise after pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with U73122 did not change thimerosal-induced [Ca2+]i rises. At concentrations between 5 and 10 μM, thimerosal killed cells in a concentration-dependent manner. The cytotoxic effect of 8 μM thimerosal was potentiated by prechelating cytosolic Ca2+ with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetate/acetomethyl (BAPTA/ AM). Flow cytometry data suggested that 1-7 μM thimerosal-induced apoptosis in a concentrationdependent manner. Collectively, in OC2 cells, thimerosal-induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx through non-L-type Ca2+ channels. Thimerosal killed cells in a concentrationdependent manner through apoptosis. [ABSTRACT FROM AUTHOR]

Leukoplakia is the most common precancerous lesion in oral mucous, but exist many factors that promote their development. The objective of our work was identify and quantify the cytogenetics alterations (micronucleus) and nuclear atypia (binucleation, karyorrhexis, condensed chromatin and karyolysis) in oral cells collected of subjects health and with leukoplakia, and to evaluate their possible relation with exogenous factors (life style). We carry out a case-control study with 43 adults subjects, with a range of 23-75 years old, distributed in two groups, healthy and no healthy subjects. They were asked to determine personal aspects like cigars and alcoholic drink consume and vegetables and fruits consumption. The results suggest that in our study group exist in the subjects with the lesion, a significative increase of nuclear atypia, but low frequency of micronucleus. We determine that sex and age are not important endogen factors in the premalignant process, neither in alterations in frequency of micronucleus and nuclear atypia, but it was different with the tobacco habit and alcoholic drink factors. [ABSTRACT FROM AUTHOR]

For many years, liquid-based cytology (LBC) has been developed for cervical cancer screening and not oral cancer, as it requires automated devices. The aim of this study was to compare the utility of centrifugated CLBC preparation with that of direct preparation in oral lesions, by Papanicolaou (Pap) and May Grünwald-Giemsa's (MGG) methods. A total of 100 consecutive cases of oral lesions were investigated. We compared the results obtained by the CLBC performed by cytocentrifugation with those obtained by direct smear applying Pap and MGG methods. The comparison between CLBC and direct smears was based on the thickening or adequacy of the smear, distribution of cells and staining quality. All smears in CLBC and direct preparation were found adequate. For thickness of the smear, 40% and 42% were excellent, 33% and 30% were good, and 27% and 28% were acceptable by LBC and direct preparation, respectively. For the distribution of cells and scantiness of background elements, 92 (92%) smears of the CLBC have revealed clear, well distributed smears, compared to 70 (70%) of those in direct preparation. For the staining quality with the Pap method, 39% and 69% were excellent staining quality, 25% and 20% were good, and 36% and 11% were acceptable for CLBC and direct preparation, respectively. In MGG method, 9% and 22% were excellent staining quality, 23% and 36% were good and 68% and 43% were acceptable for CLBC and direct preparation respectively. CLBC performed by cytocentrifugation is inexpensive, and reduces inadequate smears and background staining. [ABSTRACT FROM AUTHOR]

Astringency and bitterness are organoleptic properties widely linked to tannin compounds. Due to their significance to food chemistry, the food industry, and to human nutrition and health, these tannins' taste properties have been a line of worldwide research. In recent years, significant advances have been made in understanding the molecular perception of astringency pointing to the contribution of different oral key players. Regarding bitterness, several polyphenols have been identified has new agonists of these receptors. This review summarizes the last data about the knowledge of these taste properties perceived by tannins. Ultimately, tannins' astringency and bitterness are hand-in-hand taste properties, and future studies should be adapted to understand how the proper perception of one taste could affect the perception of the other one. [ABSTRACT FROM AUTHOR]

Background/aim: Genotoxicity is the capacity of an agent to induce damage to DNA. Given the close relationship between genotoxicity and carcinogenesis, several assays have been developed for detecting genetic damage. Among them, the single-cell gel (comet) assay plays an important role for evaluating DNA damage in mammalian cells, including those of the oral cavity. The purpose of this article was to provide a critical review of the application of single-cell gel comet assay to buccal cells., Material and Methods: A search of the scientific literature was conducted of published studies available on single-cell gel comet assay and oral cells., Results: The results showed that the majority of studies were conducted on humans, whereas few were designed for use in rodents and in vitro., Conclusion: Further studies within the field are relevant for better understanding the underlying mechanisms of genotoxicity in oral cells, especially since the use of humans is quite complicated due to issues of ethics., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

Background: Chemo-mechanical caries removal eliminates the outermost portion of the infected layer, leaving behind healthy dentine surfaces, with scarce dental tissue damage; however, the safety of caries solvents has not been established. The aim of the present study was to investigate the possible cytotoxicity of two popular chemo-mechanical caries removal agents., Materials and Methods: The cytotoxicity of Carisolv, Papacarie Duo and control vehicle solution (0.155-20% v/v) against human oral squamous cell carcinoma cells (HCS-2, HSC-3, HSC-4, Ca9-22) human gingival fibroblast (HGF), pulp (HPC) and periodontal ligament fibroblast (HPLF) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Prostaglandin E2 (PGE2) was quantified by enzyme-linked immunosorbent assay. Changes in fine cell structure were assessed by transmission electron microscopy., Results: Carisolv exhibited neither cytotoxicity nor hormetic growth stimulation. Papacarie Duo significantly reduced the viable cell number within 30 min. HSC-4 exhibited the highest sensitivity, followed by HSC-2>HSC-3>HPLF>Ca9-22>HPC>HGF cells. Interleukin-1β (3 ng/ml) stimulated HGF, but not HPC cells to produce PGE2 in the culture medium. Papacarie Duo stimulated HGF cells to produce PGE2 in synergistic fashion with interleukin-1β., Conclusion: Carisolv had acceptable biocompatibility with both normal and cancerous oral cells. On the other hand, Papacarie Duo had a rapid but slight cytotoxicity and pro-inflammatory action against oral cells, suggesting the importance of careful application of this agent., (Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)