Autoinducer-2 produced by oral microbial flora and alveolar bone loss in periodontitis.

OBJECTIVE: The aim of this study was to investigate the association between autoinducer-2 (AI-2) of oral microbial flora and the alveolar bone destruction in periodontitis to determine if AI-2 may have the potential that monitor periodontitis and predict bone loss. BACKGROUND: Plaque biofilm was the initiating factor of periodontitis and the essential factor of periodontal tissue destruction. The formation of biofilms depended on the complex regulation of the quorum sensing (QS) system, in which bacteria could sense changes in surrounding bacterial density by secreting the autoinducer (AI) to regulate the corresponding physiological function. Most oral bacteria also communicated with each other to form biofilms administrating the QS system, which implied that the QS system of periodontal pathogens was related to periodontitis, but the specific relationship was unknown. METHOD: We collected the gingival crevicular fluid (GCF) samples and measured the concentration of AI-2 in samples using the Vibrio harveyi BB180 bioluminescent-reporter system. To explore the interaction between AI-2 and bone metabolism, we utilized AI-2 purified from Fusobacterium nucleatum to investigate the impact of F. nucleatum AI-2 on osteoclast differentiation. Moreover, we constructed murine periodontitis models and multi-species biofilm models to study the association between AI-2 and periodontal disease progression. RESULTS: The AI-2 concentration in GCF samples increased along with periodontal disease progression (p < .0001). F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner. In the periodontitis mice model, the CEJ-ABC distance in the F. nucleatum AI-2 treatment group was higher than that in the simple ligation group (p < .01), and the maxilla of the mice in the group exhibited significantly lower BMD and BV/TV values (p < .05). CONCLUSIONS: We demonstrated that the AI-2 concentration varied with the alveolar bone destruction in periodontitis, and it may have the potential for screening periodontitis. F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner and aggravated bone loss.

Experimental Inoculation of Aggregatibacter actinomycetemcomitans and Streptococcus gordonii and Its Impact on Alveolar Bone Loss and Oral and Gut Microbiomes.

Oral bacteria are implicated not only in oral diseases but also in gut dysbiosis and inflammatory conditions throughout the body. The periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) often occurs in complex oral biofilms with Streptococcus gordonii (Sg), and this interaction might influence the pathogenic potential of this pathogen. This study aims to assess the impact of oral inoculation with Aa, Sg, and their association (Aa+Sg) on alveolar bone loss, oral microbiome, and their potential effects on intestinal health in a murine model. Sg and/or Aa were orally administered to C57Bl/6 mice, three times per week, for 4 weeks. Aa was also injected into the gingiva three times during the initial experimental week. After 30 days, alveolar bone loss, expression of genes related to inflammation and mucosal permeability in the intestine, serum LPS levels, and the composition of oral and intestinal microbiomes were determined. Alveolar bone resorption was detected in Aa, Sg, and Aa+Sg groups, although Aa bone levels did not differ from that of the SHAM-inoculated group. Il-1ß expression was upregulated in the Aa group relative to the other infected groups, while Il-6 expression was downregulated in infected groups. Aa or Sg downregulated the expression of tight junction genes Cldn 1, Cldn 2, Ocdn, and Zo-1 whereas infection with Aa+Sg led to their upregulation, except for Cldn 1. Aa was detected in the oral biofilm of the Aa+Sg group but not in the gut. Infections altered oral and gut microbiomes. The oral biofilm of the Aa group showed increased abundance of Gammaproteobacteria, Enterobacterales, and Alloprevotella, while Sg administration enhanced the abundance of Alloprevotella and Rothia. The gut microbiome of infected groups showed reduced abundance of Erysipelotrichaceae. Infection with Aa or Sg disrupts both oral and gut microbiomes, impacting oral and gut homeostasis. While the combination of Aa with Sg promotes Aa survival in the oral cavity, it mitigates the adverse effects of Aa in the gut, suggesting a beneficial role of Sg associations in gut health.

