Unraveling the Oral-Gut axis: Interconnection between Periodontitis and IBD, Current Challenges, and Future Perspective.

As the opposite ends of the orodigestive tract, the oral cavity and the intestine share anatomical, microbial, and immunological ties that have bidirectional health implications. A growing body of evidence suggests an interconnection between oral pathologies and Inflammatory Bowel Disease (IBD), implying a shift from the traditional concept of independent diseases to a complex, reciprocal cycle. This review outlines the evidence supporting an "Oral-Gut" axis, marked by a higher prevalence of periodontitis and other oral conditions in IBD patients and vice versa. We present an in-depth examination of the interconnection between oral pathologies and IBD, highlighting the shared microbiological and immunological pathways, and proposing a "multi-hit" hypothesis in the pathogenesis of periodontitis-mediated intestinal inflammation. Furthermore, the review underscores the critical need for a collaborative approach between dentists and gastroenterologists to provide holistic oral-systemic healthcare.

Major Histocompatibility Complex II Expression on Oral Langerhans Cells Differentially Regulates Mucosal CD4 and CD8 T Cells.

In murine periodontitis, the T helper (Th)17 response against Porphyromonas gingivalis in cervical lymph node is abrogated by diphtheria toxin-driven depletion of Langerhans cells (LCs). We determined the impact of major histocompatibility complex class II (MHC-II) presentation in LCs on Th17 cells in the oral mucosa of mice. Using an established human-Langerin promoter-Cre mouse model, we generated LC-specific deletion of the H2-Ab1 (MHC-II) gene. MHC-II expression was ablated in 81.2% of oral-resident LCs compared with >99% of skin-resident LCs. MHC-II (LCΔMHC-II) depletion did not reduce the number of CD4 T cells nor the frequency of Th17 cells compared with that in wild-type mice. However, the frequencies of Th1 cells decreased, and Helios+ T-regulatory cells increased. In ligature-induced periodontitis, the numbers of CD4 T cells and Th17 cells were similar in LCΔMHC-II and wild-type mice. Normal numbers of Th17 cells can therefore be sustained by as little as 18.8% of MHC-II-expressing LCs in oral mucosa. Unexpectedly, oral mucosa CD8 T cells increased >25-fold in LCΔMHC-II mice. Hence, these residual MHC-II-expressing LCs appear unable to suppress the local expansion of CD8 T cells while sufficient to sustain a homeostatic CD4 T-cell response. Reducing the expression of MHC-II on specific LC subpopulations may ultimately boost CD8-mediated intraepithelial surveillance at mucosal surfaces.

Unraveling the Link between Periodontitis and Inflammatory Bowel Disease: Challenges and Outlook.

Periodontitis and Inflammatory Bowel Disease (IBD) are chronic inflammatory conditions, characterized by microbial dysbiosis and hyper-immunoinflammatory responses. Growing evidence suggest an interconnection between periodontitis and IBD, implying a shift from the traditional concept of independent diseases to a complex, reciprocal cycle. This review outlines the evidence supporting an "Oral-Gut" axis, marked by a higher prevalence of periodontitis in IBD patients and vice versa. The specific mechanisms linking periodontitis and IBD remain to be fully elucidated, but emerging evidence points to the ectopic colonization of the gut by oral bacteria, which promote intestinal inflammation by activating host immune responses. This review presents an in-depth examination of the interconnection between periodontitis and IBD, highlighting the shared microbiological and immunological pathways, and proposing a "multi-hit" hypothesis in the pathogenesis of periodontitis-mediated intestinal inflammation. Furthermore, the review underscores the critical need for a collaborative approach between dentists and gastroenterologists to provide holistic oral-systemic healthcare.

Effect of scaling and root planing with and without minocycline HCl microspheres on periodontal pathogens and clinical outcomes: A randomized clinical trial.

