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Periodontal diseases (PD) are chronic infectious inflammatory diseases characterized by the destruction of tooth-supporting structures, being the presence of periodontopathogens required, but not sufficient, for disease development. As a general rule, host inflammatory mediators have been associated with tissue destruction, while anti-inflammatory mediators counteract and attenuate disease progression. With the discovery of several T-cell subsets bearing distinct immunoregulatory properties, this pro- vs. anti-inflammatory scenario became more complex, and a series of studies has hypothesized protective or destructive roles for Th1, Th2, Th17, and Treg subpopulations of polarized lymphocytes. Interestingly, the 'protective vs. destructive' archetype is usually considered in a framework related to tissue destruction and disease progression. However, it is important to remember that periodontal diseases are infectious inflammatory conditions, and recent studies have demonstrated that cytokines (TNF-[alpha] and IFN-[gamma]) considered harmful in the context of tissue destruction play important roles in the control of periodontal infection. Therefore, in this review, the state-of-the-art knowledge concerning the protective and destructive roles of host inflammatory immune response will be critically evaluated and discussed from the tissue destruction and control-of-infection viewpoints. [ABSTRACT FROM AUTHOR]

BACKGROUND AND OBJECTIVE: Inflammatory cytokines such as tumor necrosis factor-alpha are involved in the pathogenesis of periodontal diseases. A high between-subject variation in the level of tumor necrosis factor-alpha mRNA has been verified, which may be a result of genetic polymorphisms and/or the presence of periodontopathogens such as Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola (called the red complex) and Aggregatibacter actinomycetemcomitans. In this study, we investigated the effect of the tumor necrosis factor-alpha (TNFA) -308G/A gene polymorphism and of periodontopathogens on the tumor necrosis factor-alpha levels in the periodontal tissues of nonsmoking patients with chronic periodontitis (n = 127) and in control subjects (n = 177). MATERIAL AND METHODS: The TNFA -308G/A single nucleotide polymorphism was investigated using polymerase chain reaction-restriction fragment length polymorphism analysis, whereas the tumor necrosis factor-alpha levels and the periodontopathogen load were determined using real-time polymerase chain reaction. RESULTS: No statistically significant differences were found in the frequency of the TNFA -308 single nucleotide polymorphism in control and chronic periodontitis groups, in spite of the higher frequency of the A allele in the chronic periodontitis group. The concomitant analyses of genotypes and periodontopathogens demonstrated that TNFA -308 GA/AA genotypes and the red-complex periodontopathogens were independently associated with increased levels of tumor necrosis factor-alpha in periodontal tissues, and no additive effect was seen when both factors were present. P. gingivalis, T. forsythia and T. denticola counts were positively correlated with the level of tumor necrosis factor-alpha. TNFA -308 genotypes were not associated with the periodontopathogen detection odds or with the bacterial load. CONCLUSION: Our results demonstrate that the TNFA -308 A allele and red-complex periodontopathogens are independently associated with increased levels of tumor necrosis factor-alpha in diseased tissues of nonsmoking chronic periodontitis patients and consequently are potentially involved in determining the disease outcome. [ABSTRACT FROM AUTHOR]

Introduction. Tissue conditioners modified with antifungals are a potential alternative to denture stomatitis (DS) treatment. Gap Statement. Information on tissue response to this treatment before its clinical application is lacking. Aim. This study aimed to evaluate the tissue response of a tissue conditioner modified with antifungals in a rat model of DS. Methodology. After DS induction for 4 days under antibiotic therapy, Wistar rats had their intraoral devices (IODs) relined with the tissue conditioner Softone without (Soft) or with the MICs against Candida albicans of nystatin (Nys) or chlorhexidine (Chx) complexed or not with β-cyclodextrin (Nys:βCD and Chx:βCD). Three controls were included: healthy rats [negative control (Nc)], rats using a sterile IOD [sterile device (Sd)] and rats with DS that did not receive treatment (DS). After 4 days of treatment, the palatal mucosa under the IODs underwent histological processing for morphohistopathological and histometric analyses, morphology of collagen fibres (birefringence), immunohistochemistry for the expression of cell proliferation (proliferating cell nuclear antigen) and cytokine (IL-1β). Results. The Nc and Sd groups were similar ( P >0.05), displaying epithelial and connective tissues without any discernible changes in the parameters assessed. The DS and Soft groups exhibited pronounced epithelial alterations, cell proliferation and expression of the cytokine IL-1β. In groups treated with drug incorporation (Nys, Chx, Nys:βCD and Chx:βCD), all samples demonstrated a reduction in tissue inflammation or complete tissue recovery, with an epithelium compatible with health. For the immunohistochemical parameters, the Chx, Nys:βCD and Chx:βCD groups were comparable with Nc ( P >0.05). Conclusion. The proposed treatment could be promising for DS, as it led to the tissue recovery of the palatal mucosa. Nevertheless, much lower concentrations of complexed antifungals were required to achieve a similar or higher degree of tissue response compared with uncomplexed drugs in a modified tissue conditioner formulation.

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.