Streptococcus gordonii Supragingival Bacterium Oral Infection-Induced Periodontitis and Robust miRNA Expression Kinetics.

Streptococcus gordonii (S. gordonii, Sg) is one of the early colonizing, supragingival commensal bacterium normally associated with oral health in human dental plaque. MicroRNAs (miRNAs) play an important role in the inflammation-mediated pathways and are involved in periodontal disease (PD) pathogenesis. PD is a polymicrobial dysbiotic immune-inflammatory disease initiated by microbes in the gingival sulcus/pockets. The objective of this study is to determine the global miRNA expression kinetics in S. gordonii DL1-infected C57BL/6J mice. All mice were randomly divided into four groups (n = 10 mice/group; 5 males and 5 females). Bacterial infection was performed in mice at 8 weeks and 16 weeks, mice were euthanized, and tissues harvested for analysis. We analyzed differentially expressed (DE) miRNAs in the mandibles of S. gordonii-infected mice. Gingival colonization/infection by S. gordonii and alveolar bone resorption (ABR) was confirmed. All the S. gordonii-infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, and a significant increase in mandible and maxilla ABR (p < 0.0001). miRNA profiling revealed 191 upregulated miRNAs (miR-375, miR-34b-5p) and 22 downregulated miRNAs (miR-133, miR-1224) in the mandibles of S. gordonii-infected mice at the 8-week mark. Conversely, at 16 weeks post-infection, 10 miRNAs (miR-1902, miR-203) were upregulated and 32 miRNAs (miR-1937c, miR-720) were downregulated. Two miRNAs, miR-210 and miR-423-5p, were commonly upregulated, and miR-2135 and miR-145 were commonly downregulated in both 8- and 16-week-infected mice mandibles. Furthermore, we employed five machine learning (ML) algorithms to assess how the number of miRNA copies correlates with S. gordonii infections in mice. In the ML analyses, miR-22 and miR-30c (8-week), miR-720 and miR-339-5p (16-week), and miR-720, miR-22, and miR-339-5p (combined 8- and 16-week) emerged as the most influential miRNAs.

Oral infection with periodontal pathogens induced chronic obstructive pulmonary disease-like lung changes in mice.

BACKGROUND: Epidemiological studies have demonstrated that periodontitis is an independent risk factor for chronic obstructive pulmonary disease (COPD). However, the mechanism underlying the association between these two diseases remains unclear. The lung microbiota shares similarities with the oral microbiota, and there is growing evidence to suggest that the lung microbiome could play a role in the pathogenesis of COPD. This study aimed to investigate whether periodontal pathogens could contribute to the pathogenesis of COPD in a mouse model. METHODS: We established mouse models with oral infection by typical periodontal pathogens, porphyromonas gingivalis (Pg group) or fusobacterium nucleatum (Fn group), over a three-month period. Mice that did not receive oral infection were set as the control group (C group). We assessed the level of alveolar bone resorption, lung function, and histological changes in the lungs of the mice. Additionally, we measured the levels of inflammatory factors and tissue damage associated factors in the lung tissues. RESULTS: Lung function indices, including airway resistance, peak inspiratory/expiratory flow and expiratory flow-50%, were significantly reduced in the Fn group compared to the C group. Additionally, histological examination revealed an increased number of inflammatory cells and bullae formation in the lung tissue sections of the Fn group. Meanwhile, levels of inflammatory factors such as IL-1ß, IL-6, IFN-γ, and TNF-α, as well as tissue damage associated factors like matrix metalloproteinase-8 and neutrophil elastase, were significantly elevated in the lung tissue of the Fn group in comparison to the C group. The Pg group also showed similar but milder lung changes compared to the Fn group. Pg or Fn could be detected in the lungs of both oral infected groups. CONCLUSION: The results indicated that oral periodontal pathogens infection could induce COPD-like lung changes in mice, and they may play a biological role in the association between periodontitis and COPD.

