Abstract Aim To evaluate the clinical effectiveness of ozonated sunflower oil (Oz) as an adjunctive of non-surgical periodontal therapy in patients with type 2 diabetes mellitus (DM2), on fibroblast cell viability and migration and the effectiveness of Oz on a Candida albicans (C. albicans) culture. Methodology In total, 32 sites in 16 DM2 with moderate to advanced periodontal disease with periodontal pocket depths ≥5mm were selected. The treatments were divided into two groups: control, saline solution (SS) as an adjunctive of scaling and root planing (SRP+SS), and test, Oz as an adjunctive of SRP (SRP+Oz). Hematological [fasting glucose level (FGL) and hemoglobin A1c (HbA1c)] and microbiological samples were collected from the participants at baseline and three months after periodontal treatment and the microbiological samples were analyzed by PCR. C. albicans was previously tested by the agar diffusion test. The effect of Oz was tested on cell viability and fibroblast migration. Results The groups showed no statistically significant differences (paired t-test-p>0.05) regarding hematological parameters, FGL (median - baseline 171.41, 3 months 164mg/dL), and HbA1c (baseline 8%, 3 months 7.5%) (Kruskal-Wallis One-Way Nonparametric-p>0.05) after periodontal therapy. The groups showed statistical differences for periodontal parameters between baseline and three months (paired t-test-p<0.05). PCR analysis showed a reduction in the percentage of C. albicans in the SRP+Oz group after three months (McNemar’s test-p=0.002). Cell viability was lower in the high glucose Dulbecco’s modified Eagle’s medium (4500 mg/L) than in low glucose (1000 mg/L) (RM-ANOVA-p<0.0001). The wound healing test showed reduced fibroblast migration (one-way ANOVA with Dunnett’s post-test-p<0.01). Oz showed high C. albicans antifungal inhibition (Kruskal-Wallis test-p=0.0001). Conclusions SRP+Oz effectively reduced C. albicans in-vitro and in-vivo but showed no clinical improvements compared to the control. Cell viability and wound healing of fibroblasts showed no improvements.

Abstract Objectives: Depth of invasion (DOI) in oral squamous cell carcinoma (OSCC) guides treatment and prognosis but lacks three-dimensional (3D) insight. Thus, this study aimed to investigate the feasibility and accuracy of Lugol's iodine-enhanced micro-computed tomography (CT) for the 3D measurement of DOI in OSCC samples. Methodology: In total, 50 in vitro OSCC samples from Nanjing Stomatological Hospital (July 2022 to January 2024) were subjected to micro-CT imaging with a slice thickness of 50 μm following 3% Lugol iodine staining for 12 h, followed by pathological examination and staining. The panoramic diagnostic scanner digitally measured pathological DOI. The micro-CT DOI was measured by evaluating the voxel value of the boundary of the tumor lesion and comparing it with the pathological examination results. Experienced physicians analyzed both measurements, and statistical analyses were performed to determine their correlation. Results: Lugol iodine-enhanced micro-CT imaging distinguishes various tissue structures, such as tumor tissue, epithelial tissue, muscle tissue, blood vessel structure, and other major tissue structures in 3D space. This imaging technique found and localized micro-tumor lesions (1.82×1.5×1 mm3) when in conjunction with pathological sections. Statistical analysis indicated a strong correlation between pathological DOI and micro-CT DOI (P<.001; r=0.986). During DOI measurement, Lugol iodine-enhanced micro-CT imaging effectively compensated for the loss of 3D space information in the pathological measurements, improving the accuracy of the DOI measurement. Conclusions: Lugol iodine-enhanced micro-CT improves OSCC DOI 3D measurements, enhances pathological staging accuracy, and aids treatment decisions and prognosis.

