Pain Intensity of Skeletally Anchored Maxillary Molar Distalization in Conjunction with Micro-osteoperforations: A Randomized Clinical Trial.

Objective To assess pain intensity levels during orthodontic therapy of Class II malocclusion patients undergoing skeletally anchored maxillary molar distalization assisted with different micro-osteoperforation (MOP) approaches. Methods Twenty-seven patients (12 males and 18 females) with a mean age of 16.1 ± 0.3 years were randomized into three equal groups (n=9): Group 1 comprised MOPs on buccal surface, Group 2 comprised MOPs on buccal and palatal surface, and Group 3 comprised the control or no-MOP group. The patients underwent maxillary molar distalization using skeletally anchored distal jet appliance assisted with or without MOPs. The MOPs were applied repeatedly on the buccal and buccal and palatal sides, or no MOP (control). Pain intensity was assessed using a 10 cm visual analog scale after each device activation at 24, 48, 72 hours, and at seven days. Data were analyzed using one-way ANOVA and repeated measures ANOVA for non-paired and paired means. Results Both approaches of buccal and buccal and palatal application of MOPs showed statistically significant (p< 0.01) higher levels of pain intensity after the first activation at 24 hours. Nevertheless, pain intensity levels decreased significantly in both MOP groups and between the two activations. Conclusion The repeated application of MOPs on either the buccal side only or on both buccal and palatal sides during maxillary molar distalization did not affect the levels of pain experienced; however, these levels were reported to be higher than that obtained in the control group. Moreover, it is observed that these pain levels tend to gradually reduce to mild levels over the subsequent days.

Clinical Evaluation of Single Versus Repeated Micro-Osteoperforations During Orthodontic Canine Retraction: A Randomized Clinical Trial.

INTRODUCTION: In the majority of orthodontic premolar extraction cases, the canine retraction phase is the most laborious procedure. This randomized clinical trial aimed to assess the effect of single versus repeated micro-osteoperforations (MOPs) during orthodontic canine retraction. METHODS: In this split-mouth study, two equal groups of 18 patients who required maxillary first premolar extractions and fixed orthodontic therapy were randomly assigned (n=9). In Group I, MOPs were only performed once on one site before retraction, whereas in Group II, MOPs were performed on one site repeatedly once a month for four months. In both groups, the contralateral control sites received no MOPs. The canines were retracted using mini-screws and closed-coil nickel-titanium springs. Using the patients' 3D models, the primary outcome measure at four months was the amount of orthodontic canine distal movement. The amount of anchorage loss (AL), degree of molar rotation (MR) and canine rotation (CR), and degree of canine tipping (CT) were measured as the secondary outcomes. The comparison of mean changes in the primary and secondary outcomes between the groups was done using the independent sample t-test (p<0.05). RESULTS: The rate of canine retraction, degree of CT, and rotation were not significantly different between the two groups (p>0.05). Additionally, there were no statistically significant variations in the maxillary first MR and the degree of AL (p>0.05). CONCLUSIONS: When maxillary canine retraction was performed with a single and repeated regimen of MOPs, comparable levels of distal CR and tipping were observed, along with an identical minimal degree of MR and AL.

Microbially-Induced Exosomes from Dendritic Cells Promote Paracrine Immune Senescence: Novel Mechanism of Bone Degenerative Disease in Mice.

As the aging population grows, chronic age-related bone degenerative diseases become more prevalent and severe. One such disease, periodontitis (PD), rises to 70.1% prevalence in Americans 65 years and older. PD has been linked to increased risk of other age-related diseases with more serious mortality and morbidity profiles such as Alzheimer's disease and cardiovascular disease, but the cellular and biological mechanisms remain unclear. Recent in vitro studies from our group indicate that murine dendritic cells (DCs) and T cells are vulnerable to immune senescence. This occurs through a distinct process involving invasion of DCs by dysbiotic pathogen Porphyromonas gingivalis (Pg) activating the senescence associated secretory phenotype (SASP). Exosomes of the Pg-induced SASP transmit senescence to normal bystander DC and T cells, ablating antigen presentation. The biological significance of these findings in vivo and the mechanisms involved were examined in the present study using young (4-5mo) or old (22-24mo) mice subjected to ligature-induced PD, with or without dysbiotic oral pathogen and injection of Pg-induced DC exosomes. Senescence profiling of gingiva and draining lymph nodes (LN) corroborates role of advanced age and PD in elevation of senescence biomarkers beta galactosidase (SA-ß-Gal), p16 INK4A p21Waf1/Clip1, IL6, TNFα, and IL1ß, with attendant increase in alveolar bone loss, reversed by senolytic agent rapamycin. Immunophenotyping of gingiva and LN revealed that myeloid CD11c+ DCs and T cells are particularly vulnerable to senescence in vivo under these conditions. Moreover, Pg-induced DC exosomes were the most potent inducers of alveolar bone loss and immune senescence, and capable of overcoming senescence resistance of LN T cells in young mice. We conclude that immune senescence, compounded by advanced age, and accelerated by oral dysbiosis and its induced SASP exosomes, plays a pivotal role in the pathophysiology of experimental periodontitis.