Luteolin inhibits Porphyromonas gingivalis growth and alleviates alveolar bone destruction in experimental murine periodontitis.

Periodontal disease is a major oral infectious disease that destroys alveolar bones and causes tooth loss. Porphyromonas gingivalis is a key pathogen that plays a crucial role in periodontitis. In our previous study on the anti-P. gingivalis activity of flavonoid, luteolin, a major flavonoid in edible plants, inhibited the proteolytic activity of gingipains, the major virulence factor in P. gingivalis. This study demonstrated luteolin in vitro and in vivo anti-bacterial activities. Thus, luteolin inhibits planktonic growth and biofilm formation in P. gingivalis. Furthermore, oral administration of luteolin alleviated maxillary alveolar bone resorption (ABR) in murine periodontitis induced by P. gingivalis infection. These results indicate that luteolin may be a potential therapeutic compound that targets P. gingivalis by hindering its growth, biofilm formation, and ABR in the oral cavity.

Interleukin-33 Deficiency Exacerbates Bone Loss Associated with Porphyromonas Gingivalis-Induced Experimental Periodontitis in Female Mice.

BACKGROUND/AIMS: Interleukin 33 (IL-33) plays a significant role in immunity but its role in bone physiology and periodontitis needs to be further investigated. The aim of this study was to decipher the contribution of IL-33 to bone homeostasis under physiological conditions, and to alveolar bone loss associated with experimental periodontitis (EP) in IL-33 knockout (KO) mice and their wildtype (WT) littermates. METHODS: The bone phenotype of IL-33 KO mice was studied in the maxilla, femur, and fifth lumbar vertebra by micro-computed tomography (micro-CT). EP was induced by a ligature soaked with the periopathogen Porphyromonas gingivalis (Pg) around a maxillary molar. Alveolar bone loss was quantified by micro-CT. The resorption parameters were assessed via toluidine blue staining on maxillary sections. In vitro osteoclastic differentiation assays using bone marrow cells were performed with or without lipopolysaccharide from Pg (LPS-Pg). RESULTS: First, we showed that under physiological conditions, IL-33 deficiency increased the trabecular bone volume/total volume ratio (BV/TV) of the maxillary bone in male and female mice, but not in the femur and fifth lumbar vertebra, suggesting an osteoprotective role for IL-33 in a site-dependent manner. The severity of EP induced by Pg-soaked ligature was increased in IL-33 KO mice but in female mice only, through an increase in the number of osteoclasts. Moreover, osteoclastic differentiation from bone marrow osteoclast progenitors in IL-33-deficient female mice is enhanced in the presence of LPS-Pg. CONCLUSION: Taken together, our data demonstrate that IL-33 plays a sex-dependent osteoprotective role both under physiological conditions and in EP with Pg.

Antimicrobial-induced oral dysbiosis exacerbates naturally occurring alveolar bone loss.

Periodontitis-mediated alveolar bone loss is caused by dysbiotic shifts in the commensal oral microbiota that upregulate proinflammatory osteoimmune responses. The study purpose was to determine whether antimicrobial-induced disruption of the commensal microbiota has deleterious effects on alveolar bone. We administered an antibiotic cocktail, minocycline, or vehicle-control to sex-matched C57BL/6T mice from age 6- to 12 weeks. Antibiotic cocktail and minocycline had catabolic effects on alveolar bone in specific-pathogen-free (SPF) mice. We then administered minocycline or vehicle-control to male mice reared under SPF and germ-free conditions, and we subjected minocycline-treated SPF mice to chlorhexidine oral antiseptic rinses. Alveolar bone loss was greater in vehicle-treated SPF versus germ-free mice, demonstrating that the commensal microbiota drives naturally occurring alveolar bone loss. Minocycline- versus vehicle-treated germ-free mice had similar alveolar bone loss outcomes, implying that antimicrobial-driven alveolar bone loss is microbiota dependent. Minocycline induced phylum-level shifts in the oral bacteriome and exacerbated naturally occurring alveolar bone loss in SPF mice. Chlorhexidine further disrupted the oral bacteriome and worsened alveolar bone loss in minocycline-treated SPF mice, validating that antimicrobial-induced oral dysbiosis has deleterious effects on alveolar bone. Minocycline enhanced osteoclast size and interface with alveolar bone in SPF mice. Neutrophils and plasmacytoid dendritic cells were upregulated in cervical lymph nodes of minocycline-treated SPF mice. Paralleling the upregulated proinflammatory innate immune cells, minocycline therapy increased TH 1 and TH 17 cells that have known pro-osteoclastic actions in the alveolar bone. This report reveals that antimicrobial perturbation of the commensal microbiota induces a proinflammatory oral dysbiotic state that exacerbates naturally occurring alveolar bone loss.