Aim: To assess the efficacy of heterologous fibrin biopolymer (HFB) in promoting alveolar bone healing after tooth extraction in rats., Materials and Methods: The upper right incisors of 48 Wistar rats were extracted. Toothless sockets were filled with HFB (HFBG, n = 24) or blood clot (BCG, n = 24). The tooth extraction sites were subjected to micro-computed tomography (micro-CT), histological, histomorphometric and immunohistochemical (for Runt-related transcription factor 2/Runx2 and tartrate-resistant acid phosphatase/TRAP) analyses on days 0, 7, 14 and 42 after extraction., Results: Socket volume remained similar between days 0 and 14 (69 ± 5.4 mm 3 ), except in the BCG on day 14, when it was 10% lower (p = .043). Although the number of Runx2+ osteoblasts was high and similar in both groups (34 × 10 2  cells/mm 2 ), the HFBG showed lower inflammatory process and osteoclast activity than BCG at 7 days. On day 14, the number of Runx2+ osteoblasts remained high and similar to the previous period in both groups. However, osteoclast activity increased. This increase was 55% lower in the HFBG than BCG. In the BCG, the presence of an inflammatory process and larger and numerous osteoclasts on day 14 led to resorption of the alveolar bone ridge and newly formed bone. On day 42, numbers of Runx2+ osteoblast and TRAP+ osteoclasts decreased dramatically in both groups. Although the BCG exhibited a more mature cortical bone formation, it exhibited a higher socket reduction (28.3 ± 6.67%) and smaller bone volume (37 ± 5.8 mm 3 ) compared with HFBG (socket reduction of 14.8 ± 7.14% and total bone volume of 46 ± 5.4 mm 3 )., Conclusions: HFB effectively suppresses osteoclast activity and reduces alveolar bone resorption compared with blood clot, thus preventing three-dimensional bone loss, particularly during the early healing period. HFB emerges as a promising biopharmaceutical material for enhancing healing processes after tooth extraction., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Background: Natural killer (NK) cells are one of the most crucial immune cells that mediate the antitumoral response due to their ability to immediately recognize and eliminate transformed cells. Because of their great cytotoxic activity, the function of NK cells must be robustly regulated to avoid tissue damage. Such regulation is mediated by a coordinated engagement of activating (NKp46) and inhibitory (CD158b) receptors, which tumor cells may use to escape from immunosurveillance. Also, NK cells are generally divided based on surface molecules, such as CD16 and CD56, and can be classified as CD56 bright CD16 - (regulatory) and CD56 dim CD16 + (cytotoxic) NK cells. Here, we aimed to evaluate the frequency and phenotype of circulating NK cells in patients with advanced carcinomas, as well as their systemic cytokine/chemokine and growth factors production., Methods: Peripheral blood was collected from 24 patients with advanced solid cancer during or after treatment and from 10 healthy donors. The frequency and the expression of activating (NKp46) and inhibitory (CD158b) molecules of CD56 bright CD16 - and CD56 dim CD16 + NK cells were assessed by flow cytometry and the multiplex Luminex platform was used to quantify the secreted factors in peripheral blood serum., Results: Cancer patients had a lower frequency of the cytotoxic CD56 dim CD16 + NK cells subset in comparison with healthy controls. Also, the regulatory CD56 bright CD16 - NKs isolated from cancer patients exhibited a significantly lower expression of NKp46. Among 29 immunological and growth factors analyzed in the peripheral blood of oncologic patients, MCP-1, IP-10, and eotaxin, and VEGF they have presented a higher proportion. The Pearson correlation test showed that IL-12p40 positively correlates with CD56 bright CD16 - NK cells. We also observed a positive correlation between MCP-1 and the activating marker NKp46, as well as a negative correlation between IP-10 and TNF-α and NKp46. CD158b expression in CD56 dim CD16 + was positively correlated with EGF and negatively correlated with MIP-1β., Conclusions: Taken together, these results suggest that cancer patients present a shift towards a poorly cytotoxic and less activated NK profile which may contribute to tumor development and progression. The understanding of NK cell biology and soluble factors during tumor development could aid in the design of possible targeting therapeutic approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: Periodontitis and coronavirus disease (COVID-19) share risk factors and activate similar immunopathological pathways, intensifying systemic inflammation. This study investigated the clinical, immunological and microbiological parameters in individuals with COVID-19 and controls, exploring whether periodontitis-driven inflammation contributes to worsening COVID-19 endpoints., Methods: Case (positive RT-PCR for SARS-CoV-2) and control (negative RT-PCR) individuals underwent clinical and periodontal assessments. Salivary levels of TNF-α, IL-6, IL-1β, IL-10, OPG, RANKL, neutrophil extracellular traps, and subgingival biofilm were analyzed at two timepoints. Data on COVID-19-related outcomes and comorbidity information were evaluated from medical records., Results: Ninety-nine cases of COVID-19 and 182 controls were included for analysis. Periodontitis was associated with more hospitalization (p = 0.009), more days in the intensive care unit (ICU) (p = 0.042), admission to the semi-ICU (p = 0.047), and greater need for oxygen therapy (p = 0.042). After adjustment for confounders, periodontitis resulted in a 1.13-fold increase in the chance of hospitalization. Salivary IL-6 levels (p = 0.010) were increased in individuals with COVID-19 and periodontitis. Periodontitis was associated with increased RANKL and IL-1β after COVID-19. No significant changes were observed in the bacterial loads of the periodontopathogens Porphyromona gingivalis, Aggregatibacter actinomycetemcomitans, Tanerella forsythia, and Treponema denticola., Conclusions: Periodontitis was associated with worse COVID-19 outcomes, suggesting the relevance of periodontal care to reduce the burden of overall inflammation. Understanding the crosstalk between SARS-CoV-2 infection and chronic conditions such as periodontitis that can influence disease outcome is important to potentially prevent complications of COVID-19., (© 2023. The Author(s), under exclusive licence to The Society of The Nippon Dental University.)

Osteoimmune diseases, such as apical periodontitis, are prevalent, often painful, inflammatory conditions resulting in bone loss and reduced quality of life. There is growing evidence that the nociceptive fibers densely innervating affected tissues regulate disease progression; therefore, we hypothesized that nociceptors regulate the transcriptomic profile of the periapical osteolytic lesion in a mouse model of apical periodontitis. Male control and nociceptor-ablated mice underwent pulp exposures, and after 0, 7, or 14 days, total RNA from periapical tissues was submitted for sequencing and bioinformatic analysis. Pulp exposure triggers the differential expression of hundreds of genes over the course of infection. At 14 days post pulp exposure, 422 genes, including Tnf, Il1a, and Il1b, were differentially expressed between nociceptor-ablated and control mice with greater enrichment of biological processes related to inflammation in nociceptor-ablated mice. Nociceptor ablation regulates the transcriptomic profile of periapical lesions in a mouse model of apical periodontitis, shifting the gene expression profile to a greater enrichment of inflammatory genes, suggesting nociceptors play a role in the kinetics of the immune response. This newly uncovered neuro-immune axis and its mechanisms in apical periodontitis can be an important therapeutic target for the treatment of this prevalent disease., (© 2023. Springer Nature Limited.)

Leprosy is a chronic and infectious disease that primarily affects the skin and peripheral nervous system, presenting a wide spectrum of clinical forms with different degrees of severity. The distinct host immune response patters developed in the response to the bacillus Mycobacterium leprae, the leprosy etiologic agent, are associated with the spectral clinical forms and outcome of the disease. In this context, B cells are allegedly involved in the disease immunopathogenesis, usually as antibody-producing cells, but also as potential effector or regulatory elements. In order to determine the regulatory B cells role in experimental leprosy, this study evaluated the outcome of M. leprae infection in B cell deficient mice (BKO) and WT C57Bl/6 control, by means of microbiological/bacilloscopic, immunohistochemical and molecular analysis, performed 8 months after M. leprae inoculation. The results demonstrated that infected BKO showed a higher bacilli number when compared with WT animals, demonstrating the importance of these cells in experimental leprosy. The molecular analysis demonstrates that the expression of IL-4, IL-10 and TGF-β was significantly higher in the BKO footpads when compared to WT group. Conversely, there was no difference in IFN-γ, TNF-α and IL-17 expression levels in BKO and WT groups. IL-17 expression was significantly higher in the lymph nodes of WT group. The immunohistochemical analysis revealed that M1 (CD80 + ) cells counts were significantly lower in the BKO group, while no significant difference was observed to M2 (CD206 + ) counts, resulting a skewed M1/M2 balance. These results demonstrated that the absence of B lymphocytes contribute to the persistence and multiplication of M. leprae, probably due to the increased expression of the IL-4, IL-10 and TGF-β cytokines, as well as a decrease in the number of M1 macrophages in the inflammatory site., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ana Paula F Trombone reports financial support was provided by State of Sao Paulo Research Foundation. Larissa S Benelli reports financial support was provided by National Council for Scientific and Technological Development. Heloisa Marques reports financial support was provided by Coordination of Higher Education Personnel Improvement. Mariana S V Malange reports financial support was provided by State of Sao Paulo Research Foundation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Methodology: 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. 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.

