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 At low concentrations used for in-office bleaching gels, such as 6% HP, gingival barrier continues to be performed. If we take into account that, in the at-home bleaching technique, no barrier is indicated, it seems that the use of a gingival barrier fails to make much sense when bleaching gel in low concentration is used for in-office bleaching. Objective This double-blind, split-mouth, randomized clinical trial evaluated the gingival irritation (GI) of in-office bleaching using 6% hydrogen peroxide (HP) with and without a gingival barrier in adolescents, as well as color change and the impact of oral condition on quality of life. Methodology Overall, 60 participants were randomized into which side would or would not receive the gingival barrier. In-office bleaching was performed for 50 minutes with 6% HP in three sessions. The absolute risk and intensity of GI were assessed with a visual analogue scale. Color change was assessed using a digital spectrophotometer and color guides. The impact of oral condition on quality of life was assessed using the Brazilian version of the Oral Health Impact Profile (α=0.05). Results The proportion of patients who presented GI for the “with barrier” group was 31.6% and for the “without barrier” group, 30% (p=1.0). There is an equivalence for the evaluated groups regarding GI intensity (p<0.01). Color change was detected with no statistical differences (p>0.29). There was a significant impact of oral condition on quality of life after bleaching (p<0.001). Conclusions The use or not of the gingival barrier for in-office bleaching with 6% HP was equivalent for GI, as well as for bleaching efficacy, with improvement in the impact of oral condition on quality of life. inoffice office gels 6 athome at home technique indicated doubleblind, doubleblind double blind, blind double-blind splitmouth, splitmouth split mouth, mouth split-mouth (GI (HP adolescents Overall Inoffice In 5 sessions scale guides α=0.05. α005 α α=0.05 . 0 05 (α=0.05) 316 31 31.6 30 p=1.0. p10 p p=1.0 1 (p=1.0) p<0.01. p001 p<0.01 01 (p<0.01) p>0.29. p029 p>0.29 29 (p>0.29) p<0.001. p0001 p<0.001 001 (p<0.001) efficacy α00 α=0.0 (α=0.05 3 31. p1 p=1. (p=1.0 p00 p<0.0 (p<0.01 p02 p>0.2 2 (p>0.29 p000 p<0.00 00 (p<0.001 α0 α=0. (α=0.0 p=1 (p=1. p0 p<0. (p<0.0 p>0. (p>0.2 (p<0.00 α=0 (α=0. p= (p=1 p<0 (p<0. p>0 (p>0. α= (α=0 (p= p< (p<0 p> (p>0 (α= (p (p< (p> (α

Abstract Objective To determine whether intra-mucosal injection of injectable platelet-rich fibrin (i-PRF) can promote healing after Diode Laser Gingival Depigmentation (DLGD). Methodology A total of 20 arch sites of hyperpigmented gingiva of 10 patients underwent DLGD. For each patient, two arch sites were randomly assigned for either intra-mucosal injection of i-PRF (G1-i-PRF) (n=10 sites) or no treatment (G2-Control): (n=10 sites). Wound Healing Score (WHS), patient satisfaction, and Pigmentation Index (DOPI) were measured at 1 week and 1 and 3 months postoperatively. Histological assessment of tissue specimens was performed at baseline and 1 week. Results The percentage change in WHS at 1 week was significantly higher in G1 (58.34±15.43) compared to G2 (37.50±11.79). At day 1, 50% of patients in G1 were pain free compared with 75% in G2, who had mild pain. Mean DOPI decreased significantly at 3 months in both groups (P-value <0.001), without significant differences between groups. G1 specimens showed significantly higher epithelial thickness (P-value <0.001), as well as a higher number of blood vessels and less percentage of inflammatory cells. Conclusions i-PRF demonstrated better clinical and histological healing potential and less patient discomfort compared to sites without treatment after DLGD. Registered at https://clinicaltrials.gov/ as (NCT05283668). intramucosal intra mucosal plateletrich platelet rich iPRF i PRF (i-PRF DLGD . (DLGD) 2 G1iPRF GiPRF G (G1-i-PRF n=10 n10 n (n=1 G2Control GControl Control (G2-Control) sites. WHS, , (WHS) satisfaction (DOPI postoperatively 58.34±15.43 58341543 58 34 15 43 (58.34±15.43 37.50±11.79. 