Impaired salivary SIgA antibodies elicit oral dysbiosis and subsequent induction of alveolar bone loss.

OBJECTIVE: Secreted IgA (SIgA) plays a central role in preventing bacterial and viral infections on mucosal surfaces by neutralizing toxins and viruses and inhibiting bacterial attachment to epithelial cells. However, the role of salivary SIgA antibodies (Abs) in regulating oral flora is still unknown. This study aimed to evaluate the association among oral bacteria, their metabolites and periodontitis in IgA-deficient (IgA KO) and wild-type (WT) control mice. METHODS: Microcomputed tomography (micro-CT) analysis was used to assess alveolar bone resorption as a development of periodontitis. The bacterial profiles of saliva were determined using the next-generation sequencing assays. Furthermore, the metabolites in saliva were measured and compared using CE-TOFMS. RESULTS: Salivary microbiota of IgA KO mice revealed a remarkably decreased frequency of Streptococcus, and increased percentages of Aggregatibacer, Actinobacillus, and Prevotella at the genus level when compared with those of WT. Compared to WT control mice of the same age, the level of alveolar bone loss was significantly increased in IgA KO mice, and infiltration of osteoclasts was found on the surface of the alveolar bone. The metabolome profile indicated that the metabolites of IgA KO mice had greater variability in carbon metabolic, urea cycle, and lipid pathways than WT mice. CONCLUSION: These results suggest that salivary SIgA plays an important role in regulating and maintaining normal oral microflora to prevent the development of periodontal disease.

Tyrosine-protein phosphatase non-receptor type 2 inhibits alveolar bone resorption in diabetic periodontitis via dephosphorylating CSF1 receptor.

Tyrosine-protein phosphatase non-receptor type 2 (PTPN2) is an important protection factor for diabetes and periodontitis, but the underlying mechanism remains elusive. This study aimed to identify the substrate of PTPN2 in mediating beneficial effects of 25-Hydroxyvitamin D3 (25(OH)2D3 ) on diabetic periodontitis. 25(OH)2D3 photo-affinity probe was synthesized with the minimalist linker and its efficacy to inhibit alveolar bone loss, and inflammation was evaluated in diabetic periodontitis mice. The probe was used to pull down the lysates of primary gingival fibroblasts. We identified PTPN2 as a direct target of 25(OH)2D3 , which effectively inhibited inflammation and bone resorption in diabetic periodontitis mice. In addition, we found that colony-stimulating factor 1 receptor (CSF1R) rather than JAK/STAT was the substrate of PTPN2 to regulate bone resorption. PTPN2 direct interacted with CSF1R and dephosphorylated Tyr807 residue. In conclusion, PTPN2 dephosphorylates CSF1R at Y807 site and inhibits alveolar bone resorption in diabetic periodontitis mice. PTPN2 and CSF1R are potential targets for the therapy of diabetic periodontitis or other bone loss-related diseases.

Bacterial modulators of bone remodeling in the periodontal pocket.