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.

The alveolar bone repair process may be influenced by multiple local and systemic factors, which include immune system cells and mediators. Macrophages allegedly play important roles in the repair process, and the transition of an initial inflammatory M1 profile into a pro-reparative M2 profile theoretically contributes to a favorable repair outcome. In this context, considering immunoregulatory molecules as potential targets for improving bone repair, this study evaluated the role of the immunoregulatory molecule FTY720, previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups submitted to tooth extraction and maintained under control conditions or treated with FTY720 were evaluated by microtomographic (μCT), histomorphometric, immunohistochemical and molecular analysis to characterize healing and host response features at 0, 1, 3, 7 and 14 days. Our results demonstrated that the FTY720 group presented higher bone tissue density, higher bone tissue volume, greater tissue volume fraction, greater number and thickness of trabeculae and a higher number of osteoblasts and osteoclasts than the control group. Accordingly, the bone markers BMP2, BMP7, ALPL, SOST and RANK mRNA expressions increased in the FTY720 treated group. Furthermore, the levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206 + cells, suggesting that the boost of bone formation mediated by FTY720 involves an increased polarization and activity of M2 macrophages in healing sites. Thus, our results demonstrate that FTY720 favored the process of alveolar bone repair, probably trough a strengthened M2 response, associated with an increased expression of markers osteogenic differentiation and activity markers. Immunoregulatory strategies based in the modulation of macrophage polarization profile can comprise effective tools to improve the bone repair process., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dicationic imidazolium-based ionic liquids with amino acid anions, such as IonL-phenylalanine (IonL-Phe), have been proposed as a multifunctional coating for titanium (Ti) dental implants. However, there has been no evaluation of the biocompatibility of these Ti coatings in the oral environment. This study aims to evaluate the effects of IonL-Phe on early healing and osseointegration of Ti in multiple rat demographics. IonL-Phe-coated and uncoated Ti screws were implanted into four demographic groups of rats to represent biological variations that could affect healing: young males (YMs) and females (YFs), ovariectomized (OVXFs) females, and old males (OMs). Samples underwent histopathological and histomorphometric analysis to evaluate healing at 7 and 30 days around IonL-coated and uncoated Ti. The real-time quantitative polymerase chain reaction was also conducted at the 2- and 7-day YM groups to evaluate molecular dynamics of healing while the IonL-Phe was present on the surface. IonL-coated and uncoated implants demonstrated similar histological signs of healing, while coated samples' differential gene expression of immunological and bone markers was compared with uncoated implants at 2 and 7 days in YMs. While YMs presented suitable osseointegration for both uncoated and IonL-Phe-coated groups, decreased success rate in other demographics resulted from lack of supporting bone in YFs and poor bone quality in OVXFs and OMs. Overall, it was found that IonL-coated samples had increased bone-to-implant contact across all demographic groups. IonL-Phe coating led to successful osseointegration across all animal demographics and presented the potential to prevent failures in scenarios known to be challenged by bacteria.

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.

Host inflammatory immune response comprises an essential element of the bone healing process, where M2 polarization allegedly contributes to a favorable healing outcome. In this context, immunoregulatory molecules that modulate host response, including macrophage polarization, are considered potential targets for improving bone healing. This study aims to evaluate the role of the immunoregulatory molecules VIP (Vasoactive intestinal peptide) and PACAP (Pituitary adenylate cyclase activating polypeptide), which was previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups were submitted to tooth extraction and maintained under control conditions or treated with VIP or PACAP were evaluated by microtomographic (µCT), histomorphometric, immunohistochemical, and molecular analysis at 0, 3, 7, and 14 days to quantify tissue healing and host response indicators at the healing site. Gene expression analysis demonstrates the effectiveness of VIP or PACAP in modulating host response, evidenced by the early dominance of an M2-type response, which was paralleled by a significant increase in M2 (CD206 + ) in treated groups. However, despite the marked effect of M1/M2 balance in the healing sites, the histomorphometric analysis does not reveal an equivalent/corresponding modulation of the healing process. µCT reveals a slight increase in bone matrix volume and the trabecular thickness number in the PACAP group, while histomorphometric analyzes reveal a slight increase in the VIP group, both at a 14-d time-point; despite the increased expression of osteogenic factors, osteoblastic differentiation, activity, and maturation markers in both VIP and PACAP groups. Interestingly, a lower number of VIP and PACAP immunolabeled cells were observed in the treated groups, suggesting a reduction in endogenous production. In conclusion, while both VIP and PACAP treatments presented a significant immunomodulatory effect with potential for increased healing, no major changes were observed in bone healing outcome, suggesting that the signals required for bone healing under homeostatic conditions are already optimal, and additional signals do not improve an already optimal process. Further studies are required to elucidate the role of macrophage polarization in the bone healing process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Azevedo, Fonseca, Colavite, Melchiades, Tabanez, Codo, de Medeiros, Trombone and Garlet.)

Bisphenol A (BPA) is an endocrine disrupting chemical able to promote hormone-responsive tumors. The major route of BPA contamination being oral, the aim of the present study was to investigate BPA effects on oral cells. Here, we evaluated the impact of sub-chronic in vivo exposure to BPA and its in vitro effects on neoplastic and non-neoplastic oral cells. We evaluated the oral mucosa of mice chronically exposed to BPA (200 mg/L). The response of keratinocytes (NOK-SI) and Head and Neck (HN) Squamous Cell Carcinoma (SCC), HN12 and HN13 cell lines to BPA was examined. In vivo, BPA accumulated in oral tissues and caused an increase in epithelial proliferative activity. BPA disrupted the function of keratinocytes by altering pro-survival and proliferative pathways and the secretion of cytokines and growth factors. In tumor cells, BPA induced proliferative, invasive, pro-angiogenic, and epigenetic paths. Our data highlight the harmful effects of BPA on oral mucosa and, tumorigenic and non-tumorigenic cells. Additionally, BPA may be a modifier of oral cancer cell behavior by prompting a functional shift to a more aggressive phenotype., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: There is a need to improve the predictability of osseointegration in implant dentistry. Current literature uses a variety of in vivo titanium (Ti) implantation models to investigate failure modes and test new materials and surfaces. However, these models produce a variety of results, making comparison across studies difficult. The purpose of this study is to validate an oral osseointegration in the Lewis rat to provide a reproducible baseline to track the inflammatory response and healing of Ti implants., Methods: Ti screws (0.76 mm Ø × 2 mm length) were implanted into the maxillary diastema of 52 adult male Lewis rats. Peri-implant tissues were evaluated 2, 7, 14, and 30 days after implantation ( n = 13). Seven of the 13 samples underwent microtomographic analysis, histology, histomorphometry, and immunohistochemistry to track healing parameters. The remaining six samples underwent quantitative polymerase chain reaction (qPCR) to evaluate gene expression of inflammation and bone remodeling markers over time., Results: This model achieved a 78.5% success rate. Successful implants had a bone to implant contact (BIC)% of 68.86 ± 3.15 at 30 days on average. Histologically, healing was similar to other rodent models: hematoma and acute inflammation at 2 days, initial bone formation at 7, advanced bone formation and remodeling at 14, and bone maturation at 30. qPCR indicated the highest expression of bone remodeling and inflammatory markers 2-7 days, before slowly declining to nonsurgery control levels at 14-30 days., Conclusion: This model combines cost-effectiveness and simplicity of a rodent model, while maximizing BIC, making it an excellent candidate for evaluation of new surfaces.