BACKGROUND: This study tests the effects of scaling and root planing (SRP) versus SRP plus minocycline hydrochloride microspheres (SRP+MM) on 11 periodontal pathogens and clinical outcomes in Stage II-IV Grade B periodontitis participants. METHODS: Seventy participants were randomized to receive SRP (n = 35) or SRP+MM (n = 35). Saliva and clinical outcomes were collected for both groups at baseline before SRP, 1-month reevaluation, and at 3- and 6-month periodontal recall. MM were delivered to pockets ≥5 mm immediately after SRP and immediately after the 3-month periodontal maintenance in the SRP+MM group. A proprietary saliva test* was utilized to quantitate 11 putative periodontal pathogens. Microorganisms and clinical outcomes were compared between groups using generalized linear mixed-effects models with fixed effects and random effects terms. Mean changes from baseline were compared between groups via group-by-visit interaction tests. RESULTS: Significant reduction in Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, Prevotella intermedia, Parvimonas micra, and Eikenella corrodens were identified at the 1-month reevaluation after SRP+MM. Six months after SRP with a re-application of MM 3 months after SRP, Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus, and Eikenella corrodens were significantly reduced. SRP+MM participants had significant clinical outcome reductions in pockets ≥5 mm at the reevaluation, 3- and 6-month periodontal maintenance, and clinical attachment loss gains at the 6-month periodontal maintenance. CONCLUSION: MM delivered immediately after SRP and reapplication at 3 months appeared to contribute to improved clinical outcomes and sustained decreased numbers of Fusobacterium nucleatum, Prevotella intermedia, Campylobacter rectus, and Eikenella corrodens at 6 months.

The root canal microbiome diversity and function. A whole-metagenome shotgun analysis.

AIM: To evaluate the root canal microbiome composition and bacterial functional capability in cases of primary and secondary apical periodontitis utilizing whole-metagenome shotgun sequencing. METHODOLOGY: Twenty-two samples from patients with primary root canal infections, and 18 samples obtained from previously treated teeth currently diagnosed with apical periodontitis were analysed with whole-metagenome shotgun sequencing at a depth of 20 M reads. Taxonomic and functional gene annotations were made using MetaPhlAn3 and HUMAnN3 software. The Shannon and Chao1 indices were utilized to measure alpha diversity. Differences in community composition were evaluated utilizing analysis of similarity (ANOSIM) using Bray-Curtis dissimilarities. The Wilcoxon rank sum test was used to compare differences in taxa and functional genes. RESULTS: Microbial community variations within a community were significantly lower in secondary relative to primary infections (alpha diversity p = .001). Community composition was significantly different in primary versus secondary infection (R = .11, p = .005). The predominant taxa observed among samples (>2.5%) were Pseudopropionibacterium propionicum, Prevotella oris, Eubacterium infirmum, Tannerella forsythia, Atopobium rimae, Peptostreptococcus stomatis, Bacteroidetes bacterium oral taxon 272, Parvimonas micra, Olsenella profusa, Streptococcus anginosus, Lactobacillus rhamnosus, Porphyromonas endodontalis, Pseudoramibacter alactolyticus, Fusobacterium nucleatum, Eubacterium brachy and Solobacterium moorei. The Wilcoxon rank test revealed no significant differences in relative abundances of functional genes in both groups. Genes with greater relative abundances (top 25) were associated with genetic, signalling and cellular processes including the iron and peptide/nickel transport system. Numerous genes encoding toxins were identified: exfoliative toxin, haemolysins, thiol-activated cytolysin, phospholipase C, cAMP factor, sialidase, and hyaluronic glucosaminidase. CONCLUSIONS: Despite taxonomic differences between primary and secondary apical periodontitis, the functional capability of the microbiomes was similar.

Taxonomic abundance in primary and secondary root canal infections.

