B lymphocytes deficiency results in altered immune response and increased susceptibility to Mycobacterium leprae in a murine leprosy model.

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-ß 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-ß cytokines, as well as a decrease in the number of M1 macrophages in the inflammatory site.

Local Sustained Delivery of Anti-IL-17A Antibodies Limits Inflammatory Bone Loss in Murine Experimental Periodontitis.

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.

The effect of orthodontic separator and short-term fixed orthodontic appliance on inflammatory mediators and somatosensory function.

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-1ß,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-1ß 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.

ST2 regulates bone loss in a site-dependent and estrogen-dependent manner.

Interleukin-33 (IL-33) and its receptor, ST2, are implicated in bone remodeling. The lack of estrogen after menopause results in an accelerated bone loss. Here we investigated the role of ST2 in the bone loss induced by estrogen deficiency. ST2-deficient mice (ST2-/- ) and their littermates (wildtype [WT]) were ovariectomized (OVX), while ovary-intact mice were used as controls. Bone sites were analyzed by microcomputed tomography, histomorphometry, and quantitative real-time polymerase chain reaction (qPCR). Deletion of IL-33 or ST2 resulted in a similar bone loss in the femur and maxilla. Ovariectomy in WT mice caused bone loss in the same areas. The lack of ST2 in OVX mice did not alter bone remodeling in the femur but prevented bone loss in the maxilla. Consistently, ovariectomy increased the IL-33 messenger RNA (mRNA) levels in the maxilla but not in the femur. Under mechanical stimulation, ovariectomy and ST2 deletion independently increased bone remodeling induced by orthodontic tooth movement, which was also associated with a greater number of osteoclasts and a reduced number of osteoblasts in the maxillary bone. ST2-/- OVX mice, however, displayed twice as many osteoblasts as that of WT OVX mice. Ovariectomy and ST2 deletion differently altered the cytokine mRNA levels in the maxilla. Remarkably, interleukin-10 expression was decreased in both WT OVX and ST2-/- mice, and this reduction was completely restored in ST2-/- OVX mice. The results demonstrate that estrogen and IL33/ST2 independently protect against bone loss. However, the ovariectomy-induced bone loss is IL-33/ST2-dependent in the maxilla but not in the femur, indicating a bimodal and site-specific role of ST2 in bone remodeling.

CCR5Δ32 (rs333) polymorphism is associated with decreased risk of chronic and aggressive periodontitis: A case-control analysis based in disease resistance and susceptibility phenotypes.

Chronic and aggressive periodontitis are infectious diseases characterized by the irreversible destruction of periodontal tissues, which is mediated by the host inflammatory immune response triggered by periodontal infection. The chemokine receptor CCR5 play an important role in disease pathogenesis, contributing to pro-inflammatory response and osteoclastogenesis. CCR5Δ32 (rs333) is a loss-of-function mutation in the CCR5 gene, which can potentially modulate the host response and, consequently periodontitis outcome. Thus, we investigated the effect of the CCR5Δ32 mutation over the risk to suffer periodontitis in a cohort of Brazilian patients (total N=699), representative of disease susceptibility (chronic periodontitis, N=197; and aggressive periodontitis, N=91) or resistance (chronic gingivitis, N=193) phenotypes, and healthy subjects (N=218). Additionally, we assayed the influence of CCR5Δ32 in the expression of the biomarkers TNFα, IL-1ß, IL-10, IL-6, IFN-γ and T-bet, and key periodontal pathogens P. gingivalis, T. forsythia, and T. denticola. In the association analysis of resistant versus susceptible subjects, CCR5Δ32 mutant allele-carriers proved significantly protected against chronic (OR 0.49; 95% CI 0.29-0.83; p-value 0.01) and aggressive (OR 0.46; 95% CI 0.22-0.94; p-value 0.03) periodontitis. Further, heterozygous subjects exhibited significantly decreased expression of TNFα in periodontal tissues, pointing to a functional effect of the mutation in periodontal tissues during the progression of the disease. Conversely, no significant changes were observed in the presence or quantity of the periodontal pathogens P. gingivalis, T. forsythia, and T. denticola in the subgingival biofilm that could be attributable to the mutant genotype.

Melanocortin agonism as a viable strategy to control alveolar bone loss induced by oral infection.

