Abstract This study aimed to assess the influence of streptozotocin (STZ)-induced diabetes on the nociceptive behavior evoked by the injection of hypertonic saline (HS) into the masseter muscle of rats. Forty male rats were equally divided into four groups: a) isotonic saline control, which received 0.9% isotonic saline (IS), (Ctrl-IS); b) hypertonic saline control, which received 5% HS (Ctrl-HS); c) STZ-induced diabetic, which received IS, (STZ-IS); d) STZ-induced diabetic, which received HS (STZ-HS). Experimental diabetes was induced by a single intraperitoneal injection of STZ at dose of 60 mg/kg dissolved in 0.1 M citrate buffer, and 100 μL of HS or IS were injected into the left masseter to measure the nociceptive behavior. Later on, muscle RNA was extracted to measure the relative expression of the following cytokines: cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukins (IL)-1β, -2, -6, and -10. One-way analysis of variance (ANOVA) was applied to the data (p < 0.050). We observed a main effect of group on the nociceptive response (ANOVA: F = 11.60, p < 0.001), where the Ctrl-HS group presented the highest response (p < 0.001). However, nociceptive response was similar among the Ctrl-IS, STZ-IS, and STZ-HS group (p > 0.050). In addition, the highest relative gene expression of TNF-α and IL-6 was found in the masseter of control rats following experimental muscle pain (p < 0.050). In conclusion, the loss of somatosensory function can be observed in deep orofacial tissues of STZ-induced diabetic rats. STZinduced (HS groups 09 0 9 0.9 , (IS) CtrlIS Ctrl (Ctrl-IS) b 5 CtrlHS (Ctrl-HS) c STZIS (STZ-IS) d STZHS. STZHS . (STZ-HS) 6 mgkg mg kg 01 1 0. buffer 10 cytokines cyclooxygenase2 cyclooxygenase 2 cyclooxygenase- COX2, COX2 COX (COX-2) TNFα, TNFα TNF α (TNF-α) IL1β, IL1β ILβ IL 1β, 1β β (IL)-1β 2, -2 6, -6 10. -10 Oneway One way ANOVA (ANOVA 0.050. 0050 0.050 050 0.050) 1160 11 11.60 0.001, 0001 0.001 001 0.001) 0.001. However CtrlIS, Ctrl-IS STZIS, STZ-IS addition IL6 IL- conclusion (IS (Ctrl-IS (Ctrl-HS (STZ-IS (STZ-HS (COX-2 (TNF-α - -1 005 0.05 05 116 11.6 000 0.00 00 (COX- 0.0 11. (COX

ABSTRACT Purpose: To evaluate collagen fibers during the bone repair process in critical defects created in the tibias of rats, treated with zoledronic acid (AZ) associated with low-level laser therapy (LLLT). Methods: Ten rats were distributed according to treatment: group 1) saline solution; group 2) LLLT; group 3) AZ; group 4) AZ and LLLT. AZ was administered at the dose of 0.035 mg/kg at fortnightly intervals over eight weeks. Next, 2-mm bone defects were created in the tibias of all animals. The bone defects in groups 2 and 4 were irradiated LLLT in the immediate postoperative period. After periods 14 and 28 of application, the animals were euthanized, and birefringence analysis was performed. Results: Approximately 90% of the total area was occupied by collagen fibers within the red color spectrum, this area being statistically larger in relation to the area occupied by collagen fibers within the green and yellow spectrum, in the four groups. Over the 14-day period, there was no statistically significant difference between the groups. In the 28-day period, group 2 (14.02 ± 15.9%) was superior in quantifying green birefringent fibers compared to group 1 (3.06 ± 3.24%), with p = 0.009. Conclusions: LLLT associated with ZA is effective in stimulating the neoformation of collagen fibers. The LLLT group without the association with ZA showed a greater amount of immature and less organized matrix over a period of 28 days. Purpose (AZ lowlevel low level . (LLLT) Methods treatment solution 3 0035 0 035 0.03 mgkg mg kg weeks Next 2mm mm application euthanized performed Results 90 spectrum 14day day 28day 14.02 1402 02 (14.0 15.9% 159 15 9 3.06 306 06 (3.0 3.24%, 324 3.24% , 24 3.24%) 0009 009 0.009 Conclusions days (LLLT 003 03 0.0 14.0 140 (14. 15.9 3.0 30 (3. 32 3.24 000 00 0.00 0. 14. (14 15. 3. (3 3.2 (1 (

