Abstract Type VII collagen (Col7) is a major component of anchoring fibrils. Col7 plays a role in tumor development and aggressiveness of cutaneous squamous cell carcinoma of recessive dystrophic epidermolysis bullosa. However, the role of Col7 in oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL) remains largely unknown. Objective To elucidate the role of Col7 and its diagnostic potential during oral carcinogenesis. Methodology Col7 expression was immunohistochemically studied in 254 samples, including normal oral mucosa (NM), OL without dysplasia, OL with dysplasia, and OSCC. The correlation between Col7 expression and clinicopathologic parameters of OSCC was also determined. Results Col7 was present as a linear deposit at the basement membrane of NM, OL without dysplasia and OL with dysplasia, and at the tumor-stromal junction around tumor islands in OSCC. Discontinuity of expression was frequently observed in OL with dysplasia and OSCC. OSCC had the significantly lowest Col7 expression (p<0.0001). Compared with OL without dysplasia, OL with dysplasia showed significantly reduced Col7 expression. Patients in clinical stage 4 with positive nodes had low Col7 expression compared with those in clinical stage 1 and negative nodes, respectively. Conclusion Loss of Col7 is associated with tumorigenesis and aggressiveness in OSCC. A significantly reduced Col7 expression in OSCC implies that Col7 may be a useful marker for diagnosis and therapeutic targets.

Abstract The use of cocaine and its main derivative, crack, can cause some systemic effects that may lead to the development of some oral disorders. Objective To assess the oral health of people with a crack cocaine use disorder and identify salivary protein candidates for biomarkers of oral disorders. Methodology A total of 40 volunteers hospitalized for rehabilitation for crack cocaine addiction were enrolled; nine were randomly selected for proteomic analysis. Intraoral examination, report of DMFT, gingival and plaque index, xerostomia, and non-stimulated saliva collection were performed. A list of proteins identified was generated from the UniProt database and manually revised. Results The mean age (n=40) was 32 (±8.88; 18–51) years; the mean DMFT index was 16±7.70; the mean plaque and gingival index were 2.07±0.65 and 2.12±0.64, respectively; and 20 (50%) volunteers reported xerostomia. We identified 305 salivary proteins (n=9), of which 23 were classified as candidate for biomarkers associated with 14 oral disorders. The highest number of candidates for biomarkers was associated with carcinoma of head and neck (n=7) and nasopharyngeal carcinoma (n=7), followed by periodontitis (n=6). Conclusions People with a crack cocaine use disorder had an increased risk of dental caries and gingival inflammation; less than half had oral mucosal alterations, and half experienced xerostomia. As possible biomarkers for 14 oral disorders, 23 salivary proteins were identified. Oral cancer and periodontal disease were the most often associated disorders with biomarkers.

Abstract Objective To characterize activated charcoal and 2% hydrogen peroxide-based toothpastes and investigate their effects on roughness, color change, and gloss of bulk-fill composite resin. Methodology Composite resin specimens (Aura Bulk Fill, SDI) were subjected to 5,000 brushing cycles with regular toothpaste (Colgate Total 12, [RT]), activated charcoal toothpaste (Bianco Dental Carbon, [AC]), or hydrogen peroxide-containing toothpaste (Colgate Luminous White Advanced, [HP]), with or without coffee exposure. The pH, particle characterization by scanning electron microscopy (SEM), and weight% of solid particles in the toothpaste were assessed. Roughness (Ra) was evaluated using a surface profile-measuring device, color change (∆Eab/∆E00) by reflectance spectrophotometer, and gloss unit (GU) by glossmeter. Kruskal–Wallis, Dunn, Friedman, and Nemenyi tests were used, and the correlation coefficient test was performed between Ra and GU (α=0.05). Results RT presented a higher Ra after brushing and did not change after staining with coffee; moreover, the ∆Eab/∆E00 values were higher for RT than HP. Regarding gloss, AC and HP obtained higher values compared to RT. A significant negative correlation between gloss and Ra was found for RT exposed to coffee. All toothpastes had a neutral pH; however, RT had the largest percentage of solids by weight. SEM images showed particles of many sizes: irregular shape (RT), more regular particles (AC), and spherical clusters (HP). Although surface roughness, color change, and gloss may compromise the longevity of restorations, the whitening toothpastes tested did not promote more morphology changes than regular toothpastes.