Alveolar bone loss is a result of an aggressive form of periodontal disease (PD) associated with Aggregatibacter actinomycetemcomitans (Aa) infection. PD is often observed with other systemic inflammatory conditions, including arthritis. Melanocortin peptides activate specific receptors to exert antiarthritic properties, avoiding excessing inflammation and modulating macrophage function. Recent work has indicated that melanocortin can control osteoclast development and function, but whether such protection takes place in infection-induced alveolar bone loss has not been investigated. The purpose of this study was to evaluate the role of melanocortin in Aa-induced PD. Mice were orally infected with Aa and treated with the melanocortin analog DTrp8-γMSH or vehicle daily for 30 d. Then, periodontal tissue was collected and analyzed. Aa-infected mice treated with DTrp8-γMSH presented decreased alveolar bone loss and a lower degree of neutrophil infiltration in the periodontium than vehicle-treated animals; these actions were associated with reduced periodontal levels of TNF-α, IFN-γ, and IL-17A. In vitro experiments with cells differentiated into osteoclasts showed that osteoclast formation and resorptive activity were attenuated after treatment with DTrp8-γMSH. Thus, melanocortin agonism could represent an innovative way to tame overexuberant inflammation and, at the same time, preserve bone physiology, as seen after Aa infection.-Madeira, M. F. M., Queiroz-Junior, C. M., Montero-Melendez, T., Werneck, S. M. C., Corrêa, J. D., Soriani, F. M., Garlet, G. P., Souza, D. G., Teixeira, M. M., Silva, T. A., Perretti, M. Melanocortin agonism as a viable strategy to control alveolar bone loss induced by oral infection.

The role of 5-lipoxygenase in Aggregatibacter actinomycetemcomitans-induced alveolar bone loss.

AIM: Leukotrienes (LTs) are pro-inflammatory lipid mediators formed by the enzyme 5-lipoxygenase (5-LO). The involvement of 5-LO metabolites in periodontal disease (PD) is not well defined. This study aimed to assess the role of 5-LO in experimental PD induced by Aggregatibacter actinomycetemcomitans (Aa). MATERIAL AND METHODS: In vivo experiments were carried out using SV129 wild-type (WT) and 5-LO-deficient (5lo-/- ) mice inoculated with Aa. Osteoclasts were stimulated in vitro with AaLPS in the presence or not of selective inhibitors of the 5-LO pathway, or LTB4 or platelet-activating factor (PAF), as PAF has already been shown to increase osteoclast activity. RESULTS: In 5lo-/- mice, there were no loss of alveolar bone and less TRAP-positive osteoclasts in periodontal tissues, after Aa inoculation, despite local production of TNF-α and IL-6. The differentiation and activity of osteoclasts stimulated with AaLPS were diminished in the presence of BLT1 antagonist or 5-LO inhibitor, but not in the presence of cysteinyl leukotriene receptor antagonist. The osteoclast differentiation induced by PAF was impaired by the BLT1 antagonism. CONCLUSION: In conclusion, LTB4 but not CysLTs is important for Aa-induced alveolar bone loss. Overall, LTB4 affects osteoclast differentiation and activity and is a key intermediate of PAF-induced osteoclastogenesis.

Osteoprotective Effects of IL-33/ST2 Link to Osteoclast Apoptosis.

The relevance of IL-33 and its receptor ST2 for bone remodeling is not well-defined. Our aim was to assess the role and underlying mechanisms of IL-33/ST2 in mechanically induced bone remodeling. BALB/c (wild type) and ST2 deficient (St2(-/-)) mice were subjected to mechanical loading in alveolar bone. Microtomography, histology, and real-time quantitative PCR were performed to analyze bone parameters, apoptosis and bone cell counts, and expression of bone remodeling markers, respectively. MC3T3-E1 osteoblastic cells and bone marrow cells were used to verify if mechanical force triggered IL-33 and ST2 expression as well as the effects of IL-33 on osteoclast differentiation and activity. Mechanical loading increased the expression of IL-33 and ST2 in alveolar bone in vivo and in osteoblastic cells in vitro. St2(-/-) mice had increased mechanical loading-induced bone resorption, number of osteoclasts, and expression of proresorptive markers. In contrast, St2(-/-) mice exhibited reduced numbers of osteoblasts and apoptotic cells in periodontium and diminished expression of osteoblast signaling molecules. In vitro, IL-33 treatment inhibited osteoclast differentiation and activity even in the presence of receptor activator of NF-κB ligand. IL-33 also increased the expression of pro-apoptotic molecules, including Bcl-2-associated X protein (BAX), cell-surface Fas receptor (FAS), FASL, FAS-associated death domain, tumor necrosis factor-related apoptosis-inducing ligand, and BH3 interacting-domain death (BID). Overall, these findings suggest that IL-33/ST2 have anti-osteoclastogenic effects and reduce osteoclast formation and activity by inducing their apoptosis.

Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes.

The hallmark of periodontal disease is the progressive destruction of gingival soft tissue and alveolar bone, which is initiated by inflammation in response to an invasive and persistent bacterial insult. In recent years, it has become apparent that this tissue destruction is associated with a decrease in local regulatory processes, including a decrease of forkhead box P3-expressing regulatory lymphocytes. Accordingly, we developed a controlled release system capable of generating a steady release of a known chemoattractant for regulatory lymphocytes, C-C motif chemokine ligand 22 (CCL22), composed of a degradable polymer with a proven track record of clinical translation, poly(lactic-co-glycolic) acid. We have previously shown that this sustained presentation of CCL22 from a point source effectively recruits regulatory T cells (Tregs) to the site of injection. Following administration of the Treg-recruiting formulation to the gingivae in murine experimental periodontitis, we observed increases in hallmark Treg-associated anti-inflammatory molecules, a decrease of proinflammatory cytokines, and a marked reduction in alveolar bone resorption. Furthermore, application of the Treg-recruiting formulation (fabricated with human CCL22) in ligature-induced periodontitis in beagle dogs leads to reduced clinical measures of inflammation and less alveolar bone loss under severe inflammatory conditions in the presence of a diverse periodontopathogen milieu.

Nociceptors regulate osteoimmune transcriptomic response to infection.

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.

MMP3 and TIMP1 variants contribute to chronic periodontitis and may be implicated in disease progression.

AIM: Matrix metalloproteinases (MMPs) play a key role in the tissue destruction characteristic of chronic periodontitis. The purpose of this study was to investigate the association of MMP and TIMP polymorphisms with chronic periodontitis in two populations. MATERIAL AND METHODS: A total of 34 polymorphisms spanning 12 MMP and 2 TIMP genes were genotyped in 401 individuals from Brazil (99 cases with chronic periodontitis and 302 controls), and 274 individuals from the US (70 cases and 204 controls). Individuals were considered cases if presenting at least three teeth exhibiting sites of clinical attachment loss ≥ 5 mm in two different quadrants. Controls were characterized by absence of clinical attachment loss and no sites with probing depth >3 mm. MMP3 and TIMP1 mRNA expression was evaluated in healthy and diseased periodontal tissues. RESULTS: TIMP1 showed association with chronic periodontitis in the Brazilian population (for rs5906435, p = 0.0004), whereas MMP3 showed association in the US population (for rs679620, p = 0.0003; and rs650108, p = 0.002) and in the Brazilian population (for rs639752, p = 0.005). MMP3 and TIMP1 mRNA expression was significantly higher in diseased tissues when compared to control tissues. CONCLUSIONS: Our results further support a role for variations in MMP3 in chronic periodontitis and report a novel association with TIMP1. These genes may be considered additional candidate genes for chronic periodontitis.

Effects of Dicationic Imidazolium-Based Ionic Liquid Coatings on Oral Osseointegration of Titanium Implants: A Biocompatibility Study in Multiple Rat Demographics.

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.

Effect of diabetes on orthodontic tooth movement in a mouse model.

Orthodontic tooth movement is achieved by the remodeling of alveolar bone in response to mechanical loading. Type 1 diabetes results in bone remodeling, suggesting that this disease might affect orthodontic tooth movement. The present study investigated the effects of the diabetic state on orthodontic tooth movement. An orthodontic appliance was placed in normoglycemic (NG), streptozotocin-induced diabetes (DB), and insulin-treated DB (IT) C57BL6/J mice. Histomorphometric analysis and quantitative PCR of periodontium were performed. The DB mice exhibited greater orthodontic tooth movement and had a higher number of tartrate-resistant acid phosphate (TRAP) -positive osteoclasts than NG mice. This was associated with increased expression of factors involved in osteoclast activity and recruitment (Rankl, Csf1, Ccl2, Ccl5, and Tnfa) in DB mice. The expression of osteoblastic markers (Runx2, Ocn, Col1, and Alp) was decreased in DB mice. Reversal of the diabetic state by insulin treatment resulted in morphological findings similar to those of NG mice. These results suggest that the diabetic state up-regulates osteoclast migration and activity and down-regulates osteoblast differentiation, resulting in greater orthodontic tooth movement.