Abstract Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. Methodology C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (μCT) and histological/histomorphometric, birefringence, and molecular methods. Results The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. Conclusion The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains. (iNOS NO, NO , (NO) vessels leukocytes cells However unclear C57Bl6 CBl C57Bl 6 C Bl C57Bl/ (WT iNOSKO KO (iNOS-KO microcomputed micro computed μCT (μCT histologicalhistomorphometric histological histological/histomorphometric methods conditions d point 21d clots infiltration angiogenesis neoformation remodeling compared Likewise RealTime Real Time including formation resorption markers Moreover group (NO

Abstract Pain is common in orthodontic treatment, is subject to individual variation, and is associated with anxiety and stress, which can potentially become catastrophizing. The aim of the present study was to determine the variability of pain response after the insertion of orthodontic separators and to assess the association of pain levels with dental anxiety, catastrophizing, tooth sensitivity, and genetic expression of cytokines. To this end, 70 patients of both genders were divided into two equal groups according to the elastomeric separator used: G1 (Dentaurum) and G2 (Orthometric). Two separators were inserted in the mesial and distal sides of the lower right first molar. Participants were instructed to rate the level of pain at T0 (before insertion), T1 (just after insertion), and T2 (24 hours after insertion) on a visual analog scale. The gingival crevicular fluid was collected at T0 and T2. The levels of anxiety, catastrophizing, tooth sensitivity, and cytokine expression were also assessed. Statistical analysis was performed with the Fisher-Freeman-Halton, chi-squared, Spearman’s correlation, and dependent and independent t tests (α=5%). Pain intensity was higher at T2 than at T1, in both groups (P<.05). An association was established (P<.05) between pain intensity at T1 and catastrophizing, and at T2 with anxiety and catastrophizing. Within-group differences in cytokine expression were found between T0 and T2. There was no correlation between cytokine expression and pain levels, anxiety, catastrophizing, and sensitivity at T2. Tooth separation produced variable pain levels, which were influenced by anxiety and catastrophizing, however, pain level was not correlated with increased cytokine expression. treatment variation stress catastrophizing cytokines end 7 used G Dentaurum (Dentaurum Orthometric. Orthometric . (Orthometric) molar T before insertion, , just 24 (2 scale assessed FisherFreemanHalton, FisherFreemanHalton Fisher Freeman Halton, Halton Fisher-Freeman-Halton chisquared, chisquared chi squared, squared chi-squared Spearmans Spearman s α=5%. α5 α α=5% 5 (α=5%) P<.05. P05 P P<.05 05 (P<.05 Withingroup Within group however (Orthometric 2 ( α=5 (α=5% P0 P<.0 0 (P<.0 α= (α=5 P<. (P<. (α= P< (P< (α (P

Abstract: The aim of this study was to use microscopic and molecular techniques to evaluate the effects of a single session of antimicrobial photodynamic therapy (aPDT) on the alveolar repair process after tooth extraction in rats. The study sample included 84 rats divided into four groups, as follows: a) Control - untreated socket; b) Laser - socket treated using photobiomodulation; c) TBO - socket treated with topic application of the photosensitizer agent, toluidine blue O (TBO); and d) aPDT - socket treated with TBO and laser irradiation. An additional rat was used for thermal mapping during socket irradiation. The animals were euthanatized at 6, 15, and 28 days after unilateral extraction of the upper incisor. Quantitative and qualitative analyses of the connective and bone tissues, blood clot, blood vessel, and inflammatory infiltrate were performed, and real-time polymerase chain reaction was used to study the expression of genes (collagen type I, osteocalcin, alkaline phosphatase [ALP], runt-related transcription factor 2 [RUNX2], and vascular endothelial growth factor [VEGF]) involved in the bone healing process. No statistically significant differences in microscopic and molecular outcomes were observed between the groups (p > 0.05). A positive correlation was seen to exist between blood clot and VEGF (p = 0.000), and a negative correlation was observed between bone tissue and ALP (p = 0.028) and blood vessel and VEGF (p = 0.018). A single session of aPDT in the dental extraction site did not influence the alveolar repair process in rats.