Abstract Oral potentially malignant disorders (OPMD) are associated with an increased risk of oral squamous cell carcinoma (OSCC). OSCC has an aggressive profile and is the most prevalent among different head and neck malignancies. Most OSCC patients are diagnosed with advanced stage tumors and have a poor prognosis. Cancer cells are able to reprogram their metabolism, even in the presence of oxygen, enhancing the conversion of glucose to lactate via the glycolytic pathway, a phenomenon mainly regulated by hypoxia-inducible factor (HIF) signaling. Thus, several glycometabolism-related biomarkers are upregulated. Objectives This study aimed to evaluate the immunoexpression of the HIF targets GLUT1, GLUT3, HK2, PFKL, PKM2, pPDH, LDHA, MCT4, and CAIX in OPMD and OSCC samples, in order to identify potential correlations between biomarkers’ immunoexpression, clinicopathological features, and prognostic parameters. Methodology OSCC and OPMD samples from 21 and 34 patients (respectively) were retrospectively collected and stained for the different biomarkers by immunohistochemistry. Results CAIX and MCT4 expressions were significantly higher in OSCC samples when compared with OPMD samples, while the rest were also expressed by OPMD. GLUT3 and PKM2 alone, and the concomitant expression of more than four glycometabolism-related biomarkers were significantly correlated with the presence of dysplasia in OPMD. When considering OSCC cases, a trend toward increased expression of biomarkers and poor clinicopathological features was observed, and the differences regarding HK2, PFKL, LDHA and MCT4 expression were significant. Moreover, HK2 and CAIX were correlated with low survival rates. GLUT1 and GLUT3 were significantly associated with poor outcome when their expression was observed in the hypoxic region of malignant lesions. Conclusion OPMD and OSCC cells overexpress glycolysis-related proteins, which is associated with aggressive features and poor patient outcome. Further research is needed to deeply understand the glycolic phenotype in the process of oral carcinogenesis.

Abstract Objective To choose a critical animal model for assessments of bone repair with implant installation by comparing senile rats (SENIL) to young ovariectomized rats (OXV). Methodology For the ex-in vivo study, the femurs were precursors for bone marrow mesenchymal stem cells. Cellular responses were performed, including cell viability, gene expression of osteoblastic markers, bone sialoprotein immunolocalization, alkaline phosphatase activity, and mineralized matrix formation. For the in vivo study, the animals received implants in the region of the bilateral tibial metaphysis for histometric, microtomography, reverse torque, and confocal microscopy. Results Cell viability showed that the SENIL group had lower growth than OVX. Gene expression showed more critical responses for the SENIL group (p<0.05). The alkaline phosphatase activity obtained a lower expression in the SENIL group, as for the mineralization nodules (p<0.05). The in vivo histological parameters and biomechanical analysis showed lower data for the SENIL group. The confocal microscopy indicated the presence of a fragile bone in the SENIL group. The microtomography was similar between the groups. The histometry of the SENIL group showed the lowest values (p<0.05). Conclusion In experimental studies with assessments of bone repair using implant installation, the senile model promotes the most critical bone condition, allowing a better investigation of the properties of biomaterials and topographic changes.

Abstract Objective This clinical study aimed to evaluate the clinical performance of an alkasite-based bioactive material by comparing it with a resin composite (RC) in the restoration of Class II cavities over a year. Methodology A hundred Class II cavities were restored at 31 participants. Groups were as follows: Cention N (CN) (Ivoclar Vivadent, Schaan, Liechtenstein) and G-ænial Posterior (GP) (GC, Tokyo, Japan) in combination with G-Premio Bond (etch&rinse). Restorative systems were applied following manufacturers’ instructions. They were finished and polished immediately after placement and scored based on retention, marginal discoloration, marginal adaptation, sensitivity, surface texture, and color match using modified USPHS criteria after 1 week (baseline), 6 months, and 12 months. Statistical analyses were performed using chi-square, McNemar’s, and Kaplan Meier tests. Results After 12 months, the recall rate was 87%. Survival rates of CN and GP restorations were 92.5% and 97.7%, respectively. Three CN and one GP restorations lost retention. Seven CN (17.9%) and five (11.6%) GP restorations were scored as bravo for marginal adaptation and no significant difference was seen between groups (p=0.363). One (2.7%) CN and two GP (4.7%) restorations were scored as bravo for marginal discoloration, but no significant difference was observed between groups(p=1.00). For surface texture, three (8.1%) CN and three (7%) GP restorations were scored as bravo (p=1.00). None of the restorations demonstrated post-operative sensitivity or secondary caries at any examinations. Conclusion The tested restorative materials performed similar successful clinical performances after 12 months. ClinicalTrials.gov (NTC04825379).