Prader-Willi syndrome: Periodontal-prosthodontic rehabilitation in an adult patient.

INTRODUCTION: Prader-Willi syndrome (PWS) is a rare genetic multisystemic disease that is the most common inherited cause of severe childhood obesity. PWS patients are prone to significant oral and systemic health issues that detrimentally affect quality of life and decrease longevity. This report documents full-mouth pre-prosthetic surgical and restorative care in an adult PWS patient. CASE REPORT: The patient, a 29-year-old male, presented to the clinic accompanied by his guardians (parents) with the chief complaint that "My Teeth are breaking down and I would like to get them fixed". Periodontal and prosthetic comprehensive clinical and radiographic exams revealed a severely worn dentition, deep anterior overbite, altered passive eruption with generalized biofilm-induced gingivitis, and altered occlusal vertical dimension. Full mouth crown lengthening surgery combined with full mouth prosthodontic reconstruction was performed under parenteral sedation and local anesthesia. Completion of treatment was successful, and the patient was placed on a 3-month periodontal maintenance interval. DISCUSSION: Full mouth periodontal surgical and prosthodontic reconstruction on a PWS patient has not previously been reported in the literature. This case underscores the potential need for complex dental care in patients with this syndrome.

Digital evaluation of Bolton's tooth size discrepancies among different malocclusions categories of Egyptian adolescent orthodontic population: A retrospective study.

OBJECTIVE: This retrospective investigation aimed to compare Bolton's ratios among different malocclusion groups of Egyptian adolescent orthodontic patients with original Bolton's standards. MATERIALS AND METHODS: Pre-treatment dental casts of 588 Egyptian subjects, 290 males and 298 females with mean age 16.7±2.2 years, were randomly selected and classified into 220 class I (108 males and 112 females), 230 class II (112 males and 118 females), and 138 class III (68 males and 70 females) groups. Mesiodistal widths from first molar to first molar were measured on 3-dimensionally scanned models via software and ratios were calculated. Two-way analysis of variance compared ratios as a function of skeletal classification and gender. Additionally, percentages of significant discrepancy outside 2 standard deviations (SDs) were calculated. RESULTS: The anterior mean ratio for the total sample were higher (79.4±4.7) and overall mean ratio was lower (90.1±5) than Bolton's standards. The differences between the obtained and standard values were statistically significant (P<0.001). However, there were no significant differences in either anterior ratio (P=0.637) or overall ratio (P=295) regarding gender. Class I cases showed the highest mean anterior ratio of 80±5.7 whereas class II and class III cases had the lowest ratio of 78.5±4.6 and 78.7±3.5, respectively. Concerning overall ratio, class III subjects had the highest ratio of 91.8±2.6 with no substantial distinction from class II cases (90.2±4.7) but was significantly different from class I cases that demonstrated the lowest ratio (89.7±5, P=0.020). High percentages of patients displayed clinically significant tooth size discrepancies (TSD), exceeding either above or below 2SD of Bolton's values, which were more marked in the anterior ratio. These were 25.2% and 7.4% for anterior ratio and 3.4% and 15.4% for overall ratio. CONCLUSIONS: Tooth size ratios of Egyptian orthodontic patients are generally different than the original Bolton's standards. Patients with class I and class III malocclusions had greater anterior and overall ratios than those with class II malocclusions with no considerable gender differences in either ratio.

Dendritic cells a critical link to alveolar bone loss and systemic disease risk in periodontitis: Immunotherapeutic implications.