Abstract Guided bone regeneration (GBR) is an alternative treatment for craniofacial bone defects reconstruction through membrane barrier adaptation, such as demineralized dentin material membrane (DDMM). DDMM is used as a substitute for GBR material, which aligns with Green Economy principles, it has a good biological osteoinductive and osteoconductive effects, and its structure resembles bones. The balance of bone remodeling when experiencing craniofacial defects will be altered and allow changes to resorption activity, so the mechanisms of osteoclastogenesis and bone resorption are vital. Objective: this article aims to analyze the expression of TNF-α, RANKL, and osteoclast cells count after application of DDMM as GBR in mandibular bone defects. Methodology: this is an experimental study with a post-test only control group design, which began with the randomization of 120 rats into five groups: K(−), without membrane implantation; K(+), PPCM; P1, DDMM; P2, DDMM + bone graft; P3, PPCM + bone graft. The expression of TNF-α, RANKL, and osteoclast cells count were observed, followed by analysis using a one-way ANOVA and post hoc Tukey HSD comparison test. Results: there were significant differences in the expression of TNF-α, RANKL, and osteoclast cells count in all study groups (p=0.000). TNF-α showed a decreasing difference with the highest expression in the K(−) group on day 3 of 12.00±2.16. RANKL expression increased on day 14 and decreased on day 21 in all groups. The osteoclast cells count generally showed a critical period with the highest increase in the K(−) group on day 14 of 73.00±0.00. Conclusion: DDMM has the potential to be a superior membrane substitute compared to PPCM as GBR in alternative treatment for craniofacial bone defects reconstruction.

Abstract Submandibular salivary gland inflammation has been suggested as one of the mechanisms underlying impaired salivary secretion associated with sleep deprivation (SD). However, whether the salivary inflammatory response occurs to the same extent in paradoxical sleep deprivation with or without sleep recovery remains unknown. Objective: This study evaluated the extent to which inflammation influences salivary impairments associated with paradoxical sleep deprivation with or without sleep recovery. Methodology: Male Wistar rats were randomly assigned into three groups as control, partial SD (PSD) with sleep recovery for four hours a day and total SD (TSD). Paradoxical SD was carried out for seven days in the SD groups, after which saliva, blood, and submandibular gland samples were taken. Levels of interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-α), and nitrite were determined in saliva, serum, and the submandibular salivary gland. Leucocyte count and neutrophil-lymphocyte ratio were determined in all groups. One-way ANOVA and the Tukey's post hoc tests were used for data analysis. P-values < 0.05 were considered statistically significant. Results: Levels of TNF-α, IL-6, and nitrite in the submandibular salivary glands were significantly higher in the TSD groups (p=0.04,p<0.001, p=0.03, respectively) than in the control. Saliva level of TNF-α was higher in the PSD and TSD groups (p=0.003 and p=0.01 respectively) than in the control. Neutrophil-lymphocyte ratio was significantly higher in both PSD and TSD groups than in the control (p<0.01 for both). Conclusion: While total SD produced higher inflammatory response in the submandibular salivary gland, four-hour sleep recovery ameliorated this impact. This finding suggests that sleep recovery is crucial to improve inflammatory salivary gland dysfunction induced by sleep deprivation.

Abstract Objective This study aimed to investigate the role of transmembrane emp24 domain-containing protein 2 (TMED2) in oral squamous cell carcinoma (OSCC). Methodology A bioinformatics analysis was first conducted to explore TMED2 expression in OSCC and its relation with overall survival. The analysis results were further verified by assessing TMED2 expression levels in human normal oral keratinocyte cells and human OSCC cell lines using quantitative real-time polymerase chain reaction and the Western blot. Finally, the effects of TMED2 knockdown and overexpression on the expression levels of TMED2, ADP-ribosylation factor 1, extracellular signal-regulated kinase ½, and phospho-extracellular signal-regulated kinase ½ proteins were examined in cells using the Western blot. Results The GEPIA2 database showed that OSCC tissues expressed more TMED2 than normal tissues. At the cellular level, TMED2 expression significantly increased in SCC-4, HSC-3, and CAL-27 cells than in human normal oral keratinocyte cells. TMED2 knockdown reduced cell proliferation, increased the apoptosis rate in SCC-4 cells, and led to a higher proportion of cells in the G0/G1 phase and a lower proportion in the S phase. Conclusion TMED2 may promote OSCC cell proliferation and inhibit apoptosis, potentially by activation of the ADP-ribosylation factor 1/ extracellular signal-regulated kinase ½ signaling pathway.