Human periodontal ligament fibroblast (hPLF) cells play an important role in maintaining oral cavity homeostasis with special function in tissue regeneration and maintenance of dental alveoli. Although their primary cell cultures are considered a good experimental model with no genetic changes, the finite life span may limit some experimental designs. The immortalization process increases cell life span but may cause genetic changes and chromosomal instability, resulting in direct effects on physiological cell responses. In this way, we aimed to investigate the global gene expression of hPLFs after the immortalization process by the ectopic expression of the catalytic subunit of the enzyme telomerase reverse transcriptase (hTERT) through transcriptome analysis. The embryonic origin of the primary culture of hPLF cells and immortalized hPLF-hTERT was also tested by vimentin staining, hTERT synthesis evaluated by indirect immunocytochemistry, analysis of cell proliferation, and morphology. The results indicated that hPLFs and hPLF-hTERT were positive for vimentin. On the 20th cell passage, hPLFs were in senescence, while hPLF-hTERT maintained their proliferation and morphology characteristics. At the same passage, hPLF-hTERT presented a significant increase in hTERT synthesis, but transcriptome did not reveal overexpression of the hTERT gene. Fifty-eight genes had their expression altered (11 upregulated and 47 downregulated) with the absence of changes in the key genes related to these cell types and in the main cancer-associated genes. In addition, the increase in hTERT protein expression without the overexpression of its gene indicates posttranscriptional level regulation. Successful immortalization of hPLFs through the ectopic expression of hTERT encourages further studies to design experimental protocols to investigate clinical questions from a translational perspective., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nogueira, Vasconcelos, Mitre, Bittencourt, Plaça, Kataoka, Pinheiro, Garlet, De Oliveira and Lima.)

Periodontal disease (PD) is a chronic destructive inflammatory disease of the tooth-supporting structures that leads to tooth loss at its advanced stages. Although the disease is initiated by a complex organization of oral microorganisms in the form of a plaque biofilm, it is the uncontrolled immune response to periodontal pathogens that fuels periodontal tissue destruction. IL-17A has been identified as a key cytokine in the pathogenesis of PD. Despite its well documented role in host defense against invading pathogens at oral barrier sites, IL-17A-mediated signaling can also lead to a detrimental inflammatory response, causing periodontal bone destruction. In this study, we developed a local sustained delivery system that restrains IL-17A hyperactivity in periodontal tissues by incorporating neutralizing anti-IL-17A Abs in poly(lactic-coglycolic) acid microparticles (MP). This formulation allowed for controlled release of anti-IL-17A in the periodontium of mice with ligature-induced PD. Local delivery of anti-IL-17A MP after murine PD induction inhibited alveolar bone loss and osteoclastic activity. The anti-IL-17A MP formulation also decreased expression of IL-6, an IL-17A target gene known to induce bone resorption in periodontal tissues. This study demonstrates proof of concept that local and sustained release of IL-17A Abs constitutes a promising therapeutic strategy for PD and may be applicable to other osteolytic bone diseases mediated by IL-17A-driven inflammation., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Introduction: MicroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs that may orchestrate the pathogenesis of apical periodontitis (AP). This study aimed to identify differentially expressed miRNAs and investigate their target gene pathways in AP., Methods: Total RNA was extracted from 10 human AP and 2 healthy apical tissues (controls) and subjected to miRNA sequencing for the identification of differentially expressed miRNAs (>1.5-fold changes). The function of the most up-regulated miRNA was further studied in vitro. miR-10a-5p mimics and inhibitors were introduced to human stem cells from the apical papilla and K-562 cells challenged with lipopolysaccharide, and expressions of predicted target genes were examined via quantitative reverse-transcription polymerase chain reaction and RNA sequencing., Results: A total of 852 miRNAs were identified, of which 12 were significantly up-regulated (1.54- to 8.44-fold) and 94 were significantly down-regulated (0.14- to 0.67-fold) in AP. Predicted target genes of these miRNAs are involved in inflammation, pain, and related pathways. miR-10a-5p showed the highest expression levels in AP. Overexpression of miR-10a-5p in LPS-challenged stem cells from the apical papilla resulted in down-regulation of messenger RNA levels of TNFA and up-regulation of interleukin IL10. RNA sequencing of K-562 cells treated with miR-10a-5p mimics and inhibitors identified miR-10a-5p target genes associated with multiple pathways, including macrophage-mediated inflammation and coagulation pathways., Conclusions: Over 100 miRNAs were differentially expressed in AP and appeared to be involved with modulation of genes in inflammatory and immune pathways. MiR-10a-5p was the most significantly up-regulated miRNA in AP and may play a critical role in suppressing inflammation and promoting healing., (Copyright © 2020 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Periodontal and periapical lesions are infectious inflammatory osteolitytic conditions in which a complex inflammatory immune response mediates bone destruction. However, the uncertainty of a lesion's progressive or stable phenotype complicates understanding of the cellular and molecular mechanisms triggering lesion activity. Evidence from clinical and preclinical studies of both periodontal and periapical lesions points to a high receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio as the primary determinant of osteolytic activity, while a low RANKL/OPG ratio is often observed in inactive lesions. Proinflammatory cytokines directly modulate RANKL/OPG expression and consequently drive lesion progression, along with pro-osteoclastogenic support provided by Th1, Th17, and B cells. Conversely, the cooperative action between Th2 and Tregs subsets creates an anti-inflammatory and proreparative milieu associated with lesion stability. Interestingly, the trigger for lesion status switch from active to inactive can originate from an unanticipated RANKL immunoregulatory feedback, involving the induction of Tregs and a host response outcome with immunological tolerance features. In this context, dendritic cells (DCs) appear as potential determinants of host response switch, since RANKL imprint a tolerogenic phenotype in DCs, described to be involved in both Tregs and immunological tolerance generation. The tolerance state systemically and locally suppresses the development of exacerbated and pathogenic responses and contributes to lesions stability. However, immunological tolerance break by comorbidities or dysbiosis could explain lesions relapse toward activity. Therefore, this article will provide a critical review of the current knowledge concerning periodontal and periapical lesions activity and the underlying molecular mechanisms associated with the host response. Further studies are required to unravel the role of immunological responsiveness or tolerance in the determination of lesion status, as well as the potential cooperative and/or inhibitory interplay among effector cells and their impact on RANKL/OPG balance and lesion outcome.