37501179 37.50±11.79 37 50 11 79 (37.50±11.79) 75 Pvalue P value <0.001, 0001 <0.001 0 001 <0.001) cells https//clinicaltrials.gov/ httpsclinicaltrialsgov https //clinicaltrials.gov/ clinicaltrials gov https://clinicaltrials.gov NCT05283668. NCT05283668 NCT (NCT05283668) (DLGD n=1 n1 (n= (G2-Control (WHS 58.34±15.4 5834154 5 4 (58.34±15.4 3750117 37.50±11.7 7 (37.50±11.79 000 <0.00 00 https//clinicaltrials.gov clinicaltrialsgov //clinicaltrials.gov NCT0528366 (NCT05283668 n= (n 58.34±15. 583415 (58.34±15. 375011 37.50±11. (37.50±11.7 <0.0 NCT052836 (NCT0528366 58.34±15 58341 (58.34±15 37501 37.50±11 (37.50±11. <0. NCT05283 (NCT052836 58.34±1 5834 (58.34±1 3750 37.50±1 (37.50±11 <0 NCT0528 (NCT05283 58.34± 583 (58.34± 375 37.50± (37.50±1 < NCT052 (NCT0528 58.34 (58.34 37.50 (37.50± NCT05 (NCT052 58.3 (58.3 37.5 (37.50 NCT0 (NCT05 58. (58. 37. (37.5 (NCT0 (58 (37. (NCT (5 (37 ( (3

Abstract In children with poor oral hygiene, dental caries are the most common factor that causes periapical pathologies as a result of irreversible pulpal injuries and leads to the cessation of root development in immature permanent molar teeth. Objective The primary goal is to evaluate the effects of two different intracanal medicaments, calcium hydroxide [Ca(OH)2] and double antibiotic paste (DAP), on radiographic outcomes during regenerative endodontic procedures (REP) of immature permanent mandibular first molars with symptomatic irreversible pulpitis and symptomatic apical periodontitis (SIP/SAP). Additionally, the secondary goal was to evaluate MMP-8 levels during REP using two different intracanal medicaments. Methodology The study included 20 patients with immature mandibular first molars exhibiting SIP/SAP. Participants were randomly assigned into two groups based on the applied intracanal medicament. Ca(OH)2 (n=10) was prepared by mixing it with sterile distilled water, while the same amount of powdered metronidazole and ciprofloxacin were mixed and combined with sterile distilled water for DAP (n=10). MMP-8 in periapical samples were measured at baseline and on the 14th day using immunofluorometric assay. Image-J software with TurboReg plug-in was utilized to determine changes in root length, root width, radiographic root area (RRA) during the 12-month follow-up period. Data were analyzed by SPSS 25.0 (p<.05). Results Significant increase in MMP-8 on the 14th day compared to baseline in both groups (p<0.001). There was no significant difference between the two groups in terms of the increase in MMP-8 (p>0.05). Root length significantly increased in both groups (p=0.001), with Ca(OH)2 showing a greater increase (p=0.046). Root width and RRA increased similarly in both groups at 12th month. Conclusion Both Ca(OH)2 and DAP applications resulted in a significant increase in periapical MMP-8 levels. Increase in radiographic root width and root area was similar between two groups, but Ca(OH)2 led to a significantly greater increase in root length. Further studies with larger sample sizes are necessary to validate our findings during REP of vital immature permanent mandibular molars. Clinical Trials database: NCT05581706 hygiene teeth medicaments CaOH2 CaOH Ca OH 2 [Ca(OH)2 DAP, , (DAP) (REP SIPSAP SIP/SAP . SIP SAP (SIP/SAP) Additionally MMP8 MMP 8 MMP- medicament Ca(OH) n=10 n10 n 10 (n=10 n=10. th assay ImageJ Image J plugin plug (RRA 12month month 12 followup follow up period 250 25 0 25. p<.05. p05 p p<.05 05 (p<.05) p<0.001. p0001 p<0.001 001 (p<0.001) p>0.05. p005 p>0.05 (p>0.05) p=0.001, p=0.001 (p=0.001) p=0.046. p0046 p=0.046 046 (p=0.046) database NCT NCT0558170 [Ca(OH) (DAP (SIP/SAP Ca(OH n=1 n1 1 (n=1 p0 p<.0 (p<.05 p000 p<0.00 00 (p<0.001 p00 p>0.0 (p>0.05 p=0.00 (p=0.001 p004 p=0.04 04 (p=0.046 NCT055817 [Ca(OH n= (n= p<. (p<.0 p<0.0 (p<0.00 p>0. (p>0.0 p=0.0 (p=0.00 (p=0.04 NCT05581 (n p< (p<. p<0. (p<0.0 p>0 (p>0. p=0. (p=0.0 NCT0558 (p< p<0 (p<0. p> (p>0 p=0 (p=0. NCT055 (p (p<0 (p> p= (p=0 NCT05 (p= NCT0