The signaling network involved in the pathogenesis of periodontal disease is not yet fully understood. This review aims to describe possible mechanisms through which the bacterial modulators may be linked directly or indirectly to the process of alveolar bone loss in periodontitis. From the late 1970s to present, new paradigm shifts have been developed regarding our understanding of pathological bone remodeling in periodontal disease. Upcoming evidence suggests that in periodontal disease the local immune response is exacerbated and involves the existence of signaling pathways that have been shown to modulate bone-cell function leading to alveolar bone loss. Those complex signaling pathways have been observed not only between bacteria but also between bacteria and the gingival surface of the host. More specifically, it has been shown that bacteria, through their secretion molecules, may interact indirectly and directly with immune-type cells of the host, resulting in the production of osteolytic agents that enhance bone resorption. Further research is required to provide a clear understanding of the role of these molecules in the pathogenesis of periodontal disease, and the availability of new technologies, such as next-generation sequencing and metagenomic analysis, may be useful tools in achieving this.

25-Hydroxyvitamin D3 -enhanced PTPN2 positively regulates periodontal inflammation through the JAK/STAT pathway in human oral keratinocytes and a mouse model of type 2 diabetes mellitus.

BACKGROUND AND OBJECTIVE: Periodontitis is an increasingly prevalent complication of diabetes mellitus (known as diabetes mellitus-associated periodontitis), and 25-hydroxyvitamin D3 (25VD3 ) was recently found to be a critical regulator of innate immunity in this disease, but the underlying mechanisms remain poorly understood. T-cell protein tyrosine phosphatase non-receptor type 2 (PTPN2) is a potential downstream protein of the 25VD3 /vitamin D receptor pathway. The aim of this study was to investigate the regulation of PTPN2 in periodontal inflammation in diabetes mellitus-associated periodontitis. MATERIAL AND METHODS: Porphyromonas gingivalis-infected db/db mice were treated with 25VD3 . Their fasting blood glucose and body weight were monitored every other week, and the levels of alveolar bone loss and serum inflammatory cytokines (tumor necrosis factor-α, interferon-γ and interleukin-6) were determined at the time of killing. The effect of PTPN2 on human OKF6-TERT2 oral keratinocytes was examined through the knockout of PTPN2 using the CRISPR/Cas9 knockout plasmid. The expression levels of the PTPN2, vitamin D receptor and JAK1/STAT3 signaling proteins in the gingival epithelium and OKF6-TERT2 cells were determined through western blot and immunohistochemical analyses. RESULTS: After 25VD3 treatment, db/db mice exhibited alleviated serum inflammatory cytokines and alveolar bone loss, and 25VD3 -enhanced PTPN2 expression decreased the expression of the JAK1/STAT3 signaling proteins in the gingival epithelium. Analyses of human oral keratinocytes showed that 25VD3 increased the expression of PTPN2, which dephosphorylates protein substrates in the JAK1/STAT3 signaling pathway. CONCLUSION: PTPN2 contributed to a decrease in periodontal inflammation in type 2 diabetes mellitus via dephosphorylate protein substrates in the JAK1/STAT3 signaling pathway after 25VD3 treatment in human oral keratinocytes and a mouse model of type 2 diabetes mellitus. A thorough understanding of PTPN2 and its involvement in inhibiting inflammation might provide alternative therapeutic approaches for the pathogenesis and treatment of diabetes mellitus-associated periodontitis.

Mucosal Langerhans Cells Promote Differentiation of Th17 Cells in a Murine Model of Periodontitis but Are Not Required for Porphyromonas gingivalis-Driven Alveolar Bone Destruction.