The effect of a single episode of antimicrobial photodynamic therapy in the treatment of experimental periodontitis. Microbiological profile and cytokine pattern in the dog mandible.

The purpose of this study was to evaluate the effect of a single application of antimicrobial photodynamic therapy (aPDT) on microbiological profile and cytokine pattern in dogs. Periodontal disease was induced by placing 3.0 silk ligatures around the mandibular pre-molars bilaterally during 8 weeks. The dogs were randomly treated with aPDT using a dye/laser system, scaling and root planning (SRP), or with the association of treatments (SRP + aPDT). Plaque samples were collected at baseline, 1, 3, and 4 weeks, and the mean counts of 40 species were determined using DNA-DNA hybridization. Gingival biopsies were removed and the expression of tumor necrosis factor alpha (TNF-α), receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase (MMP-1), interleukin (IL) 6, IL-10 and total bacterial load by analysis of 16 S rRNA gene were evaluated through real-time PCR. The results shows that the levels of the majority of the species were reduced 1 week post-therapy for all treatments, however, an increase in counts of Prevotella intermedia (p = 0.00), Prevotella. nigrescens (p = 0.00) and Tannerella forsythia (p = 0.00) was observed for aPDT and SRP + aPDT. After 4 weeks, a regrowth of Porphyromonas gingivalis (p = 0.00) and Treponema denticola (p = 0.00), was observed for all treatments. Also, a strikingly reduction of counts on counts of Aggregatibacter actinomycetemcomitans was observed for the aPDT (p = 0.00). For the cytokine pattern, the results were similar for all treatments, and a reduction in the expression of cytokines and bacterial load was observed throughout the study. Our results suggest that SRP, aPDT in a single application, and SRP + aPDT affects different bacterial species and have similar effects on the expression of cytokines evaluated during the treatment of ligature-induced periodontitis.

Expression Profiling and Functional Characterization of MicroRNAs in Apical Periodontitis.

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.

Cellular and Molecular Dynamics during Early Oral Osseointegration: A Comprehensive Characterization in the Lewis Rat.

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.

Regulatory T cells attenuate experimental periodontitis progression in mice.

AIMS: The aim of this study was to identify the presence and characterize the function of regulatory T cells (Tregs) in experimental periodontitis in mice. MATERIAL AND METHODS: C57Bl/6 mice infected with Actinobacillus actinomycetemcomitans, treated or not with anti-glucocorticoid-inducible tumour necrosis factor receptor (anti-GITR) to inhibit Tregs function, were analysed regarding inflammatory cell and Tregs influx, alveolar bone loss and cytokine expression/production (analysed by real-time polymerase chain reaction and ELISA) throughout experimental periodontitis. RESULTS: A. actinomycetemcomitans inoculation in mice resulted in periodontal disease characterized by marked alveolar bone loss and an influx of inflammatory cells. Flow cytometry evaluation of inflammatory cells demonstrated an increased number of CD4(+)CD25(+) and CD4(+)FOXp3(+) cells, characterizing the presence of Tregs in the periodontal environment in a late stage after infection. Tregs-associated cytokines interleukin-10 (IL-10), cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) and transforming growth factor-beta (TGF-beta) were found to be expressed/produced in a kinetics that resembles Tregs migration. Treatment with anti-GITR, which inhibits Tregs function, showed increased alveolar bone loss and inflammatory cell migration. A reduction in IL-10, CTLA-4 and TGF-beta levels was also observed, while interferon-gamma, tumour necrosis factor-alpha and receptor activator for nuclear factor kappaB ligand levels were increased. However, bacterial load and C-reactive protein serum did not show any differences. CONCLUSION: Taken together, our results showed that the presence of Treg cells attenuates the severity of experimental periodontitis without impairment in the control of infection.

Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice.

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappaB ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.

Investigation of the early healing response to dicationic imidazolium-based ionic liquids: a biocompatible coating for titanium implants.

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.

Estrogen protects dental roots from orthodontic-induced inflammatory resorption.

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.