AIM: To evaluate the root canal microbiome composition in cases of primary and secondary apical periodontitis. METHODOLOGY: Thirty-nine samples from patients with primary root canal infections obtained before root canal treatment, and 40 samples obtained during root-end resection procedures from previously filled cases with apical periodontitis were evaluated using 16S rRNA next-generation sequencing analysis (NGS). Demographic and clinical factors included age, sex, infection type, percussion sensitivity, and presence of pain. Differences in abundances of genera were evaluated using Kruskal-Wallis test. Alpha and beta diversity indices were calculated using mothur. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity was used to measure beta diversity. Differences in community composition were evaluated using analysis of similarity (ANOSIM) with Bonferroni correction for multiple comparisons. RESULTS: Significantly fewer operational taxonomic units values were observed from samples from secondary infections (p < .0001). While no significant differences were observed in the Chao 1 index between primary and secondary infections, the Shannon alpha diversity was significantly lower in secondary relative to primary infections (p = .008). Among samples, sex, age (adult vs. older adult), percussion sensitivity, and presence of pain all showed no significant effects on community composition via an analysis of similarity (ANOSIM). However, community composition was significantly different depending on whether the sample was from a primary or secondary infection (R = .051, p = .03). Nine microbial genera comprised the predominant taxa observed among samples (>3.3%) and included Parvimonas, Fusobacterium, Campylobacter, Arachnia, Eubacterium, Prevotella, Peptostreptococcus, Fretibacterirum, and Pseudoramibacter. Significantly greater relative abundances of Prevotella, Peptostreptococcus, Veillonella, Lactucaseibacillus, and Dialister were observed in primary infections. CONCLUSIONS: Primary endodontic infections are more diverse than secondary infections. The microbial composition is not associated with the clinical manifestations of apical periodontitis.

Disinfection and Biocompatibility of Titanium Surfaces Treated with Glycine Powder Airflow and Triple Antibiotic Mixture: An In Vitro Study.

The aim of this in vitro study was to compare three disinfection protocols of biofilm-coated machined (MAC) and acid etched (SLA) commercial pure Grade 4 Titanium disks. Samples were infected with a vial of polymicrobial biofilm to simulate peri-implantitis in vitro. Seventeen MAC and twenty SLA titanium disks were randomly assigned to: (1) glycine powder air-flow (GYPAP) for 1 min; (2) a local delivered triple paste antibiotic composed by a gel mixture with ciprofloxacin, metronidazole, and clarithromycin (3MIX) for 1 h; and (3) a combination of both (GYPAP + 3MIX). Biocompatibility of the titanium disks after each treatment protocol was assessed by measurement of adhesion and growth of adipose-derived mesenchymal stem cells (ASCs) after 24 and 72 h. A confocal laser scanning microscope (CLSM) assessed the antibacterial effect of each treatment. Data of the antibacterial efficacy and cell viability were presented as mean with standard deviation and calculated by one-way ANOVA with multiple comparisons via Bonferroni tests. Results were considered significant with p < 0.05. The higher cell viability was achieved by the 3MIX and GYPAP combination on the SLA surfaces after 72 h. CLSM analysis showed a mean ratio of dead bacteria statistically higher in the 3MIX + GYPAP group compared with the GYPAP and 3MIX subgroups (p < 0.05). In conclusion, data showed that the combination of GYPAP and 3MIX could be preferred to the other protocols, especially in presence of SLA titanium surface.

The Palatal Approach for Direct Maxillary Sinus Floor Elevation: A Case Report.

INTRODUCTION: The traditional techniques of maxillary sinus floor elevation via a direct or indirect approach are suitable for the majority of cases. However, in cases of unfavorable anatomy and/or a thick lateral bony wall, we propose here a new approach for sinus floor elevation. CASE PRESENTATION: Forty-two-year-old female presents for sinus floor elevation after a failed attempt due to anatomical limitations and intraoperative complications. During the second surgical procedure, the access to the sinus membrane was performed from the palatal side due to thickness of the buccal wall ranging from 6 to 9 mm and the presence of septa. The sinus augmentation was successful, allowing for future implant placement. CONCLUSION: In selected cases, when the anatomy of the maxillary sinus poses limitations and the lateral wall thickness requires significant bone removal, the palatal approach is a valid alternative to the traditional sinus augmentation techniques.

No evidence for colonization of oral bacteria in the distal gut in healthy adults.