Abstract Objective To evaluate the effects of fluoride (F) gels supplemented with micrometric or nano-sized sodium trimetaphosphate (TMPmicro and TMPnano, respectively) on the in vitro remineralization of caries-like lesions. Methodology Bovine enamel subsurface lesions (n=168) were selected according to their surface hardness (SH) and randomly divided into seven groups (n=24/group): Placebo (without F/TMP), 4,500 ppm F (4500F), 4500F + 2.5% TMPnano (2.5% Nano), 4500F + 5% TMPnano (5% Nano), 4500F + 5% TMPmicro (5% Micro), 9,000 ppm F (9000F), and 12,300 ppm F (Acid gel). The gels were applied in a thin layer for one minute. Half of the blocks were subjected to pH cycling for six days, whereas the remaining specimens were used for loosely- (calcium fluoride; CaF2) and firmly-bound (fluorapatite; FA) fluoride analysis. The percentage of surface hardness recovery (%SHR), area of subsurface lesion (ΔKHN), CaF2, FA, calcium (Ca), and phosphorus (P) on/in enamel were determined. Data (log10-transformed) were subjected to ANOVA and the Student-Newman-Keuls’ test (p<0.05). Results We observed a dose-response relation between F concentrations in the gels without TMP for %SHR and ΔKHN. The 2.5% Nano and 5% Micro reached similar %SHR when compared with 9000F and Acid gels. For ΔKHN, Placebo and 5% Nano gels had the highest values, and 5% Micro, 2.5% Nano, 9000F, and Acid gels, the lowest. All groups had similar retained CaF2 values, except for Placebo and Acid gel. We verified observed an increase in Ca concentrations in nano-sized TMP groups. Regarding P, TMP groups showed similar formation and retention to 9000F and Acid. Conclusion Adding 2.5% nano-sized or 5% micrometric TMP to low-fluoride gels lead to enhanced in vitro remineralization of artificial caries lesions.

Abstract Despite the recognized impact of diet on non-communicable diseases, the association between the Mediterranean diet and periodontal diseases is still uncertain. This study aimed to determine the association between adherence to the Mediterranean Diet Index (MDI) and self-reported gingival health status in Chilean adults, exploring the feasibility of using validated web-based survey questionnaires. Methodology Cross-sectional data were collected from a representative sample of a population of Chilean adults (18-60 years old) using a low-cost and time-saving methodology. By the PsyToolkit platform, anonymous survey data were downloaded and analyzed in bivariate (crude) and backward stepwise selection multivariate logistic regression models adjusted for sociodemographic determinants, smoking, and dental attendance using STATA 17. Odds ratios (OR) [95% confidence intervals] were estimated. Results In total, 351 complete statistical data were mostly obtained from female university students who had never smoked and reported having visited a dentist in the previous year. Multivariate regression models showed an association between MDI and very good/good gingival health status (OR 1.18 [95% CI 1.04-1.34], p=0.013), absence of bleeding on toothbrushing (OR 1.12 [95% CI 1.01-1.25], p=0.035), and absence of clinical signs of gingival inflammation (OR 1.24 [95% CI 1.10-1.40], p<0.001), after controlling for age, sex, educational level, smoking, and dental attendance. Conclusions We associated adherence to the Mediterranean diet with better self-reported gingival health status in a population of Chilean adults in an entirely web-based research environment. Longitudinal studies with random sampling are required to establish the effect of diet on gingival and periodontal health. Nevertheless, this evidence could contribute to the design of low-cost surveillance programs to reduce the burden of periodontal disease and related “common risk factors”.

Abstract Inflammation is a necessary step in response to injuries, being vital in restoring homeostasis and facilitating tissue healing. Among the cells that play a crucial role in inflammatory responses, stromal cells, including fibroblasts, have an undeniable significance in fine-tuning the magnitude of mediators that directly affect hyper-inflammatory responses and tissue destruction. Fibroblasts, the dominant cells in the gingival connective tissue, are a very heterogeneous population of cells, and more recently they have been receiving well deserved attention as central players and often the ‘principal dancers’ of many pathological processes ranging from inflammation and fibrosis to altered immunity and cancer. The goal of the current investigation is to dive into the exact role of the stromal fibroblast and the responsible mechanistic factors involved in both regulation and dysregulation of the inflammatory responses. This article reviews the most recent literature on how fibroblasts, in their different activation states or subtypes, play a crucial role in contributing to inflammatory outcomes. We will focus on recent findings on inflammatory diseases. We will also provide connections regarding the stromal-immune relationship, which supports the idea of fibroblast coming out from the ‘ensemble’ of cell types to the protagonist role in immunometabolism and inflammaging. Additionally, we discuss the current advances in variation of fibroblast nomenclature and division into clusters with their own suggested function and particularities in gene expression. Here, we provide a perspective for the periodontal implications, discussing the fibroblast role in the infection-driven and inflammatory mediated diseases such as periodontitis.