Abstract This study evaluated the effect of Stemregen® nutritional supplement on inflammation and resorption in apical periodontitis using a rat model. Methodology Rats were divided in three groups: negative control (n=7), positive control (n=10), and Stemregen® (Stem) (n=10). Apical periodontitis was induced in the positive control and Stem groups, and all rats were sacrificed on the 30th day. Serum phosphorus (P), calcium (Ca), and alkaline phosphatase (ALP) were analyzed. Histopathological assessments measured osteoblastic and osteoclastic activity, inflammation, fibrosis, and abscess density. Immunohistochemical analyses evaluated RANKL, TRAP, and OPG levels. Results Results showed significantly lower osteoblastic activity in the negative control compared to Stem and positive control groups (p=0.005). Osteoclastic activity was higher in the positive control (p=0.032). Inflammation and abscess formation were reduced in the Stem group compared to the positive control (p<0.001). OPG levels were lower in the negative control compared to the other groups (p=0.005). Conclusion Stemregen® effectively reduced inflammation and bone destruction, suggesting potential benefits for apical periodontitis management, though further research is needed.

Abstract Objectives The oral cavity harbors a plethora of bacterial species. Dysbiosis of oral and gut microbiota is associated with several oral and systemic pathologies, such as cancer, obesity, diabetes, atherosclerosis and gastrointestinal diseases. Imbalance in the oral-gut microbial axis has been associated with head and neck squamous cell carcinoma (HNSCC). This study aims to analyze the bacterial profile of HNSCC across various taxonomic units, investigate molecular patterns associated with prevalent bacterial phylum in HNSCC, and compare the bacterial profile in HNSCC and gastrointestinal (GI) carcinoma using computational analysis. Methodology The microbe-host transcriptomic, proteomic, and epigenetic analyses of HNSCC and GI carcinomas were performed using The Cancer Microbiome Atlas (TCMA) database. The differential expression of the host’s mRNA transcripts and proteins associated with tumor microbiome were analyzed using The University of Alabama at Birmingham Cancer data analysis (UALCAN) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) websites. Results A decrease in Actinobacteria and an enrichment of Flavobacteria at the class level, Neisseriales, Pasteurellales, and Campylobacterales at the order level, Pasteurellaceae, Flavobacteriaceae, Campylobacteraceae, and Peptoniphilaceae at the family level, and Hemophilus, Porphyromonas, and Leptotrichia at the genus level were observed in HNSCC compared to the normal mucosa. RICTOR protein, mRNA transcripts (HIST1H2BB, SCARNA11, TBC1D21 gene), and hsa-miR-200a-5p miRNA were significantly correlated with prevalent bacterial species in HNSCC. A major increase in Actinobacteria, Fusobacteria, and Spirochaetes was observed in HNSCC compared to GI carcinoma. Conclusion The oral-gut microbial dysbiosis, as reflected by the differential abundance of bacterial species in oral and GI carcinomas, suggests the implication of tumor microbiome and their genomic interactions with the host in carcinogenesis.

Abstract During oral surgery and temporomandibular joint repositioning, pain hypersensitivity often occurs due to irritation or inflammation of the nerve endings in the orofacial region. Objective: This study aimed to investigate the effects of ECa 233, a Centella asiatica–standardized extract, on the development of mechanical hyperalgesia and allodynia induced by chronic constriction injury of the infraorbital nerve in mice. Methodology: The right infraorbital nerves of the mice were ligated. Oral carbamazepine (20 mg/kg) or ECa 233 (30, 100, or 300 mg/kg) was administered daily for 21 days. Von Frey and air-puff tests were performed on both sides of the whisker pad on days 0, 7, 14, and 21. Thereafter, the expression of purinergic receptor subtype 3 (P2X3) and voltage-gated sodium channel 1.7 (NaV1.7), a transmembrane protein, in the trigeminal ganglion and c-fos immunoreactivity-positive neurons in the trigeminal nucleus caudalis was assessed. Results: After 21 days of infraorbital nerve ligation, the mice showed allodynia- and hyperalgesia-like behavior, P2X3 and NaV1.7 were upregulated in the trigeminal ganglion, and nociceptive activity increased in the trigeminal nucleus caudalis. However, the oral administration of carbamazepine (20 mg/kg), ECa 233 (100 mg/kg), or ECa 233 (300 mg/kg) mitigated these effects. Nevertheless, ECa 233 failed to affect NaV1.7 protein expression. Conclusion: Carbamazepine and ECa 233 can prevent pain hypersensitivity in mice. Considering the side effects of the long-term use of carbamazepine, ECa 233 monotherapy or combined ECa 233 and carbamazepine therapy can be used as an alternative for regulating the development of hypersensitivity in trigeminal pain. However, further detailed clinical studies should be conducted to provide comprehensive information on the use of ECa 233.