Abstract There are many suitable strategies for addressing caries, which is an ongoing worldwide problem. Although white spot lesions (WSLs) can be either remineralized naturally or treated with non- or micro-invasive strategies, their whitish and opaque appearance may persist. Objective To evaluate the effects of tooth bleaching as a complement to fluoride-enhanced remineralization or resin infiltration in masking WSLs, as well as in enamel surface roughness relative to that of the adjacent enamel. Methodology Flattened rectangular bovine enamel fragments (6×3×~2.9 mm length, width and thickness) were divided into six groups (L/N, F/N, F.BL/BL, I/N, I.BL/BL, N/N; n=15). Treatments applied to the 3×3 mm left half included: L (Lesion) - WSL simulation with 50 mM acetate buffer, 96 hours, 37ºC; F (Fluoride) - WSL treatment with 2% NaF neutral gel, 1x/week, 8 weeks; I (Infiltration) - WSL treatment with H3PO4 37%/10 s; Icon®-Dry/30 s; Icon®-Infiltrant/3 min+1 min; N (Nothing) - sound enamel/control. Treatments applied to both halves after F and I included: BL (Bleaching) - Opalescence Boost 40%, 3×/20 min each; N (Nothing) - control. The differences in color (ΔE00, ΔL, Δa, Δb) and surface roughness (ΔRa) between the left and right halves were measured. Kruskal-Wallis/post-hoc tests were applied to ΔE00, ΔL, Δa and ΔRa, and 1-way ANOVA/Tukey tests to Δb (α=0.05). Results The factor under study significantly influenced ΔE00 (p=0.0001), ΔL (p=0.0024), Δb (p=0.0015), and ΔRa (p<0.001), but not Δa (p=0.1592). Both fluoride-enhanced remineralization and resin infiltration were able to mask WSL, regardless of subsequent bleaching. However, when bleaching was performed, ΔE00 median values did not exceed the acceptability threshold for color difference. Only resin infiltration reduced ΔRa between WSL and the adjacent enamel. Conclusions Both remineralization and infiltration, particularly if complemented by bleaching, fostered satisfactory esthetic results. Only infiltration without bleaching led to really good results in surface roughness. caries problem WSLs (WSLs non microinvasive micro invasive persist fluorideenhanced fluoride enhanced 6×3×~2.9 6329 6 3 2 9 (6×3×~2. length thickness L/N, LN (L/N FN F/N FBLBL F.BL/BL IN I/N IBLBL I.BL/BL N/N NN n=15. n15 n n=15 . 15 n=15) 33 3× included Lesion (Lesion 5 buffer hours 37ºC ºC Fluoride (Fluoride gel 1xweek xweek 1x week x 1x/week weeks Infiltration (Infiltration HPO H PO H3PO 3710 37 10 37%/1 s Icon®Dry/30 IconDry30 IconDry Icon® Dry/30 Icon Dry 30 Icon®-Dry/3 Icon®Infiltrant/3 IconInfiltrant3 IconInfiltrant Infiltrant/3 Infiltrant Icon®-Infiltrant/ min1 1 min+ Nothing (Nothing enamelcontrol control enamel/control Bleaching (Bleaching 40 40% 320 20 3×/2 each ΔE (ΔE00 (ΔRa measured KruskalWallis/posthoc KruskalWallisposthoc Kruskal Wallis/post hoc Wallis post 1way way ANOVATukey ANOVA Tukey α=0.05. α005 α α=0.05 0 05 (α=0.05) ΔE0 p=0.0001, p00001 p p=0.0001 , 0001 (p=0.0001) p=0.0024, p00024 p=0.0024 0024 (p=0.0024) p=0.0015, p00015 p=0.0015 0015 (p=0.0015) p<0.001, p0001 p<0.001 001 (p<0.001) p=0.1592. p01592 p=0.1592 1592 (p=0.1592) However performed difference 6×3×~2. 632 (6×3×~2 L/N n1 n=1 371 37%/ Icon®Dry/3 IconDry3 Dry30 Dry/3 Icon®-Dry/ Icon®Infiltrant/ Infiltrant3 Infiltrant/ Icon®-Infiltrant 4 32 3×/ (ΔE0 KruskalWallis posthoc Wallispost α00 α=0.0 (α=0.05 p0000 p=0.000 000 (p=0.0001 p0002 p=0.002 002 (p=0.0024 p=0.001 (p=0.0015 p000 p<0.00 00 (p<0.001 p0159 p=0.159 159 (p=0.1592 6×3×~2 63 (6×3×~ n= 37% Icon®Dry/ Dry3 Dry/ Icon®-Dry Icon®Infiltrant (ΔE α0 α=0. (α=0.0 p=0.00 (p=0.000 (p=0.002 (p=0.001 p00 p<0.0 (p<0.00 p015 p=0.15 (p=0.159 6×3×~ (6×3× Icon®Dry α=0 (α=0. p=0.0 (p=0.00 p0 p<0. (p<0.0 p01 p=0.1 (p=0.15 6×3× (6×3 α= (α=0 p=0. (p=0.0 p<0 (p<0. (p=0.1 6×3 (6× (α= p=0 (p=0. p< (p<0 6× (6 (α p= (p=0 (p< ( (p= (p