Abstract Objective This study aims to evaluate bone repair and the development of the medication related osteonecrosis of the jaw (MRONJ) associated with the use of zoledronic acid in Wistar rats. Methodology 48 male Wistar rats were divided into four groups: ZA, treated with intraperitoneal zoledronic acid, 0.6 mg/kg every 28 days, totaling five doses; control (C), treated with 0.9% sodium chloride; ZA-surgical (SZA) and C-surgical (SC), submitted to extraction of the right upper molars 45 days after the first application. Alveolar bone repair was evaluated by macroscopic and histological analysis. Protein expression evaluations were performed by qPCR. Results Macroscopic evaluation showed that 91.66% (11) of the animals in the SZA group and 41.66% (5) from the SC group presented solution of epithelium continuity (P<0.05). All animals in the SZA group and none in the SC group had bone sequestration. The area of osteonecrosis was higher in the SZA group than in the SC group (P<0.05). In molecular evaluation, the SZA group presented changes in the expression of markers for osteoclasts, with increased RANK and RANKL, and a decrease in OPG. Conclusion The results highlighted strong and evident interference of zoledronic acid in bone repair of the socket, causing osteonecrosis and delayed bone remodeling.

Abstract Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of the limited methodological tools available in this field. Objective: In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice. Material and Methods: Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days). Results: The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers. Conclusions: This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.

Abstract Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.

Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

ABSTRACT Increased matrix metalloproteinases (MMPs) activity is a hallmark of periapical granulomas. However, the factors underlying the MMPs expression modulation in healthy and diseased periapical tissues remains to be determined. Objective In this study, we evaluated the association between the MMP1-1607 polymorphism (rs1799750) and pro-inflammatory milieu elements with MMP-1 mRNA levels in vivo. Material and Methods MMP1-1607 SNP and the mRNA levels of MMP-1, TNF-a, IFN-g, IL-17A, IL-21, IL-10, IL-4, IL-9, and FOXp3 were determined via RealTimePCR in DNA/RNA samples from patients presenting periapical granulomas (N=111, for both genotyping and expression analysis) and control subjects (N=214 for genotyping and N=26 for expression analysis). The Shapiro-Wilk, Fisher, Pearson, Chi-square ordinal least squares regression tests were used for data analysis (p<0.05 was considered statistically significant). Results The MMP1-1607 1G/2G and 1G/2G+2G/2G genotypes were significantly more prevalent in the patients than in controls, comprising a risk factor for periapical lesions development. MMP-1 mRNA levels were higher in periapical lesions than in healthy periodontal ligament samples, as well as higher in active than in inactive lesions. The polymorphic allele 2G carriers presented a significantly higher MMP-1 mRNA expression when compared with the 1G/1G genotype group. The ordered logistic regression demonstrated a significant correlation between the genetic polymorphism and the expression levels of MMP-1. Additionally, the pro- and anti-inflammatory cytokines IL-17A, IFN-g, TNF-a, IL-21, IL-10, IL-9, and IL-4 were significant as complementary explanatory variables of MMP-1 expression. Conclusion The MMP1-1607 SNP was identified as a risk factor for periapical lesions development, possibly due to its association with increased MMP-1 mRNA levels in periapical lesions. The MMP-1 expression is also under the control of the inflammatory milieu elements, being the cytokines TNF-a, IL-21, IL-17A, and IFN-g associated with increased MMP-1 levels in periapical lesions, while IL-10, IL-9, or IL-4 presented an inverse association.