Abstract Aim: Polymorphisms in the COMT gene can alter enzymatic functions, raising levels of endogenous catecholamines, which stimulates beta-adrenergic receptors related to pain. This study aimed to evaluate whether a polymorphism in the COMT gene (rs4818) is associated with dental pain in children. Methodology: A cross-sectional study was conducted with a representative sample of 731 pairs of children and parents randomly selected from a population-based sample of eight-year-old children. Reports of dental pain was evaluated using a question directed at the parents and self-reported pain using the Faces Pain Scale – Revised. Dental caries experience was determined using the Decayed, Missing, and Filled Teeth (DMFT) index. For genetic analysis, DNA was obtained from oral mucosa epithelial cells of 352 children randomly selected from the initial sample. Results: Children with the CC genotype had higher odds of reporting moderate to intense pain than those with the GG genotype (OR=3.60; 95% CI=0.80–16.20; p=0.03). These same children had greater odds of parental reports of pain (OR=1.93; 95% CI=0.91-4.08; p=0.02). Moreover, lower schooling of parents/guardians and caries experience in the primary dentition were significantly associated with greater odds of a parental report of dental pain (OR=2.06; 95% CI=1.47–2.91; p<0.001; OR=6.26; 95% CI=4.46–8.78; p<0.001). Conclusions: The rs4818 polymorphism of the COMT gene is associated with dental pain. Children with the C allele are more likely to report higher levels of pain. Clinical Relevance: Even though the experience of pain is subjective and multifactorial, this study raises the hypothesis that there is a genetic predisposition to dental pain that should be considered in clinical practice.

Abstract Objective This retrospective study aimed to analyze the clinical efficacy of two regenerative surgical methods — Bio-Oss granules combined with barrier membranes and Bio-Oss Collagen alone — and to help clinicians achieve better periodontal regeneration outcomes in the specific periodontal condition. Methodology Patients who underwent periodontal regeneration surgery from January 2018 to April 2022 were retrospectively screened, and their clinical and radiographic outcomes at 6 months postoperatively were analyzed. The probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), gingival recession (GR), distance from the cemento-enamel junction to the bottom of the bone defect (CEJ-BD), and depth of intrabony defects (INFRA) were recorded before the operation (T0) and 6 months after it (T1), and subsequently compared. Results In total, 143 patients were included — 77 were placed in the Bio-Oss group and 66 were placed in the Bio-Oss Collagen group. All indicators, including PD and CAL at T1, showed significant differences compared to baseline, for both groups (P<0.001). PD reduction was greater in the group receiving the Bio-Oss Collagen treatment (P=0.042). Furthermore, in cases when the baseline PD range was 7-11 mm and the age range was 35-50 years, PD reduction was more significant for patients receiving the Bio-Oss Collagen treatment (P=0.031, 0.023). A linear regression analysis indicated that postoperative PD and CAL were positively correlated with baseline values, and that the efficacy tended to decrease with increasing age. Conclusion Both the use of Bio-Oss Collagen alone and the use of Bio-Oss granules combined with barrier membranes resulted in significant effects in the treatment of periodontal intrabony defects. The Bio-Oss Collagen treatment generated more improvements in PD than the Bio-Oss granules combined with barrier membranes, particularly within the baseline PD range of 7-11 mm and the 35-50 years age group. Additionally, age was the main factor influencing the effectiveness of regenerative surgery for intrabony defects: older individuals exhibited fewer improvements.