Extensive research in humans and animal models has begun to unravel the complex mechanisms that drive the immunopathogenesis of periodontitis. Neutrophils mount an early and rapid response to the subgingival oral microbiome, producing destructive enzymes to kill microbes. Chemokines and cytokines are released that attract macrophages, dendritic cells, and T cells to the site. Dendritic cells, the focus of this review, are professional antigen-presenting cells on the front line of immune surveillance. Dendritic cells consist of multiple subsets that reside in the epithelium, connective tissues, and major organs. Our work in humans and mice established that myeloid dendritic cells are mobilized in periodontitis. This occurs in lymphoid and nonlymphoid oral tissues, in the bloodstream, and in response to Porphyromonas gingivalis. Moreover, the dendritic cells mature in situ in gingival lamina propria, forming immune conjugates with cluster of differentiation (CD) 4+ T cells, called oral lymphoid foci. At such foci, the decisions are made as to whether to promote bone destructive T helper 17 or bone-sparing regulatory T cell responses. Interestingly, dendritic cells lack potent enzymes and reactive oxygen species needed to kill and degrade endocytosed microbes. The keystone pathogen P. gingivalis exploits this vulnerability by invading dendritic cells in the tissues and peripheral blood using its distinct fimbrial adhesins. This promotes pathogen dissemination and inflammatory disease at distant sites, such as atherosclerotic plaques. Interestingly, our recent studies indicate that such P. gingivalis-infected dendritic cells release nanosized extracellular vesicles called exosomes, in higher numbers than uninfected dendritic cells do. Secreted exosomes and inflammasome-related cytokines are a key feature of the senescence-associated secretory phenotype. Exosomes communicate in paracrine with neighboring stromal cells and immune cells to promote and amplify cellular senescence. We have shown that dendritic cell-derived exosomes can be custom tailored to target and reprogram specific immune cells responsible for inflammatory bone loss in mice. The long-term goal of these immunotherapeutic approaches, ongoing in our laboratory and others, is to promote human health and longevity.

Matrix-Bound Zolzoledronate Enhances the Biofilm Colonization of Hydroxyapatite: Effects on Osteonecrosis.

(1) Background: The aim of this study was to test whether matrix-bound zoledronate (zol) molecules enhanced the oral biofilm colonization of a mineralized matrix, rendering the alveolar bone more susceptible to medication-related osteonecrosis of the jaw (MRONJ) following invasive dental procedures. (2) Methods: We tested the effect of matrix-bound zol on the growth and attachment of Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Actinomyces israelii (Ai), and whether the nitrogen-containing component of zol contributed to such effect. The role of oral bacteria in the induction of osteonecrosis was then tested using an extra-oral bone defect model. (3) Results: The attachment of biofilm to hydroxyapatite discs increased when the discs were pre-treated with zol. Bacterial proliferation was not affected. Matrix-bound zol was more potent than non-nitrogen-containing etidronate in enhancing the colonization. Stimulation was dampened by pre-treating the bacteria with histidine. The delivery of oral biofilm to a tibial defect caused osteonecrosis in zol-treated rats. (4) Conclusions: We conclude that matrix-bound zol enhances the oral biofilm colonization of hydroxyapatite. This enhancement depended on the presence of the nitrogen-containing group. The oral biofilm rendered the extra-oral bone susceptible to medication-related osteonecrosis, suggesting that it has an important role in the induction of MRONJ.

Porphyromonas gingivalis Provokes Exosome Secretion and Paracrine Immune Senescence in Bystander Dendritic Cells.