Objective: To verify the photobiomodulation effect on angiogenic proteins produced and released by dental human pulpal fibroblasts (HPFs)., Material and Methods: HPFs were irradiated with 660-nm low-level laser at fluences of 2.5 J/cm 2 and 3.7 J/cm 2 . The control group was not irradiated. MTT, crystal violet, and ELISA assays respectively verified viability, proliferation, and angiogenic protein (supernatant/lysate) at 6 h, 12 h, and 24 h after photobiomodulation. Capillary-like structure formation assay verified functional role. Two-way ANOVA/Tukey's test and ANOVA/Bonferroni's multiple comparisons test respectively verified cell viability/proliferation and intragroup and intergroup comparisons of protein synthesis (p < 0.05)., Results: Irradiated and non-irradiated HPFs showed statistically similar cell viability and proliferation pattern. Intragroup comparisons showed similar patterns of protein synthesis for all groups: VEGF-A, VEGF-C, and vascular endothelial growth factor receptor 1 (VEGFR1) increased significantly in the supernatant, while FGF-2 and VEGF-A increased significantly in the lysate. The lower fluence significantly increased BMP-9 (6 h) in the supernatant and VEGFR1 (6 h and 12 h) and VEGF-D (24 h) in the lysate, while the higher fluence significantly increased BMP-9 (6 h) in the supernatant and VEGFR1 (12 h) in the lysate. Regardless of the time, both fluences statistically downregulated placental growth factor (PLGF) and PDGF secretion. Both fluences statistically decreased VEGF-A secretion (24 h) and PLGF production (6 h)., Conclusion: Photobiomodulation produced stimulatory effects on angiogenic protein secretion by pulp fibroblasts. In terms of photobiomodulation, over time, both fluences significantly increased the secretion of VEGF-A, VEGF-C, and VEGFR1 and significantly upregulated BMP-9 (6 h) in the supernatant; for capillary-like structure formation, the fluence of 2.5 J/cm 2 was better than the fluence of 3.7 J/cm 2 ., Clinical Relevance: This study results addressed effective photobiomodulation parameters tailored for pulp angiogenesis.

F1-protein fraction (F1) is a natural bioactive compound extracted from the rubber tree, Hevea brasiliensis, and has been recently studied for its therapeutic potential in wound healing. In this study, we investigated the concentration-dependent effects of F1 (0.01%, 0.025%, 0.05%, and 0.1%) incorporated into deproteinized bovine bone (DBB) and porous biphasic calcium phosphate (pBCP), on the repair of rat calvarial critical-size bone defects (CSBD). The defects were analyzed by 3D-microtomography and 2D-histomorphometry at 12 weeks postsurgery. The binding efficiency of F1 to pBCP (96.3 ± 1.4%) was higher than that to DBB (67.7 ± 3.3%). In vivo analysis showed a higher bone volume (BV) gain in all defects treated with DBB (except in 0.1% of F1) and pBCP (except in 0.05% and 0.1% of F1) compared to the CSBD without treatment/control group (9.96 ± 2.8 mm 3 ). DBB plus 0.025% F1 promoted the highest BV gain (29.7 ± 2.2 mm 3 , p < .0001) compared to DBB without F1 and DBB plus 0.01% and 0.1% of F1. In the pBCP group, incorporation of F1 did not promote bone gain when compared to pBCP without F1 (15.9 ± 4.2 mm 3 , p > .05). Additionally, a small BV occurred in defects treated with pBCP plus 0.1% F1 (10.4 ± 1.4 mm 3, p < .05). In conclusion, F1 showed a higher bone formation potential in combination with DBB than with pBCP, in a concentration-dependent manner. Incorporation of 0.25% F1 into DBB showed the best results with respect to bone formation/repair in CSBD. These results suggest that DBB plus 0.25% F1 can be used as a promising bioactive material for application in bone tissue engineering., (© 2020 Wiley Periodicals, Inc.)

Objective: The aim of this study was to assess the correlation of inflammatory and pain genes polymorphisms with the presence of temporomandibular disorder (TMD) patients and with pressure pain sensitivity., Design: Data was collected from 268 consecutive subjects at Bauru School of Dentistry. Subjects aged younger than 20 years, with dental and neuropathic pain, sinusitis, cognitive and neurologic disorder were excluded. Included subjects were evaluated using the Research Diagnostic Criteria for Temporomandibular disorders and divided into two groups: TMD cases and healthy controls. Groups were submitted to pressure pain threshold (PPT) test for the temporomandibular joint, anterior temporalis and masseter muscles and genotyped for Val158Met, IL6-174, IL-1β-3954 and TNFA-308. Student's t-test and Pearson chi-square test were used to comparisons between groups. A linear multiple regression was used to evaluate the influence of genetics variables on the PPT and a bivariate analysis was used to assesses the influence of genetics variables on pain sensitivity below the PPT cut off of the structures in TMD group., Results: TMD group showed significantly lower PPT values for all structures when compared with control group (p < 0.001). SNP IL6-174 predicted higher pain sensitivity in the temporomandibular joint (p < 0.005) and in anterior temporalis muscle (p < 0.044) and SNP Val158Met in the masseter muscle (p < 0.038); when TMD group was divided according to PPT cut-off values the SNP Val158Met influenced increase pain sensibility in the masseter muscle., Conclusion: TNFA-308 was associated with TMD and SNP IL6-174 and SNP Val158Met influenced pain sensitivity of patients with TMD., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