Abstract Objective: Although autogenous grafting is accepted as the gold standard in intraoral grafting, xenogenous grafts are frequently used in sinus lift surgeries due to their osteoinductive and osteoconductive properties. This study aimed to investigate the efficacy of fish spine-derived xenogenic grafts in sinus augmentation surgery. Material and Methods: In this study, a fish spine-derived xenogenic graft was produced for comparison with autogenous graft and bovine derived xenogenic grafts. Twenty-one New Zealand rabbits were used. Autogenous grafts (AG- Group 1), as well as bovine-derived (bHAP - Group 2) and fish spine-derived (fHAP - Group 3) xenogenic grafts were placed in the right and left sinuses of rabbits. The animals were sacrificed at the 4th and 8th weeks. New bone formation (NBF) was evaluated through histological examination, while bone volume (BV), new bone surface/bone volume (BS-BV), new bone surface/tissue volume (BS-TV), and trabecular separation (Tb-Sp) were assessed via Micro-CT. Statistical significance was considered at p<0.05. Results: Histological examination revealed a significant difference in NBF between AG-bHAP (p<0.001), as well as between fHAP-bHAP (p<0.001) in the fourth-week group. No significant difference was found in the eighth-week group (p=0.130). In the eighth-week group, a statistically significant difference was found between fHAP and bHAP in terms of BV. (p=0.007). Conclusion: Although both graft materials used in this study showed positive effects on bone regeneration, fHAP and AG presented similar effects on bone regeneration and were superior to bHAP. Objective properties spinederived spine surgery Methods Twentyone Twenty one (AG 1, 1 , 1) bovinederived 2 3 th weeks (NBF BV, BV (BV) surfacebone surface BSBV, BSBV BS (BS-BV) surfacetissue tissue BSTV, BSTV TV (BS-TV) TbSp Tb Sp (Tb-Sp MicroCT. MicroCT Micro CT. CT Micro-CT p005 p 0 05 p<0.05 Results AGbHAP p<0.001, p0001 p<0.001 001 fHAPbHAP (p<0.001 fourthweek fourth week eighthweek eighth p=0.130. p0130 p=0.130 . 130 (p=0.130) p=0.007. p0007 p=0.007 007 (p=0.007) Conclusion (BV (BS-BV (BS-TV p00 p<0.0 p000 p<0.00 00 (p<0.00 p013 p=0.13 13 (p=0.130 p=0.00 (p=0.007 p0 p<0. (p<0.0 p01 p=0.1 (p=0.13 p=0.0 (p=0.00 p<0 (p<0. p=0. (p=0.1 (p=0.0 p< (p<0 p=0 (p=0. (p< p= (p=0 (p (p=

Abstract Objective: We aimed to investigate the regulatory effects of HMGB1/TLR4 signaling pathway on the expression of IL-10 and VEGF in human bone marrow mesenchymal stem cells. Methodology: Human JBMSCs were isolated and cultured. Then, HMGB1 was added into the JBMSCs culture medium, and the protein and mRNA expression levels of IL-10 and VEGF were assessed. Moreover, cells were pretreated with a specific TLR4 inhibitor (TAK-242), and the expression changes of IL-10 and VEGF were compared. Results: Compared with the control group, exposure to HMGB1 in human JBMSCs up-regulated TLR4, IL-10, and VEGF secretion at both protein and mRNA levels (P<0. 05). In addition, the increased expression of IL-10 and VEGF could be restrained in TAK-242 group compared with the HMGB1 group (P<0.05). Conclusions: The results indicated that HMGB1 activate TLR4 signaling pathway in Human JBMSCs, which plays a regulatory role in cytokines expression.