Periodontitis is a chronic oral inflammatory disease affecting one in five individuals that can lead to tooth loss. CD4(+) Th cells activated by a microbial biofilm are thought to contribute to the destruction of alveolar bone surrounding teeth by influencing osteoclastogenesis through IL-17A and receptor activator for NF-κB ligand effects. The relative roles of mucosal Ag presentation cells in directing Th cell immune responses against oral pathogens and their contribution to destruction of alveolar bone remain unknown. We tested the contribution of mucosal Langerhans cells (LCs) to alveolar bone homeostasis in mice following oral colonization with a well-characterized human periodontal pathogen, Porphyromonas gingivalis We found that oral mucosal LCs did not protect from or exacerbate crestal alveolar bone destruction but were responsible for promoting differentiation of Th17 cells specific to P. gingivalis. In mice lacking LCs the Th17 response was suppressed and a Th1 response predominated. Bypassing LCs with systemic immunization of P. gingivalis resulted in a predominantly P. gingivalis-specific Th1 response regardless of whether LCs were present. Interestingly, we find that in vivo clonal expansion of P. gingivalis-specific Th cells and induced regulatory T cells does not depend on mucosal LCs. Furthermore, destruction of crestal alveolar bone induced by P. gingivalis colonization occurred regardless of the presence of mucosal LCs or P. gingivalis-specific Th17 cells. Our data indicate that both LCs and Th17 cells are redundant in contributing to alveolar bone destruction in a murine model of periodontitis.

Osteocytes play an important role in experimental periodontitis in healthy and diabetic mice through expression of RANKL.

AIM: Periodontitis results from bacteria-induced inflammation. A key cytokine, RANKL, is produced by a number of cell types. The cellular source of RANKL critical for periodontitis has not been established. METHODS: We induced periodontal bone loss by oral inoculation of Porphyromonas gingivalis and Fusobacterium nucleatum in both normoglycaemic and streptozotocin-induced type 1 diabetic mice. Experimental transgenic mice had osteocyte-specific deletion of floxed receptor activator of nuclear factor kappa-B ligand (RANKL) mediated by DMP-1-driven Cre recombinase. Outcomes were assessed by micro-CT, histomorphometric analysis, immunofluorescent analysis of RANKL and tartrate-resistant acid phosphatase staining for osteoclasts and osteoclast activity. RESULTS: Oral infection stimulated RANKL expression in osteocytes of wild-type mice, which was increased by diabetes and blocked in transgenic mice. Infected wild-type mice had significant bone loss and increased osteoclast numbers and activity, which were further enhanced by diabetes. No bone loss or increase in osteoclastogenesis or activity was detected in transgenic mice with RANKL deletion in osteocytes that were normoglycaemic or diabetic. CONCLUSIONS: This study demonstrates for the first time the essential role of osteocytes in bacteria-induced periodontal bone loss and in diabetes-enhanced periodontitis.

Early histological, microbiological, radiological, and clinical response to cemented and screw-retained all-ceramic single crowns.

OBJECTIVES: To assess the early histological, microbiological, radiological, and clinical response to cemented and screw-retained all-ceramic single-tooth implant-supported reconstructions. MATERIALS AND METHODS: Patients with single-tooth implants were randomly allocated to receive a cemented lithium disilicate crown on a customized zirconia abutment (CEM) or a screw-retained crown with a directly veneered zirconia abutment (SCREW). At the screening visit, at crown insertion and at the 6-month follow-up, clinical parameters were measured at the implant and the contralateral tooth. Marginal bone levels, technical parameters, and esthetic outcomes were measured at the implants. At the 6-month follow-up, a microbiological test was performed and a soft tissue biopsy was harvested at the implants for histological analysis. Inflammatory cells and fibroblasts/-cytes were analyzed at the level of the sulcular epithelium, junctional epithelium, and connective tissue. The histological parameters were analyzed by means of a linear mixed model. RESULTS: Thirty-three patients completed the study, and implant and crown survival rates were 100% at 6 months. Histologically, the number of inflammatory cells tended to be higher in group CEM (p > 0.05). Moreover, significantly less inflammatory cells and fibroblasts/-cytes were found in the sulcular epithelium compared to the junctional epithelium and supracrestal connective tissue (p < 0.001). Four patients were tested positive for periodontal marker pathogens at the 6-month follow-up, and three of them belonged to group CEM. From crown insertion to the 6-month follow-up, median marginal bone levels changed only minimally and measured 0.31 and 0.32 mm in group CEM and 0.47 and 0.36 mm in group SCREW, respectively. Clinical and esthetic parameters remained stable over time and were comparable between natural teeth and implants as well as between the groups. CONCLUSIONS: Cemented reconstructions were associated with more inflammatory cells, and more patients were diagnosed with periodonto-pathogens. Both types of reconstructions resulted in similar radiological (marginal bone levels) and clinical outcomes (bleeding on probing and probing depth).

Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontal disease, which is characterized by inflammation-driven alveolar bone loss. A. actinomycetemcomitans activates the p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) stress pathways in macrophages that are involved in host responses. During the inflammatory process in periodontal disease, chemokines are upregulated to promote recruitment of inflammatory cells. The objective of this study was to determine the role of MK2 signaling in chemokine regulation during A. actinomycetemcomitans pathogenesis. Utilizing a murine calvarial model, Mk2+/+ and Mk2-/- mice were treated with live A. actinomycetemcomitans bacteria at the midsagittal suture. MK2 positively regulated the following macrophage RNA: Emr1 (F4/80), Itgam (CD11b), Csf1r (M-CSF Receptor), Itgal (CD11a), Tnf, and Nos2 Additionally, RNA analysis revealed that MK2 signaling regulated chemokines CCL3 and CCL4 in murine calvarial tissue. Utilizing the chimeric murine air pouch model, MK2 signaling differentially regulated CCL3 and CCL4 in the hematopoietic and nonhematopoietic compartments. Bone resorption pits in calvaria, observed by micro-computed tomography, and osteoclast formation were decreased in Mk2-/- mice compared to Mk2+/+ mice after A. actinomycetemcomitans treatment. In conclusion, these data suggest that MK2 in macrophages contributes to regulation of chemokine signaling during A. actinomycetemcomitans-induced inflammation and bone loss.

Vaccination with recombinant RgpA peptide protects against Porphyromonas gingivalis-induced bone loss.

OBJECTIVE: Following Porphyromonas gingivalis infection in mice, the efficacy of vaccination by recombinant and native RgpA in modulating the early local anti-inflammatory and immune responses and periodontal bone loss were examined. MATERIAL AND METHODS: Using the subcutaneous chamber model, exudates were analyzed for cytokines after treatment with native RgpA and adjuvant (test), or adjuvant and saline alone (controls). Mice were also immunized with recombinant RgpA after being orally infected with P. gingivalis. After 6 wk, serum was examined for anti-P. gingivalis IgG1 and IgG2a titers and for alveolar bone resorption. RESULTS: Immunization with native RgpA shifted the immune response toward an anti-inflammatory response as demonstrated by decreased proinflammatory cytokine IL-1ß production and greater anti-inflammatory cytokine IL-4 in chamber exudates. Systemically, immunization with recombinant RgpA peptide prevented alveolar bone loss by 50%, similar to immunization with heat-killed whole bacteria. Furthermore, recombinant RgpA shifted the humoral response toward high IgG1 and low IgG2a titers, representing an in vivo anti-inflammatory response. CONCLUSIONS: The present study demonstrates the potential of RgpA to shift the early local immune response toward an anti-inflammatory response while vaccination with recRgpA protected against P. gingivalis-induced periodontitis.

Oral infection with Porphyromonas gingivalis induces peri-implantitis in a murine model: Evaluation of bone loss and the local inflammatory response.