Objective: Root resorption is a side effect of orthodontic tooth movement (OTM). Despite the recognized role of estrogen on bone, there is little information about their effects on orthodontic-induced inflammatory root resorption (OIIRR). We aimed to investigate if estrogen deficiency affects OIIRR in two mice strains., Methods: Female Balb/C (Balb) and C57BL6/J (C57) mice were ovariectomized (OVX) and replaced with estradiol (E2). Tooth samples subjected or not to OTM were collected and analyzed by microCT, histomorphometry and qPCR., Results: OVX resulted in decreased root volume (RV/TV) and root mineral density (RMD) in Balb mice without OTM. In contrast, OVX did not modify physiological root structure of C57 mice. OTM and OIIRR were increased after OVX in both mice strains after 30 days. E2 replacement reversed this phenotype in Balb, but not in C57 mice. Due to the significant increase of OIIRR in OVX Balb mice, the expression of key molecules was investigated in periodontium. Accordingly, these mice showed increased expression of receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor alpha, matrix metalloproteinases-2 and -13 and decreased osteoprotegerin (OPG) and interleukin-10 expression after OTM. E2 replacement reversed the changes of these markers., Conclusion: The lack of estrogen in Balb mice without OTM triggered loss of root structure which was positively correlated to RANKL/OPG ratio. Regardless of mouse strain, the absence of estrogen following OTM induced OIIRR. Mechanisms involve the imbalance of RANKL/OPG system, inflammatory and osteoclastic makers., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of this study was to analyze the effect of rapid maxillary expansion (RME) on hard tissues. Opening loops bonded to the first and second maxillary molars on both sides were used to apply distracting forces of 0.28 N, 0.42 N and 0.56 N at the midpalatal suture for 7 and 14 days. Microcomputed tomography (MicroCT), histomorphometry and quantitative polymerase chain reaction (qPCR) analysis were performed to evaluate RME effectiveness, midpalatal suture remodeling, cell counting of osteoblasts, osteoclasts and chondrocytes and the expression of bone remodeling markers, respectively. All forces at the two different time points resulted in similar RME and enhanced of bone remodeling. Accordingly, increased number of osteoblasts and reduced chondrocytes counting and no difference in osteoclasts were seen after all RME protocols. RME yielded increased expression of bone remodeling markers as osteocalcin (Ocn), dentin matrix acidic phosphoprotein-1 (Dmp1), runt-related transcription factor 2 (Runx2), collagen type I Alpha 1 (Col1a1), alkaline phosphatase (ALP), receptor activator of nuclear factor kappa B (RANK), receptor activator of nuclear factor kappa B ligand (Rankl), osteoprotegerin (Opg), cathepsin K (Ctsk), matrix metalloproteinases 9 and 13 (Mmp9 and 13), transforming growth fator beta 1, 2 and 3 (Tgfb 1, Tgfb 2 and Tgfb3), bone morphogenetic protein 2 (Bmp-2), sclerostin (Sost), beta-catenin-like protein 1 (Ctnnbl) and Wnt signaling pathways 3, 3a and 5a (Wnt 3, Wnt 3a and Wnt 5a). These findings characterize the cellular changes and potential molecular pathways involved in RME, proving the reliability of this protocol as a model for mechanical-induced bone remodeling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dicationic Imidazolum-based ionic liquids with amino acid anions (IonL) have been proposed as a multifunctional coating for titanium dental implants, as their properties have been shown to address multiple early complicating factors while maintaining host cell compatibility. This study aims to evaluate effects of this coating on host response in the absence of complicating oral factors during the early healing period using a subcutaneous implantation model in the rat. IonLs with the best cytocompatibility and antimicrobial properties (IonL-Phe, IonL-Met) were chosen as coatings. Three different doses were applied to cpTi disks and subcutaneously implanted into 36 male Lewis rats. Rats received 2 implants: 1 coated implant on one side and an uncoated implant on the contralateral sides (n=3 per formulation, per dose). Peri-implant tissue was evaluated 2 and 14 days after implantation with H&E staining and IHC markers associated with macrophage polarization as well as molecular analysis (qPCR) for inflammatory and healing markers. H&E stains revealed the presence of the coating, blood clots and inflammatory infiltrate at 2 days around all implants. At 14 days, inflammation had receded with more developed connective tissue with fibroblasts, blood vessels in certain doses of coated and uncoated samples with no foreign body giant cells. This study demonstrated that IonL at the appropriate concentration does not significantly interfere with and healing and Ti foreign body response. Results regarding optimal dose and formulation from this study will be applied in future studies using an oral osseointegration model., Competing Interests: Disclosures: The authors declare there are no conflicts of interest in this study.

Purpose: The purpose of this study was to evaluate bone repair with two bone substitutes, deproteinized bovine bone and biphasic calcium phosphate ceramics, associated with autogenous bone., Materials and Methods: The experimental groups were as follows: autogenous bone only (AB), autogenous bone/deproteinized bovine bone (1:1), and autogenous bone/biphasic calcium phosphate ceramic (1:1). After 30, 60, and 90 days, animals were euthanized and samples were collected for microcomputed tomography (micro-CT), histologic, histomorphometric, and expression analyses of VEGFA, RUNX2, ALP, COL1A1, OCN, PHEX, RANKL, and OPG genes by real-time polymerase chain reaction (PCR) array., Results: Histomorphometric analysis showed no difference in the amount of immature bone between AB and AB/biphasic calcium phosphate ceramic at 30 and 60 days. There was less mature bone formation in the AB/deproteinized bovine bone at 60 days compared with AB/biphasic calcium phosphate ceramic and AB, and a lower amount of immature bone in the AB/deproteinized bovine bone at 30 and 60 days compared with the AB (P ≤ .05). Micro-CT analysis showed higher immature bone volume (BVI) in the AB/biphasic calcium phosphate ceramic at 60 days and lower BVI at 90 days (P ≤ .05). Molecular analysis showed a lower expression of all genes in the AB/deproteinized bovine bone and AB/biphasic calcium phosphate ceramic compared with AB at all time points. A greater expression of RANKL was found in the AB/deproteinized bovine bone compared with AB/biphasic calcium phosphate ceramic at 30 days (P ≤ .05), and a lower expression of the OC, RUNX2, and ALP genes in AB/deproteinized bovine bone and AB/biphasic calcium phosphate ceramic was found compared with AB at all time points (P ≤ .05)., Conclusion: The use of biphasic calcium phosphate ceramic resulted in greater immature bone formation than deproteinized bovine bone at an early assessment. The studied bone regeneration genes were downregulated in comparison to the control.

Single nucleotide polymorphisms (SNPs) in WNT genes may impact gene/protein function and contribute to individual predisposition to apical periodontitis (AP). Here, we investigated the association of SNPs in/nearby WNT3, WNT3A, WNT5A, WNT8A, WNT9B and WNT11 genes with AP using a case-control dataset. Cases were defined as individuals with deep caries and AP (n = 188); controls had deep caries and no AP (n = 230). Genotyping was performed using Taqman chemistry in real time PCR. Data analyses was performed using Fisher Exact tests assuming a Bonferroni correction threshold value of 0.005. Single-SNP association analysis revealed a trend for association with WNT3 rs9890413 genotypes (P = 0.009) under a dominant model and allelic association for WNT3A rs1745420 (P = 0.009). Haplotypes involving WNT3-WNT9B-WNT3A alleles were also significantly associated with AP (P ≤ 0.003). Luciferase reporter assays showed higher transcriptional activity (1.4-fold) with the alternate G allele in rs1745420. Expression of WNT3, WNT3A and WNT5A in AP tissues was significantly higher than in control tissues, and inversely correlated with the expression of SERPINB1, COL1A1 and TIMP1 (P < 0.05). Our results suggest that WNT genes have a role in modulating AP and polymorphisms in these genes may increase susceptibility to AP.