Abstract Associations between the WNT5A rs566926 variant and non-syndromic orofacial cleft (NSOC) have been reported in different populations. Objective This study aimed to investigate the role of the rs566926 single nucleotide polymorphism (SNP) in WNT5A and its interactions with SNPs in BMP4, FGFR1, GREM1, MMP2, and WNT3 in the occurrence of NSOC in a Brazilian population. Methodology A case-control genetic association study was carried out involving participants from four regions of Brazil, totaling 801 patients with non-syndromic cleft lip with or without cleft palate (NSCL±P), 273 patients with cleft palate only (NSCPO), and 881 health volunteers without any congenital condition (control). Applying TaqMan allelic discrimination assays, we evaluated WNT5A rs566926 in an ancestry-structured multiple logistic regression analysis, considering sex and genomic ancestry as covariates. Interactions between rs566926 and variants in genes involved in the WNT5A signaling pathway (BMP4, FGFR1, GREM1, MMP2, and WNT3) were also explored. Results WNT5A rs566926 was significantly associated with an increased risk of NSCL±P, particularly due to a strong association with non-syndromic cleft lip only (NSCLO), in which the C allele increased the risk by 32% (OR: 1.32, 95% CI: 1.04–1.67, p=0.01). According to the proportions of European and African genomic ancestry, the association of rs566926 reached significant levels only in patients with European ancestry. Multiple interactions were detected between WNT5A rs566926 and BMP4 rs2071047, GREM1 rs16969681 and rs16969862, and FGFR1 rs7829058. Conclusion The WNT5A rs566926 polymorphism was associated with NSCL±P, particularly in individuals with NSCLO and high European ancestry. Epistatic interactions involving WNT5A rs566926 and variants in BMP4, GREM1, and FGFR1 may contribute to the risk of NSCL±P in the Brazilian population.

Abstract Objective: The aim of this population-based retrospective study was to compare the osteogenic effect of newly formed bone after maxillary sinus floor elevation (MSFE) and simultaneous implantation with or without bone grafts by quantitatively analyzing trabecular bone parameters. Methodology: A total of 100 patients with missing posterior maxillary teeth who required MSFE and implantation were included in this study. Patients were divided into two groups: the non-graft group (n=50) and the graft group (n=50). Radiographic parameters were measured using cone beam computed tomography (CBCT), and the quality of newly formed bone was analyzed by assessing trabecular bone parameters using CTAn (CTAnalyzer, SkyScan, Antwerp, Belgium) software. Results: In the selected regions of interest, the non-graft group showed greater bone volume/total volume (BV/TV), bone surface/total volume (BS/TV), trabecular number (Tb. N), and trabecular thickness (Tb. Th) than the graft group (p<0.001). The non-graft group showed lower trabecular separation (Tb. Sp) than the graft group (p<0.001). The incidence of perforation and bleeding was higher in the graft group than in the non-graft group (p<0.001), but infection did not significantly differ between groups (p>0.05). Compared to the graft group, the non-graft group showed lower postoperative bone height, gained bone height and apical bone height (p<0.001). Conclusion: MSFE with and without bone grafts can significantly improve bone formation. In MSFE, the use of bone grafts hinders the formation of good quality bone, whereas the absence of bone grafts can generate good bone quality and limited bone mass.

Abstract Objective: This study aimed to investigate the effects of systemic administration of P. eurycarpa Yalt. plant extract on alveolar bone loss and oxidative stress biomarkers in gingival tissue in a rat model of experimental periodontitis. Methodology: 32 male Wistar albino rats, weighing 200–250 g, were divided into four groups (n=8): Healthy control (HC), Experimental periodontitis control (EPC), Experimental periodontitis 400 mg/kg (EP400), Experimental periodontitis 800 mg/kg (EP800). Experimental periodontitis was induced using the ligating method. Distilled water was administered to the HC and EPC groups and the plant extract was administered to the EP400 and EP800 groups by oral gavage at doses of 400 mg/kg and 800 mg/kg, respectively. The rats were sacrificed on the 15th day. The values of glutathione peroxidase GSH-Px, malondialdehyde (MDA), superoxide dismustase (SOD), interleukin-1β (IL-1β), interleukin-10 (IL-10), total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI) in the gingival tissues were analyzed by ELISA tests. Alveolar bone loss was assessed using micro-CT images of the maxilla. Results: Although the IL-1β, TOS, OSI results of the healthy control group were lower than those of the other groups, the TAS values were higher (p<0.05). No significant difference was found in the biochemical parameters among the EPC, EP400, and EP800 groups (p>0.05). Alveolar bone loss was significantly reduced in the extract groups compared to the EPC group (p<0.001). Conclusion: Within the limitations of this study, it was observed that the systemic P. eurycarpa extract application reduced alveolar bone loss in a rat model of experimental periodontitis. Further studies are needed to elucidate the beneficial effects of P. eurycarpa.