AIM: Peri-implantitis is a major health concern, with unclear pathogenesis, and with no accessible animal models. Our aim was to establish a mouse model for peri-implantitis and to investigate mediators of inflammation. MATERIALS AND METHODS: Mice were divided into implanted versus non-implanted groups. Implants were inserted immediately following the extraction of the upper first molar. Four weeks following implantation, implanted and non-implanted mice were challenged with either Porphyromonas gingivalis or vehicle (eight mice in each subgroup, 32 mice in total). Alveolar bone loss and expression of inflammatory mediators in the soft tissue were assessed 42 days following infection. RESULTS: Porphyromonas gingivalis infection induced greater bone loss around implants than around teeth. In non-infected animals, the presence of the implant correlated with elevated expression of Il-10, Foxp3 and Rankl/Opg ratio, while Tnf-α levels were decreased relative to tissue around teeth. Six weeks following infection, Tnf-α increased significantly while the expression of Foxp3 decreased in the tissue around the implants. No significant differences in anti- or pro-inflammatory mediators were found around teeth of infected, relative to non-infected mice. CONCLUSIONS: Oral infection with P. gingivalis of mice with implants induced bone loss and a shift in gingival cytokine expression. This mouse model enables exploration of the pathogenesis of peri-implantitis and testing of novel treatments.

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.

Exploring the microbiome of healthy and diseased peri-implant sites using Illumina sequencing.

AIM: To compare the microbiome of healthy (H) and diseased (P) peri-implant sites and determine the core peri-implant microbiome. MATERIALS AND METHODS: Submucosal biofilms from 32 H and 35 P sites were analysed using 16S rRNA sequencing (MiSeq, Illumina), QIIME and HOMINGS. Differences between groups were determined using principal coordinate analysis (PCoA), t tests and Wilcoxon rank sum test and FDR-adjusted. The peri-implant core microbiome was determined. RESULTS: PCoA showed partitioning between H and P at all taxonomic levels. Bacteroidetes, Spirochetes and Synergistetes were higher in P, while Actinobacteria prevailed in H (p < .05). Porphyromonas and Treponema were more abundant in P while Rothia and Neisseria were higher in H (p < .05). The core peri-implant microbiome contained Fusobacterium, Parvimonas and Campylobacter sp. T. denticola, and P. gingivalis levels were higher in P, as well as F. alocis, F. fastidiosum and T. maltophilum (p < .05). CONCLUSION: The peri-implantitis microbiome is commensal-depleted and pathogen-enriched, harbouring traditional and new pathogens. The core peri-implant microbiome harbours taxa from genera often associated with periodontal inflammation.

The predictive value of microbiological findings on teeth, internal and external implant portions in clinical decision making.

AIM: The primary aim of this study was to evaluate 23 pathogens associated with peri-implantitis at inner part of implant connections, in peri-implant and periodontal pockets between patients suffering peri-implantitis and participants with healthy peri-implant tissues; the secondary aim was to estimate the predictive value of microbiological profile in patients wearing dental implants using data mining methods. MATERIAL AND METHODS: Fifty participants included in the present case─control study were scheduled for collection of plaque samples from the peri-implant pockets, internal connection, and periodontal pocket. Real-time polymerase chain reaction was performed to quantify 23 pathogens. Three predictive models were developed using C4.5 decision trees to estimate the predictive value of microbiological profile between three experimental sites. RESULTS: The final sample included 47 patients (22 healthy controls and 25 diseased cases), 90 implants (43 with healthy peri-implant tissues and 47 affected by peri-implantitis). Total and mean pathogen counts at inner portions of the implant connection, in peri-implant and periodontal pockets were generally increased in peri-implantitis patients when compared to healthy controls. The inner portion of the implant connection, the periodontal pocket and peri-implant pocket, respectively, presented a predictive value of microbiologic profile of 82.78%, 94.31%, and 97.5% of accuracy. CONCLUSION: This study showed that microbiological profile at all three experimental sites is differently characterized between patients suffering peri-implantitis and healthy controls. Data mining analysis identified Parvimonas micra as a highly accurate predictor of peri-implantitis when present in peri-implant pocket while this method generally seems to be promising for diagnosis of such complex infections.