The microbial communities in the mouth and colon are anatomically connected via the saliva. However, the extent to which oral microbes reach and successfully colonize the distal gut has been debated. To resolve this long-standing controversy, we used exact amplicon sequence variants generated from concurrently collected saliva/stool microbiota in 66 healthy adults from two countries to show that, with one exception (Dialister invisus), the two niches are completely distinct. Thus, there is no evidence for colonization of oral bacteria in the distal gut. This defines the healthy state to which pathological states could be compared. Finding the same bacteria in the mouth and stool may warrant clinical investigation for an underlying pathology.

WNT-5a and SOST Levels in Gingival Crevicular Fluid Depend on the Inflammatory and Osteoclastogenic Activities of Periodontal Tissues.

Background and Objectives: Wnt signaling leads to stimulation of osteoblasts and it reduces osteoclastogenesis and bone resorption via the regulation of the osteprotegrin and receptor activator of nuclear factor kappa-Β ligan (RANKL). Wnt signaling pathways are regulated by their physiological antagonists such as sclerostin (SOST) as well as WNT-5a. The aim of this study was to determine the total amount of Sclerostin and WNT-5a in the gingival crevicular fluid (GCF) in sites with a continuum from a healthy to diseased periodontium. Materials and Methods: In this cross-sectional study, a total of 20 patients with generalized periodontitis, 10 subjects with gingivitis as well as 14 individuals with a healthy periodontium were recruited upon clinical and radiographic periodontal examination. In patients diagnosed with periodontitis, GCF samples were collected from periodontitis, gingivitis and healthy sites, while gingivitis patients provided samples from gingivitis and healthy sites. In healthy patients, only healthy sites were sampled. Protein total amount of SOST and WNT-5a were quantified by sandwich enzyme-linked immunosorbent assay (ELISA). Results: A total of 108 GCF samples were collected from a total of 44 individuals. When all periodontitis (n = 51), gingivitis (n = 12) and healthy (n = 45) sites were analyzed regardless of the patient diagnosis, periodontitis sites demonstrated significantly elevated WNT-5a total amounts (p = 0.03) when compared to gingivitis sites. Gingivitis sites demonstrated a trend of more total SOST (p = 0.09) when compared to periodontitis and healthy sites. Within each patient diagnostic category, sites showed similar SOST and WNT-5a total amounts (p > 0.05). Conclusions: WNT-5a levels in GCF depend on the stage of periodontitis sites. SOST trended higher in the GCF of gingivitis sites but similar in chronic periodontitis and healthy sites. WNT-5a and SOST play a crucial role in periodontal tissue remodeling and depend on the inflammatory and osteoclastogenic activities.

Calprotectin (S100A8/A9) Is an Innate Immune Effector in Experimental Periodontitis.

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and also as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9-/- and wild-type (WT; S100A9+/+) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9-/- mice; by day 5, the immune cell numbers were similar. At 5 days post ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9-/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9-/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pretreatment, differences disappeared between WT and S100A9-/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity, including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss and protecting against experimental periodontitis.

Transient Expression of IL-17A in Foxp3 Fate-Tracked Cells in Porphyromonas gingivalis-Mediated Oral Dysbiosis.

In periodontitis Porphyromonas gingivalis contributes to the development of a dysbiotic oral microbiome. This altered ecosystem elicits a diverse innate and adaptive immune response that simultaneously involves Th1, Th17, and Treg cells. It has been shown that Th17 cells can alter their gene expression to produce interferon-gamma (IFN-γ). Forkhead box P3 (Foxp3) is considered the master regulator of Treg cells that produce inhibitory cytokines like IL-10. Differentiation pathways that lead to Th17 and Treg cells from naïve progenitors are considered antagonistic. However, it has been reported that Treg cells expressing IL-17A as well as IFN-γ producing Th17 cells have been observed in several inflammatory conditions. Each scenario appears plausible with T cell transdifferentiation resulting from persistent microbial challenge and consequent inflammation. We established that oral colonization with P. gingivalis drives an initial IL-17A dominated Th17 response in the oral mucosa that is dependent on intraepithelial Langerhans cells (LCs). We hypothesized that Treg cells contribute to this initial IL-17A response through transient expression of IL-17A and that persistent mucosal colonization with P. gingivalis drives Th17 cells toward an IFN-γ phenotype at later stages of infection. We utilized fate-tracking mice where IL-17A- or Foxp3-promoter activity drives the permanent expression of red fluorescent protein tdTomato to test our hypothesis. At day 28 of infection timeline, Th17 cells dominated in the oral mucosa, outnumbering Th1 cells by 3:1. By day 48 this dominance was inverted with Th1 cells outnumbering Th17 cells by nearly 2:1. Tracking tdTomato+ Th17 cells revealed only sporadic transdifferentiation to an IFN-γ-producing phenotype by day 48; the appearance of Th1 cells at day 48 was due to a late de novo Th1 response. tdTomato+ Foxp3+ T cells were 35% of the total live CD4+T cells in the oral mucosa and 3.9% of them developed a transient IL-17A-producing phenotype by day 28. Interestingly, by day 48 these IL-17A-producing Foxp3+ T cells had disappeared. Therefore, persistent oral P. gingivalis infection stimulates an initial IL-17A-biased response led by Th17 cells and a small but significant number of IL-17A-expressing Treg cells that changes into a late de novo Th1 response with only sporadic transdifferentiation of Th17 cells.