Introduction: The balance between the host proinflammatory immune response and the counteracting anti-inflammatory and reparative responses supposedly determine the outcome of periapical lesions. In this scenario, the vasoactive intestinal peptide (VIP) may exert a protective role because of its prominent immunoregulatory capacity. In this study, we investigated (in a cause-and-effect manner) the potential involvement of VIP in the development of human and experimental periapical lesions., Methods: Periapical granulomas (n = 124) and control samples (n = 48) were comparatively assessed for VIP and multiple immunologic/activity marker expression through real-time polymerase chain reaction. Experimental periapical lesions (C57Bl/6 wild-type mice) were evaluated regarding endogenous VIP expression correlation with lesion development and the effect of recombinant VIP therapy in lesion outcome. CCR4KO and IL4KO strains and anti-glucocorticoid-induced TNFR-related protein inhibition were used to test the involvement of Treg and Th2 cells in VIP-mediated effects., Results: VIP expression was more prevalent in periapical granulomas than in controls, presenting a positive association with immunoregulatory factors and an inverse association/correlation with proinflammatory mediators and the receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio. Endogenous VIP expression up-regulation was temporally associated with lesion immunoregulation and a decline of bone loss. VIP therapy in mice prompted the arrest of lesion development, being associated with an anti-inflammatory and proreparative response that limits the proinflammatory, Th1, Th17, and osteoclastogenic response in the periapex. The VIP protective effect was dependent of Treg migration and activity and independent of interleukin 4., Conclusions: Our results show that VIP overexpression in human and experimental periapical lesions is associated with lesion inactivity and that VIP therapy results in the attenuation of experimental lesion progression associated with the immunosuppressive response involving Treg cells., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Short-chain fatty acids (SCFAs) exert a variety of immune and metabolic functions by binding to G-protein-coupled receptors, mainly free fatty acid receptor 2 (FFAR2). However, the effects of SCFAs and FFARs on bone remodeling, especially in alveolar bone, have been less explored. In this study, we investigated the influence of the SCFA/FFAR2 axis on alveolar bone. Bone samples from wild-type (WT) and FFAR2-deficient mice (FFAR2-/-) were analyzed using micro-CT, histology and qPCR. WT and FFAR2-/- animals received a high-fiber diet (HFD) reported to increase circulating levels of SCFAs. Additionally, we analyzed the effects of SCFAs and a synthetic FFAR2 agonist, phenylacetamide-1 (CTMB), on bone cell differentiation. The participation of histone deacetylase inhibitors (iHDACs) in the effects of SCFAs was further assessed in vitro. CTMB treatment was also evaluated in vivo during orthodontic tooth movement (OTM). FFAR2-/- mice exhibited deterioration of maxillary bone parameters. Consistent with this, FFAR2-/- mice exhibited a significant increase of OTM and changes in bone cell numbers and in the expression of remodeling markers. The HFD partially reversed bone loss in the maxillae of FFAR2-/- mice. In WT mice, the HFD induced changes in the bone markers apparently favoring a bone formation scenario. In vitro, bone marrow cells from FFAR2-/- mice exhibited increased differentiation into osteoclasts, while no changes in osteoblasts were observed. In line with this, differentiation of osteoclasts was diminished by SCFAs and CTMB. Moreover, CTMB treatment significantly reduced OTM. Pretreatment of osteoclasts with iHDACs did not modify the effects of SCFAs on these cells. In conclusion, SCFAs function as regulators of bone resorption. The effects of SCFAs on osteoclasts are dependent on FFAR2 activation and are independent of the inhibition of HDACs. FFAR2 agonists may be useful to control bone osteolysis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Type 1 diabetes (T1D) is associated with delayed tissue healing and bone loss. Periodontal tissues during tooth movement (OTM) in T1D and under diabetic treatment are poorly understood. We aimed to study the effect of metformin as an add-on to insulin therapy on periodontal structures during OTM in T1D rats., Methods: Rats were divided into normoglycemic (NG, n = 20) and streptozotocin-induced diabetic groups that were untreated (T1D, n = 20), treated with insulin (I-T1D, n = 20), or treated with insulin plus metformin (IM-T1D, n = 20). After 7 days of treatment, the first right upper molar (M1) was moved mesially. At days 0, 3, 7 and 14, the pattern of OTM and the periodontal tissues were analyzed by micro-CT, histomorphometry, and immunohistochemistry for TRAP., Results: In T1D, major osteoclastogenic activity and bone loss versus other groups were confirmed by a greater TRAP-positive cell number and reabsorption surface on both the pressure and tension sides for 14 days (p < 0.01). Additionally, we observed low bone volume density. Metformin plus insulin resulted in a daily insulin dose reduction and major glycemic control versus I-T1D. Although no significant differences were observed between I-T1D and IM-T1D, the tooth displacement and inclination, periodontal ligament thickness, and alveolar bone density on the pressure side in IM-T1D were similar to that of NG (p > 0.05)., Conclusion: Antidiabetic treatment reduces severe periodontal damage during applied orthodontic force in T1D untreated rats. Metformin as an add-on to insulin therapy resulted in glycemic control and a periodontal tissue response to orthodontic forces that was similar to that of normoglycemic rats., (© 2019 American Academy of Periodontology.)

The maxillofacial skeleton is highly dynamic and requires a constant equilibrium between the bone resorption and bone formation. The field of osteoimmunology explores the interactions between bone metabolism and the immune response, providing a context to study the complex cellular and molecular networks involved in oro-maxillofacial osteolytic diseases. In this review, we present a framework for understanding the potential mechanisms underlying the immuno-pathobiology in etiologically-diverse diseases that affect the oral and maxillofacial region and share bone destruction as their common clinical outcome. These otherwise different pathologies share similar inflammatory pathways mediated by central cellular players, such as macrophages, T and B cells, that promote the differentiation and activation of osteoclasts, ineffective or insufficient bone apposition by osteoblasts, and the continuous production of osteoclastogenic signals by immune and local stromal cells. We also present the potential translational applications of this knowledge based on the biological mechanisms involved in the inflammation-induced bone destruction. Such applications can be the development of immune-based therapies that promote bone healing/regeneration, the identification of host-derived inflammatory/collagenolytic biomarkers as diagnostics tools, the assessment of links between oral and systemic diseases; and the characterization of genetic polymorphisms in immune or bone-related genes that will help diagnosis of susceptible individuals.

Background: Chemokines and chemokine receptors are critical in oral tumourigenesis. The atypical chemokine receptor ACKR2 is a scavenger of CC chemokines controlling the availability of these molecules at tumour sites, but the role of ACKR2 in the context of oral carcinogenesis is unexplored., Methods: In this study, wild-type (WT) and ACKR2 deficient mice (ACKR2 -/- ) were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) for induction of oral carcinogenesis. Tongues were collected for macro and microscopic analysis and to evaluate the expression of ACKRs, CC chemokines and its receptors, inflammatory cytokines, angiogenic factors, adhesion molecules and extracellular matrix components., Results: An increased expression of ACKR2 in squamous cell carcinoma (SCC) lesions of 4NQO-treated WT mice was observed. No significant differences were seen in the ACKR1, ACKR3 and ACKR4 mRNA expression comparing SCC lesions from WT and ACKR2 -/- treated mice. Significantly higher expression of CCL2, IL-6 and IL-17 was detected in ACKR2 -/- treated mice. In contrast, the expression of other CC-chemokines, and receptors, angiogenic factors, adhesion molecules and extracellular matrix components were similarly increased in SCC lesions of both groups. Clinical and histopathological analysis revealed no differences in inflammatory cell recruitment and in the SCC incidence comparing WT and ACKR2 -/- treated mice., Conclusion: The results suggest that ACKR2 expression regulates inflammation in tumour-microenvironment but the absence of ACKR2 does not impact chemically-induced oral carcinogenesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Zika virus (ZIKV) infection during pregnancy may cause major congenital defects, including microcephaly, ocular, articular and muscle abnormalities, which are collectively defined as Congenital Zika Syndrome. Here, we performed an in-depth characterization of the effects of congenital ZIKV infection (CZI) in immunocompetent mice., Methods: Pregnant dams were inoculated with ZIKV on embryonic day 5.5 in the presence or absence of a sub-neutralizing dose of a pan-flavivirus monoclonal antibody (4G2) to evaluate the potential role of antibody-dependent enhancement phenomenon (ADE) during short and long outcomes of CZI., Findings: ZIKV infection induced maternal immune activation (MIA), which was associated with occurrence of foetal abnormalities and death. Therapeutic administration of AH-D antiviral peptide during the early stages of pregnancy prevented ZIKV replication and death of offspring. In the post-natal period, CZI was associated with a decrease in whole brain volume, ophthalmologic abnormalities, changes in testicular morphology, and disruption in bone microarchitecture. Some alterations were enhanced in the presence of 4G2 antibody., Interpretation: Our results reveal that early maternal ZIKV infection causes several birth defects in immunocompetent mice, which can be potentiated by ADE phenomenon and are associated with MIA. Additionally, antiviral treatment with AH-D peptide may be beneficial during early maternal ZIKV infection. FUND: This work was supported by the Brazilian National Science Council (CNPq, Brazil), Minas Gerais Foundation for Science (FAPEMIG), Funding Authority for Studies and Projects (FINEP), Coordination of Superior Level Staff Improvement (CAPES), National Research Foundation of Singapore and Centre for Precision Biology at Nanyang Technological University., (Copyright © 2019. Published by Elsevier B.V.)