Abstract Microcosm biofilms can reproduce the complexity of a dental biofilm. However, different forms of cultivation have been used. The impact of the culture atmosphere on the development of microcosm biofilms and their potential to cause tooth demineralization has not yet been deeply studied. Objective This study analyzes the effects of three experimental cultivation models (microaerophile vs. anaerobiosis vs. experimental mixed) on the colony-forming units (CFU) of the cariogenic microorganisms and tooth demineralization. Methodology 90 bovine enamel and 90 dentin specimens were distributed into different atmospheres: 1) microaerophilia (5 days, 5% CO2); 2) anaerobiosis (5 days, jar); 3) mixed (2 days microaerophilia and 3 days anaerobiosis), which were treated with 0.12% chlorhexidine (positive control – CHX) or Phosphate-Buffered Saline (negative control – PBS) (n=15). Human saliva and McBain’s saliva containing 0.2% sucrose were used for microcosm biofilm formation, for 5 days. From the second day to the end of the experiment, the specimens were treated with CHX or PBS (1x1 min/day). Colony-forming units (CFU) were counted, and tooth demineralization was analyzed using transverse microradiography (TMR). Data were subjected to two-way ANOVA and Tukey’s or Sidak’s test (p<0.05). Results CHX was able to reduce total microorganism’s CFU compared to PBS (differences of 0.3–1.48 log10 CFU/mL), except for anaerobiosis and microaerophilia in enamel and dentin biofilm, respectively. In the case of dentin, no effect of CHX on Lactobacillus spp. was observed. CHX significantly reduced enamel demineralization compared to PBS (78% and 22% reductions for enamel and dentin, respectively). Enamel mineral loss did not differ when compared with the other atmospheres; however, the enamel lesion depth was greater under anaerobiosis. Dentin mineral loss was lower under anaerobiosis when compared with the other atmospheres. Conclusion The type of atmosphere has, in general, little influence on the cariogenic ability of the microcosm biofilm.

Abstract Irrigation solutions might affect dentin surface characteristics and, consequently, endodontic sealers adhesion. Objective This study analyzed the effect of different final irrigation protocols on push-out bond strength (BS) of AH Plus to dentin seven days and 20 months after obturation. Scanning electron micrographs were obtained from the dentin surface of one sample/group after final irrigation. Methodology Canals of bovine incisors were instrumented and received final irrigation with (n=21): G1 – 2.5% sodium hypochlorite (NaOCl) + distilled water; G2 – 2.5% NaOCl + 17% EDTA; G3 – 2.5% NaOCl + 17% EDTA + 2.5% NaOCl; G4 – 2.5% NaOCl + 17% EDTA + 2% chlorhexidine (CHX); G5 – mixture 5% NaOCl + 18% etidronate (HEDP); and G6 – mixture 5% NaOCl + 10% tetrasodium EDTA (Na4EDTA). After irrigation, one root/group was split and images were obtained by scanning electron microscopy (SEM). The other 20 roots/group were filled with only AH Plus sealer. Three slices/root were used for push-out assessment seven days and 20 months after obturation. One-way analysis of variance and Tukey (α<0.05) were used to compare the results among experimental groups, and unpaired t-test (α<0.05) was used to compare the results of the same group over time. Results The photomicrographs showed that, excepting G1, all groups completely removed the smear layer from the samples. In G2 and G4, the opening of the dentin tubules enlarged. In G3, erosion was observed in the peritubular and intertubular dentin. Values of the BS in the seven days were G2=G3=G4=G5>G6=G1 and in the 20 months were G3=G5>G6=G4>G1=G2. G3, G5, and G6 presented values of BS in 20 months similar to the values of seven days (P>0.05). Conclusions The final irrigation protocols tested produced dentin surfaces with different characteristics. Only G3 and G5 presented high BS values that were stable over time.