Cre-loxP Reporter Mouse Reveals Stochastic Activity of the Foxp3 Promoter.

Mouse models that combine specific loxP-flanked gene sequences with Cre recombinase expressed from cell-regulated promoters have become important tools to investigate gene function. Critically however, expression of Cre recombinase may not always be restricted to the target cell or tissue of interest due to promiscuous activity of the driving promoter. Expression of Cre recombinase and, by extension, excision of the loxP-flanked gene may occur in non-target cells and may not be readily apparent. Here we report on the fidelity of Cre recombinase expressed from the il17a or Foxp3 promoters by combining them with a constitutively expressed floxed-stopped tdTomato reporter gene. Foxp3-driven Cre recombinase in F1 mice induced tdTomato red fluorescent protein in Treg cells but also in a range of other immune cells. Frequency of tdTomato expression was variable but positively correlated (p < 0.0001) amongst lymphoid (B cells and CD8 T cells) and blood-resident myeloid cells (dendritic cells, monocytes, neutrophils) suggesting stochastic activity of the Foxp3 promoter rather than developmental regulation in common ancestral progenitors. Interestingly, frequency of tdTomato+ dendritic cells, monocytes and neutrophils did not correlate with the tdTomato+ fraction in eosinophils, indicating that activity of the Foxp3 promoter in eosinophils occurred after the split from a common multipotent progenitor. When these F1 mice were crossed to achieve homozygosity of the promoter and reporter gene, a novel visually red phenotype was observed segregating amongst littermates. The red coloration was widespread and prevalent in non-immune tissues. Thymocytes examined from these red mice showed that all four subsets of immature thymocytes (CD4- CD8-) based on differential expression of CD25 and CD44 were expressing tdTomato. Finally, we show evidence of Foxp3 Cre recombinase independent tdTomato expression, suggesting germ line transmission of an activated tdTomato reporter gene. Our data highlights potential issues with conclusions drawn from using specifically the B6.129(Cg)-Foxp3tm4(YFP/Cre)Ayr/J mice.

Fetal Weight Outcomes in C57BL/6J and C57BL/6NCrl Mice after Oral Colonization with Porphyromonas gingivalis.

Porphyromonas gingivalis is considered a keystone pathogen that contributes to the initiation and progression of periodontitis in humans. P. gingivalis has also been detected in human placentas associated with adverse pregnancy outcomes. The spread of P. gingivalis from the oral cavity to the reproductive tract thus represents a potential mechanism whereby periodontitis can lead to adverse pregnancy outcomes. In a murine model of pregnancy and oral infection with P. gingivalis, C57BL/6J mice developed low fetal weight, whereas C57BL/6NCrl mice did not. Although C57BL/6NCrl mice harbor segmented filamentous bacteria that drive a Th17 response, fetal weight was independent of frequency of Th17 or Th1 in either substrain. Low fetal weight was instead correlated with increasing amounts of P. gingivalis DNA in the placentas of the C57BL/6J dams. In contrast, fetal weight in C57BL/6NCrl mice was independent of P. gingivalis in the placenta. Differences in genetics or microbiome that influence the ability of P. gingivalis to colonize the placenta may drive differential fetal weight outcomes between C57BL/6J and C57BL/6NCrl mice and, potentially, between diverse human populations.