Periodontitis is a disease of ageing or inflammaging, and is comorbid with other more severe age-related chronic diseases. With advanced age comes an increase in accumulation of senescent cells that release soluble and insoluble pro-inflammatory factors collectively termed the senescence associated secretory phenotype (SASP). In the present report, we examined whether immune cells typical of those at the oral mucosa-microbe interface, are vulnerable to cellular senescence (CS) and the role of dysbiotic oral pathogen Porphyromonas gingivalis. Bone marrow-derived dendritic cells (DCs) from young (yDCs) and old (oDCs) mice were co-cultured in vitro with CS inducer doxorubicin or P.gingivalis (Pg), plus or minus senolytic agent rapamycin. CS profiling revealed elevated CS mediators SA-ß-Gal, p16 INK4A, p53, and p21Waf1/Clip1 in oDCs, or yDCs in response to doxorubicin or P. gingivalis, reversible with rapamycin. Functional studies indicate impaired maturation function of oDCs, and yDC exposed to P. gingivalis; moreover, OVA-driven proliferation of CD4+ T cells from young OTII transgenic mice was impaired by oDCs or yDCs+Pg. The SASP of DCs, consisting of secreted exosomes and inflammasome-related cytokines was further analyzed. Exosomes of DCs cocultured with P. gingivalis (PgDCexo) were purified, quantitated and characterized. Though typical in terms of size, shape and phenotype, PgDCexo were 2-fold greater in number than control DCs, with several important distinctions. Namely, PgDCexo were enriched in age-related miRNAs, and miRNAs reported to disrupt immune homeostasis through negative regulation of apoptosis and autophagy functions. We further show that PgDCexo were enriched in P. gingivalis fimbrial adhesin protein mfa1 and in inflammasome related cytokines IL-1ß, TNFα and IL-6. Functionally PgDCexo were readily endocytosed by recipient yDCs, amplifying functional impairment in maturation and ability to promote Ova-driven proliferation of OTII CD4+ T cells from young mice. In conclusion P. gingivalis induces premature (autocrine) senescence in DCs by direct cellular invasion and greatly amplifies senescence, in paracrine, of bystander DCs by secretion of inflammatory exosomes. The implications of this pathological pathway for periodontal disease in vivo is under investigation in mouse models.

Influence of T2DM and prediabetes on blood DC subsets and function in subjects with periodontitis.

OBJECTIVE: To compare the myeloid and plasmacytoid DC counts and maturation status among subjects with/without generalized periodontitis (GP) and type 2 diabetes mellitus (T2DM). METHODS: The frequency and maturation status of myeloid and plasmacytoid blood DCs were analyzed by flow cytometry in four groups of 15 subjects: healthy controls, T2DM with generalized CP (T2DM + GP), prediabetes with GP (PD + GP), and normoglycemics with GP (NG + GP). RT-PCR was used to determine levels of Porphyromonas gingivalis in the oral biofilms and within panDCs. The role of exogenous glucose effects on differentiation and apoptosis of healthy human MoDCs was explored in vitro. RESULTS: Relative to controls and to NG + GP, T2DM + GP showed significantly lower CD1c + and CD303 + DC counts, while CD141 + DCs were lower in T2DM + GP relative to controls. Blood DC maturation required for mobilization and immune responsiveness was not observed. A statistically significant trend was observed for P. gingivalis levels in the biofilms of groups as follows: controls 

Systemic Antibiotic Therapy Reduces Circulating Inflammatory Dendritic Cells and Treg-Th17 Plasticity in Periodontitis.

Periodontitis (PD) is a common dysbiotic inflammatory disease that leads to local bone deterioration and tooth loss. PD patients experience low-grade bacteremias with oral microbes implicated in the risk of heart disease, cancer, and kidney failure. Although Th17 effectors are vital to fighting infection, functional imbalance of Th17 effectors and regulatory T cells (Tregs) promote inflammatory diseases. In this study, we investigated, in a small pilot randomized clinical trial, whether expansion of inflammatory blood myeloid dendritic cells (DCs) and conversion of Tregs to Th17 cells could be modulated with antibiotics (AB) as part of initial therapy in PD patients. PD patients were randomly assigned to either 7 d of peroral metronidazole/amoxicillin AB treatment or no AB, along with standard care debridement and chlorhexidine mouthwash. 16s ribosomal RNA analysis of keystone pathogen Porphyromonas gingivalis and its consortium members Fusobacterium nucleatum and Streptococcus gordonii confirmed the presence of all three species in the reservoirs (subgingival pockets and blood DCs) of PD patients before treatment. Of the three species, P. gingivalis was reduced in both reservoirs 4-6 wk after therapy. Further, the frequency of CD1C+CCR6+ myeloid DCs and IL-1R1 expression on IL-17A+FOXP3+CD4+ T cells in PD patients were reduced to healthy control levels. The latter led to decreased IL-1ß-stimulated Treg plasticity in PD patients and improvement in clinical measures of PD. Overall, we identified an important, albeit short-term, beneficial role of AB therapy in reducing inflammatory DCs and Treg-Th17 plasticity in humans with PD.

Polymicrobial synergy within oral biofilm promotes invasion of dendritic cells and survival of consortia members.