The release of the prototypic DAMP High Mobility Group Box 1 (HMGB1) into extracellular environment and its binding to the Receptor for Advanced Glycation End Products (RAGE) has been described to trigger sterile inflammation and regulate healing outcome. However, their role on host response to Ti-based biomaterials and in the subsequent osseointegration remains unexplored. In this study, HMGB1 and RAGE inhibition in the Ti-mediated osseointegration were investigated in C57Bl/6 mice. C57Bl/6 mice received a Ti-device implantation (Ti-screw in the edentulous alveolar crest and a Ti-disc in the subcutaneous tissue) and were evaluated by microscopic (microCT [bone] and histology [bone and subcutaneous]) and molecular methods (ELISA, PCR array) during 3, 7, 14, and 21 days. Mice were divided into 4 groups: Control (no treatment); GZA (IP injection of Glycyrrhizic Acid for HMGB1 inhibition, 4 mg/Kg/day); RAP (IP injection of RAGE Antagonistic Peptide, 4 mg/Kg/day), and vehicle controls (1.5% DMSO solution for GZA and 0.9% saline solution for RAP); treatments were given at all experimental time points, starting 1 day before surgeries. HMGB1 was detected in the Ti-implantation sites, adsorbed to the screws/discs. In Control and vehicle groups, osseointegration was characterized by a slight inflammatory response at early time points, followed by a gradual bone apposition and matrix maturation at late time points. The inhibition of HMGB1 or RAGE impaired the osseointegration, affecting the dynamics of mineralized and organic bone matrix, and resulting in a foreign body reaction, with persistence of macrophages, necrotic bone, and foreign body giant cells until later time points. While Control samples were characterized by a balance between M1 and M2-type response in bone and subcutaneous sites of implantation, and also MSC markers, the inhibition of HMGB1 or RAGE caused a higher expression M1 markers and pro-inflammatory cytokines, as well chemokines and receptors for macrophage migration until later time points. In conclusion, HMGB1 and RAGE have a marked role in the osseointegration, evidenced by their influence on host inflammatory immune response, which includes macrophages migration and M1/M2 response, MSC markers expression, which collectively modulate bone matrix deposition and osseointegration outcome.

Background: Although inflammation can alter cytokines release and nerve function, it is not yet fully established if orthodontic-induced inflammation can cause significant extraoral trigeminal somatosensory alterations and release of inflammatory chemical mediators., Objective: The primary aim of this study was to investigate the impact of orthodontic separator and short-term fixed orthodontic appliance on the extraoral trigeminal somatosensory function and concentrations of cytokines in the gingival crevicular fluid (GCF)., Methods: Twenty-two female patients were evaluated as follow: baseline, 24 hour-after elastomeric separator (-aES), 24 hour- and 1 month-after bonding brackets (-aBB) at both arches. The outcome variables were as follows: self-reported pain (Visual Analog Scale), QSTs (current perception threshold-CPT, cold detection threshold-CDT, warm detection threshold-WDT, mechanical detection threshold-MDT, mechanical suprathreshold-MST and wind-up ratio-WUR. All QSTs were performed at infra-orbital and mental nerve entry zone at patient`s dominant side. In addition, GCF samples in order to assess cytokines profile (IL-1β,IL-8,IL-6 and TNF-α) were collected. ANOVA and Tukey's post hoc analyses were performed (a = 5%)., Results: Patients reported higher pain intensity 24 hour-aBB compared to baseline and 24 hour-aES (P < 0.050). Patients were less sensitive to pin-prick pain (MST) at 24 hour-aBB and 1 month-aBB compared to baseline (P < 0.006). Significant increases in IL-6 levels were observed 24 hour-aBB (P < 0.001). Multiple comparison analysis showed significant increase in IL-1β levels (P < 0.001) and TNF-α (P < 0.001) 1 month-aBB compared to baseline., Conclusion: Elastomeric separators only induced mild pain and were not able to significantly increase proinflammatory cytokines level in the GCF. In addition, orthodontic fixed appliance may induce only minor somatosensory changes at extraoral trigeminal locations., (© 2018 John Wiley & Sons Ltd.)

TBX21-1993T/C (rs4794067) polymorphism increases the transcriptional activity of the Tbx21, essential for interferon gamma (IFNg) transcription, but its functional impact on development Th1- response in vivo remains unclear, as well its potential influence over inflammatory osteolytic conditions, such as periapical lesions. Therefore, this study comprises a case-control and functional investigation of Tbx21 genetic variations impact on Th1 response in vivo and in vitro, and its impact on periapical lesions risk and outcome, performed with a population of healthy controls (H; N = 283) and patients presenting periapical lesions (L; N = 188) or deep caries (DC; N = 152). TBX21-1993T/C genotyping demonstrated that the polymorphic allele C, as well TC/TC+CC genotypes, was significantly less frequent in the L patients compared to H and DC groups. Additionally, gene expression analysis demonstrates that T-cell-specific T-box transcription factor (Tbet) and IFNg transcripts levels were downregulated whereas IL-17 levels were upregulated in the TBX21-1993 C carriers (TC/TC+CC) in comparison with the TT group. Also, while TT and TC+CC genotypes are equally prevalent in the lesions presenting low IFN/IL17 ratio, a significant decrease in polymorphic TC+CC genotypes was observed in lesions presenting intermediate and high IFN/IL17 ratio. In vitro experiments confirmed the predisposition to Th1 polarization associated with TBX21-1993, since PBMC CD4 T cells from T allele carriers produce higher IFNg levels upon CD3/CD28 stimulation than the C group, in both standard/neutral and Th1-polarizing culture conditions. In conclusion, the TBX21-1993 T allele and TC/CC genotypes predispose to Th1-type immune response development in vitro, influence immune response polarization in vivo, and consequently account for the risk for apical periodontitis development., (©2018 Society for Leukocyte Biology.)