Placental colonization with periodontal pathogens: the potential missing link.

Observational studies demonstrate that women with severe periodontitis have a higher risk of adverse pregnancy outcomes like preterm birth and low birthweight. Standard treatment for periodontitis in the form of scaling and root planing during the second trimester failed to reduce the risk of preterm or low birthweight. It is premature to dismiss the association between periodontitis and adverse pregnancy outcomes because one explanation for the failure of scaling and root planing to reduce the risk of adverse pregnancy outcomes is that periodontal pathogens spread to the placental tissue prior to periodontal treatment. In the placenta, orally derived organisms could cause direct tissue damage or mediate a maternal immune response that impairs the growth of the developing fetus. Sequencing studies demonstrate the presence of organisms derived from the oral microbiome in the placenta, but DNA-based sequencing studies should not be the only technique to evaluate the placental microbiome because they may not detect important shifts in the metabolic capability of the microbiome. In humans, polymerase chain reaction and histology have detected periodontal pathogens in placental tissue in association with multiple adverse pregnancy outcomes. We conclude that both placental and oral microbiomes may play a role in periodontitis-associated adverse pregnancy outcomes. However, the measure to determine the association between periodontal pathogens in the placenta and adverse pregnancy outcomes should be the amount and prevalence, not the mere presence of such microorganisms. Placental colonization with periodontal pathogens thus potentially represents the missing link between periodontitis and adverse pregnancy outcomes.

Sclerostin and WNT-5a gingival protein levels in chronic periodontitis and health.

BACKGROUND AND OBJECTIVE: Wnt signaling pathways regulate osteoblast differentiation and bone formation and are associated with inflammatory responses driven by innate and adaptive immunity via the NF-κB pathway. The aim of this study was to compare the levels of sclerostin (SOST), WNT-5a, and TNF-α between chronic periodontitis and periodontally healthy sites and determine their value as diagnostic markers of chronic periodontitis. MATERIAL AND METHODS: In a cross-sectional assessment 25 chronic periodontitis cases and 25 periodontally healthy controls were selected upon clinical and radiographic periodontal evaluation. Gingival crevicular fluid (GCF) was collected cross-sectionally from diseased and healthy sites in periodontitis patients and from healthy sites in each control subject. In a subgroup analysis, ten patients with generalized moderate and severe chronic periodontitis and ten generalized periodontally healthy individuals were included. The protein levels of SOST, WNT-5a, and TNF-α in GCF were measured by sandwich ELISA. The Shapiro-Wilk test was utilized to assess the normality of the distribution and non-parametric comparisons were performed. RESULTS: The protein levels of SOST were significantly higher in the generalized moderate and severe chronic periodontitis subgroup when compared to the generalized healthy (P = 0.002), while the WNT-5a and TNF-α GCF total amounts were similar (P > 0.05). Diseased sites in the periodontitis patients exhibited significantly higher total protein levels of WNT-5a than in healthy sites (P = 0.017), whereas no differences were detected for SOST and TNF-α (P > 0.05). The total protein levels of SOST, WNT-5a, and TNF-α in GCF were similar in periodontitis and non-periodontitis patients (P > 0.05). CONCLUSIONS: Sclerostin and WNT-5a gingival protein levels demonstrated a high diagnostic value for generalized moderate and severe chronic periodontitis, while a low accuracy was detected for localized chronic periodontitis.

Peptide coatings enhance keratinocyte attachment towards improving the peri-implant mucosal seal.