Abstract Objective Many genes and signaling molecules are involved in orthodontic tooth movement, with mechanically and hypoxically stabilized HIF-1α having been shown to play a decisive role in periodontal ligament signaling during orthodontic tooth movement. Thus, this in vitro study aimed to investigate if genetic polymorphisms in HIF1A (Hypoxia-inducible factor α-subunits) influence the expression pattern of HIF-1α protein during simulated orthodontic compressive pressure. Methodology Samples from human periodontal ligament fibroblasts were used and their DNA was genotyped using real time Polymerase chain reaction for the genetic polymorphisms rs2301113 and rs2057482 in HIF1A . For cell culture and protein expression experiments, six human periodontal ligament fibroblast cell lines were selected based on the patients’ genotype. To simulate orthodontic compressive pressure in fibroblasts, a 2 g/cm2 force was applied under cell culture conditions for 48 hours. Protein expression was evaluated by Western Blot. Paired t-tests were used to compare HIF-1α expression with and without compressive pressure application and unpaired t-tests were used to compare expression between the genotypes in rs2057482 and rs2301113 (p<0.05). Results The expression of HIF-1α protein was significantly enhanced by compressive pressure application regardless of the genotype (p<0.0001). The genotypes in the genetic polymorphisms rs2301113 and rs2057482 were not associated with HIF-1α protein expression (p>0.05). Conclusions Our study confirms that compressive pressure application enhances HIF-1α protein expression. We could not prove that the genetic polymorphisms in HIF1A affect HIF-1α protein expression by periodontal ligament fibroblasts during simulated orthodontic compressive force.

Abstract Fluoride (F) has been widely used to control dental caries, and studies suggest beneficial effects against diabetes when a low dose of F is added to the drinking water (10 mgF/L). Objectives This study evaluated metabolic changes in pancreatic islets of NOD mice exposed to low doses of F and the main pathways altered by the treatment. Methodology In total, 42 female NOD mice were randomly divided into two groups, considering the concentration of F administered in the drinking water for 14 weeks: 0 or 10 mgF/L. After the experimental period, the pancreas was collected for morphological and immunohistochemical analysis, and the islets for proteomic analysis. Results In the morphological and immunohistochemical analysis, no significant differences were found in the percentage of cells labelled for insulin, glucagon, and acetylated histone H3, although the treated group had higher percentages than the control group. Moreover, no significant differences were found for the mean percentages of pancreatic areas occupied by islets and for the pancreatic inflammatory infiltrate between the control and treated groups. Proteomic analysis showed large increases in histones H3 and, to a lesser extent, in histone acetyltransferases, concomitant with a decrease in enzymes involved in the formation of acetyl-CoA, besides many changes in proteins involved in several metabolic pathways, especially energy metabolism. The conjunction analysis of these data showed an attempt by the organism to maintain protein synthesis in the islets, even with the dramatic changes in energy metabolism. Conclusion Our data suggests epigenetic alterations in the islets of NOD mice exposed to F levels comparable to those found in public supply water consumed by humans.

Abstract Objective To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1β, in cultured human dental pulp cells. Methodology Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1β in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. Results Stimulation of the pulp cells with IL-1β resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1β (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1β was also blocked by incubation with the extract. Conclusions Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1β in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

Abstract Regular use of toothpaste with fluoride (F) concentrations of ≥ 1000 ppm has been shown to contribute to reducing caries increment. However, when used by children during the period of dental development, it can lead to dental fluorosis. Objective: In this study, we aimed to evaluate the in vitro effect of a toothpaste formulation with reduced fluoride (F) concentration (200 ppm) supplemented with sodium trimetaphosphate (TMP: 0.2%), Xylitol (X:16%), and Erythritol (E: 4%) on dental enamel demineralization. Methodology: Bovine enamel blocks were selected according to initial surface hardness (SHi) and then divided into seven experimental toothpaste groups (n=12). These groups included 1) no F-TMP-X-E (Placebo); 2) 16% Xylitol and 4% Erythritol (X-E); 3) 16% Xylitol, 4% Erythritol and 0.2%TMP (X-E-TMP); 4) 200 ppm F (no X-E-TMP: (200F)); 5) 200 ppm F and 0.2% TMP (200F-TMP); 200 ppm F, 16% Xylitol, 4% Erythritol, and 0.2% TMP (200F-X-E-TMP); and 7) 1,100 ppm F (1100F). Blocks were individually treated 2×/day with slurries of toothpastes and subjected to a pH cycling regimen for five days (DES: 6 hours and RE: 18 hours). Then, the percentage of surface hardness loss (%SH), integrated loss of subsurface hardness (ΔKHN), fluoride (F), calcium (Ca), and phosphorus (P) in enamel were determined. The data were analyzed by ANOVA (1-criterion) and the Student-Newman-Keuls test (p<0.001). Results: We found that the 200F-X-E-TMP treatment reduced %SH by 43% compared to the 1100F treatments (p<0.001). The ΔKHN was ~ 65% higher with 200F-X-E-TMP compared to 1100F (p<0.001). The highest concentration of F in enamel was observed on the 1100F treatment (p<0.001). The 200F-X-E-TMP treatment promote higher increase of Ca and P concentration in the enamel (p<0.001). Conclusion: The association of 200F-X-E-TMP led to a significant increase of the protective effect on enamel demineralization compared to the 1100F toothpaste.