Years of human microbiome research have confirmed that microbes rarely live or function alone, favoring diverse communities. Yet most experimental host-pathogen studies employ single species models of infection. Here, the influence of three-species oral microbial consortium on growth, virulence, invasion and persistence in dendritic cells (DCs) was examined experimentally in human monocyte-derived dendritic cells (DCs) and in patients with periodontitis (PD). Cooperative biofilm formation by Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis was documented in vitro using growth models and scanning electron microscopy. Analysis of growth rates by species-specific 16s rRNA probes revealed distinct, early advantages to consortium growth for S. gordonii and F. nucleatum with P. gingivalis, while P. gingivalis upregulated its short mfa1 fimbriae, leading to increased invasion of DCs. F. nucleatum was only taken up by DCs when in consortium with P. gingivalis. Mature consortium regressed DC maturation upon uptake, as determined by flow cytometry. Analysis of dental plaques of PD and healthy subjects by 16s rRNA confirmed oral colonization with consortium members, but DC hematogenous spread was limited to P. gingivalis and F. nucleatum. Expression of P. gingivalis mfa1 fimbriae was increased in dental plaques and hematogenous DCs of PD patients. P. gingivalis in the consortium correlated with an adverse clinical response in the gingiva of PD subjects. In conclusion, we have identified polymicrobial synergy in a three-species oral consortium that may have negative consequences for the host, including microbial dissemination and adverse peripheral inflammatory responses.

Oral Pathobiont Activates Anti-Apoptotic Pathway, Promoting both Immune Suppression and Oncogenic Cell Proliferation.

Chronic periodontitis (CP) is a microbial dysbiotic disease linked to increased risk of oral squamous cell carcinomas (OSCCs). To address the underlying mechanisms, mouse and human cell infection models and human biopsy samples were employed. We show that the 'keystone' pathogen Porphyromonas gingivalis, disrupts immune surveillance by generating myeloid-derived dendritic suppressor cells (MDDSCs) from monocytes. MDDSCs inhibit CTLs and induce FOXP3 + Tregs through an anti-apoptotic pathway. This pathway, involving pAKT1, pFOXO1, FOXP3, IDO1 and BIM, is activated in humans with CP and in mice orally infected with Mfa1 expressing P. gingivalis strains. Mechanistically, activation of this pathway, demonstrating FOXP3 as a direct FOXO1-target gene, was demonstrated by ChIP-assay in human CP gingiva. Expression of oncogenic but not tumor suppressor markers is consistent with tumor cell proliferation demonstrated in OSCC-P. gingivalis cocultures. Importantly, FimA + P. gingivalis strain MFI invades OSCCs, inducing inflammatory/angiogenic/oncogenic proteins stimulating OSCCs proliferation through CXCR4. Inhibition of CXCR4 abolished Pg-MFI-induced OSCCs proliferation and reduced expression of oncogenic proteins SDF-1/CXCR4, plus pAKT1-pFOXO1. Conclusively, P. gingivalis, through Mfa1 and FimA fimbriae, promotes immunosuppression and oncogenic cell proliferation, respectively, through a two-hit receptor-ligand process involving DC-SIGN+hi/CXCR4+hi, activating a pAKT+hipFOXO1+hiBIM-lowFOXP3+hi and IDO+hi- driven pathway, likely to impact the prognosis of oral cancers in patients with periodontitis.

Erbium, Chromium:Yttrium-Scandium-Gallium-Garnet Laser Effectively Ablates Single-Species Biofilms on Titanium Disks Without Detectable Surface Damage.

BACKGROUND: Increasing evidence implicates biofilms, consisting of species such as Porphyromonas gingivalis (Pg), in the etiology of peri-implantitis. Multiple approaches to ablate biofilms on failing implants have been proposed and include use of lasers, most recently the erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser. The purpose of this study is to establish an in vitro single-species biofilm model on implant surfaces and determine power settings of the Er,Cr:YSGG laser that remove biofilm without causing physical damage to disks. METHODS: Single-species biofilms consisting of Pg strain 381 were grown on titanium disks, including: 1) sandblasted, large-grit, acid-etched (SLA); 2) calcium phosphate nano-coated (CaP); 3) anodized; or 4) machined surfaces. Power settings from 0 to 1.5 W using an Er,Cr:YSGG laser equipped with radial firing tip were used. Biofilm formation/removal was quantitated using confocal and scanning electron microscopy. Surface changes in temperature, microroughness, and water contact angle were analyzed. RESULTS: Results show confluent Pg biofilm coating all disk surfaces. The laser removed biofilms from all surfaces, with CaP and SLA surfaces requiring power setting of 1.0 to 1.5 W for ablation of bacteria coating the disks. Within this power range, and with water spray, there were no changes in surface temperature, surface roughness, or contact angle on any surfaces tested. CONCLUSION: The Er,Cr:YSGG laser with radial firing tip and water spray was able to effectively ablate ≥95% of biofilm on all types of tested titanium surfaces, using clinically relevant power settings, without causing measurable physical changes to surfaces.