There is a critical need for preventing peri-implantitis as its prevalence has increased and dental implants lack features to prevent it. Research strategies to prevent peri-implantitis have focused on modifying dental implants to incorporate different antimicrobial agents. An alternative strategy consists of barring the expansion of the biofilm subgingivally by forming a long-lasting permucosal seal between the soft tissue and the implant surface. Here, we innovatively biofunctionalized titanium with bioinspired peptide coatings to strengthen biological interactions between epithelial cells and the titanium surface. We selected laminin 332- and ameloblastin-derived peptides (Lam, Ambn). Laminin 332 participates in the formation of hemidesmosomes by keratinocytes and promotes epithelial attachment around teeth; and ameloblastin, an enamel derived protein, is involved in tissue regeneration events following disruption of the periodontium. Lam, Ambn or combinations of both peptides were covalently immobilized on titanium discs. Successful immobilization of the peptides was confirmed by contact angle goniometry, X-ray photoelectron spectroscopy and fluorescent labelling of the peptides. Additionally, we confirmed the mechanical and thermochemical stability of the peptides on Ti substrates. Proliferation and hemidesmosome formation of human keratinocytes (TERT-2/OKF-6) were assessed by immunofluorescence labelling. The peptide-coated surfaces increased cell proliferation for up to 48 h in culture compared to control surfaces. Most importantly, formation of hemidesmosomes by keratinocytes was significantly increased on surfaces coated with Ambn + Lam peptides compared to control (p < 0.01) and monopeptide coatings (p < 0.005). Together, these results support the Ambn + Lam multipeptide coating as a promising candidate for inducing a permucosal seal around dental implants.

Discriminating between Interstitial and Circulating Leukocytes in Tissues of the Murine Oral Mucosa Avoiding Nasal-Associated Lymphoid Tissue Contamination.

Periodontitis is a chronic inflammatory response to a microbial biofilm that destroys bone and soft tissues supporting the teeth. Murine models of periodontitis based on Porphyromonas gingivalis (Pg) colonization have shown that extravasation of leukocytes into oral tissue is critical to driving alveolar bone destruction. Identifying interstitial leukocytes is key to understanding the immunopathogenesis of periodontitis. Here, we describe a robust flow cytometry assay based on intravenous FITC-conjugated anti-mouse CD45 mAb that distinguishes interstitial leukocytes in the oral mucosa of mice from those circulating within the vasculature or in post-dissection contaminating blood. Unaccounted circulating leukocytes skewed the relative frequency of B cells and granulocytes and inflated the numbers of all leukocyte cell types. We also describe a dissection technique that avoids contamination of oral mucosal tissues with nasal-associated lymphoid tissues (NALT), a B cell rich organ that can inflate leukocyte numbers at least 10-fold and skew the assessment of interstitial CD4 T cell phenotypes. Unlike circulating CD4 T cells, interstitial CD4 T cells were almost exclusively antigen-experienced cells (CD44hi). We report for the first time the presence of antigen-experienced Pg-specific CD4 T cells in NALT following oral feeding of mice with Pg. This new combined flow cytometry and dissection approach allows identification of leukocytes infiltrating the connective tissues of the murine oral mucosa and avoids confounding analyses of leukocytes not recruited to inflamed oral mucosal tissues in disease conditions like periodontitis, candidiasis, or sialadenitis.

The innate host response in caries and periodontitis.

INTRODUCTION: Innate immunity rapidly defends the host against infectious insults. These reactions are of limited specificity and exhaust without providing long-term protection. Functional fluids and effector molecules contribute to the defence against infectious agents, drive the immune response, and direct the cellular players. AIM: To review the literature and present a summary of current knowledge about the function of tissues, cellular players and soluble mediators of innate immunity relevant to caries and periodontitis. METHODS: Historical and recent literature was critically reviewed based on publications in peer-reviewed scientific journals. RESULTS: The innate immune response is vital to resistance against caries and periodontitis and rapidly attempts to protect against infectious agents in the dental hard and soft tissues. Soluble mediators include specialized proteins and lipids. They function to signal to immune and inflammatory cells, provide antimicrobial resistance, and also induce mechanisms for potential repair of damaged tissues. CONCLUSIONS: Far less investigated than adaptive immunity, innate immune responses are an emerging scientific and therapeutic frontier. Soluble mediators of the innate response provide a network of signals to organize the near immediate molecular and cellular response to infection, including direct and immediate antimicrobial activity. Further studies in human disease and animal models are generally needed.

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.