Cytotoxicity and osteogenic potential of silicate calcium cements as potential protective materials for pulpal revascularization.

OBJECTIVES: In pulpal revascularization, a protective material is placed coronal to the blood clot to prevent recontamination and to facilitate osteogenic differentiation of mesenchymal stem cells to produce new dental tissues. Although mineral trioxide aggregate (MTA) has been the material of choice for clot protection, it is easily displaced into the clot during condensation. The present study evaluated the effects of recently introduced calcium silicate cements (Biodentine and TheraCal LC) on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by comparing with MTA Angelus. METHODS: Cell viability was assessed using XTT assay and flow cytometry. The osteogenic potential of hDPSCs exposed to calcium silicate cements was examined using qRT-PCR for osteogenic gene expressions, alkaline phosphatase enzyme activity, Alizarin red S staining and transmission electron microscopy of extracellular calcium deposits. Parametric statistical methods were employed for analyses of significant difference among groups, with α=0.05. RESULTS: The cytotoxic effects of Biodentine and TheraCal LC on hDPSCs were time- and concentration-dependent. Osteogenic differentiation of hDPSCs was enhanced after exposure to Biodentine that was depleted of its cytotoxic components. This effect was less readily observed in hDPSCs exposed to TheraCal LC, although both cements supported extracellular mineralization better than the positive control (zinc oxide-eugenol-based cement). SIGNIFICANCE: A favorable tissue response is anticipated to occur with the use of Biodentine as a blood clot-protecting material for pulpal revascularization. Further investigations with the use of in vivo animal models are required to validate the potential adverse biological effects of TheraCal LC on hDPSCs.

Dendritic cells: microbial clearance via autophagy and potential immunobiological consequences for periodontal disease.

Dendritic cells are potent antigen-capture and antigen-presenting cells that play a key role in the initiation and regulation of the adaptive immune response. This process of immune homeostasis, as maintained by dendritic cells, is susceptible to dysregulation by certain pathogens during chronic infections. Such dysregulation may lead to disease perpetuation with potentially severe systemic consequences. Here we discuss in detail how intracellular pathogens exploit dendritic cells and escape degradation by altering or evading autophagy. This novel mechanism explains, in part, the chronic, persistent nature observed in several immuno-inflammatory diseases, including periodontal disease. We also propose a hypothetical model of the plausible role of autophagy in the context of periodontal disease. Promotion of autophagy may open new therapeutic strategies in the search of a 'cure' for periodontal disease in humans.

Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

Novel Coating of Surgical Suture Confers Antimicrobial Activity Against Porphyromonas gingivalis and Enterococcus faecalis.

BACKGROUND: The oral cavity is colonized by >10(9) bacteria, many of which can increase heart disease risk when seeded into the bloodstream. Most dentoalveolar surgeries require the use of surgical sutures. Suture placement and removal can increase the risk of postoperative infection and bacteremia. The aim of this study is to evaluate the antimicrobial activity of a novel quaternary ammonium compound, K21, when coated on different suture materials. METHODS: The periodontal pathogen Porphyromonas gingivalis and the endodontic species Enterococcus faecalis were grown to early log phase and inoculated on enriched Brucella blood agar, on which were placed identical lengths of surgical suture (chromic gut, polyester suture, silk, and nylon suture) and control unwaxed dental floss impregnated with K21 at 5%, 10%, 20%, and 25% volume/volume in ethanol vehicle. Controls included the following: 1) sutures treated with vehicle; 2) untreated sutures; and 3) unwaxed floss. Zones of inhibition in millimeters were measured at five randomized sites per suture/floss for each concentration and material used. Mean ± SD of zones of inhibition were calculated, and analysis of variance (P <0.05) was used to determine whether differences were statistically significant. RESULTS: The results indicate that K21-coated suture at concentrations ranging from 5% to 25%, depending on the type of suture, have antimicrobial activity for P. gingivalis and E. faecalis. Nylon suture coated with K21 at 5%, 10%, 20%, and 25% resulted in zones ranging from 3 to 11 mm. Polyester suture was more effective at lower K21 concentrations with 5% (P = 0.0031), 10% (P = 0.0011), and 20% (P = 0.0002), yielding 7.5, 8.3, and 10.5 mm zones of inhibition. K21-coated silk suture yielded significant zones of inhibition at 25% (P <0.0001), whereas chromic gut was effective at K21 concentrations of 5% (P = 0.0081) and 25% (P <0.0001). CONCLUSION: It is concluded that K21-coated surgical sutures have antimicrobial activity for bacterial species of direct relevance to postoperative infection and bacteremia.

Efficacy of 3D conforming nickel titanium rotary instruments in eliminating canal wall bacteria from oval-shaped root canals.

OBJECTIVES: To evaluate the effectiveness of TRUShape® 3D Conforming Files, compared with Twisted Files, in reducing bacteria load from root canal walls, in the presence or absence of irrigant agitation. METHODS: Extracted human premolars with single oval-shaped canals were infected with Enterococcus faecalis. Teeth in Group I (N=10; NaOCl and QMix® 2in1 as respective initial and final irrigants) were subdivided into 4 subgroups: (A) TRUShape® instrumentation without irrigant activation; (B) TRUShape® instrumentation with sonic irrigant agitation; (C) Twisted Files without irrigant agitation; (D) Twisted Files with sonic irrigant agitation. To remove confounding factor (antimicrobial irrigants), teeth in Group II (N=10) were irrigated with sterile saline, using the same subgroup designations. Specimens before and after chemomechanical débridement were cultured for quantification of colony-forming units (CFUs). Data from each group were analyzed separately using two-factor ANOVA and Holm-Sidak multiple comparison (α=0.05). Canal wall bacteria were qualitatively examined using scanning electron microscopy (SEM) and light microscopy of Taylor-modified Brown and Brenn-stained demineralised sections. RESULTS: CFUs from subgroups in Group I were not significantly different (P=0.935). For Group II, both file type (P<0.001) and irrigant agitation (P<0.001) significantly affected log-reduction in CFU concentrations. The interaction of these two factors was not significant (P=0.601). Although SEM showed reduced canal wall bacteria, bacteria were present within dentinal tubules after rotary instrumentation, as revealed by light microscopy of longitudinal root sections. CONCLUSIONS: TRUShape® files removed significantly more canal wall bacteria than Twisted Files when used without an antibacterial irrigant; the latter is required to decontaminate dentinal tubules. CLINICAL SIGNIFICANCE: Root canal disinfection should not be focused only on a mechanistic approach. Rather, the rational choice of a rotary instrumentation system should be combined with the use of well-tested antimicrobial irrigants and delivery/agitation techniques to establish a clinically realistic chemomechanical débridement protocol.

Secondary lymphoid organ homing phenotype of human myeloid dendritic cells disrupted by an intracellular oral pathogen.

Several intracellular pathogens, including a key etiological agent of chronic periodontitis, Porphyromonas gingivalis, infect blood myeloid dendritic cells (mDCs). This infection results in pathogen dissemination to distant inflammatory sites (i.e., pathogen trafficking). The alteration in chemokine-chemokine receptor expression that contributes to this pathogen trafficking function, particularly toward sites of neovascularization in humans, is unclear. To investigate this, we utilized human monocyte-derived DCs (MoDCs) and primary endothelial cells in vitro, combined with ex vivo-isolated blood mDCs and serum from chronic periodontitis subjects and healthy controls. Our results, using conditional fimbria mutants of P. gingivalis, show that P. gingivalis infection of MoDCs induces an angiogenic migratory profile. This profile is enhanced by expression of DC-SIGN on MoDCs and minor mfa-1 fimbriae on P. gingivalis and is evidenced by robust upregulation of CXCR4, but not secondary lymphoid organ (SLO)-homing CCR7. This disruption of SLO-homing capacity in response to respective chemokines closely matches surface expression of CXCR4 and CCR7 and is consistent with directed MoDC migration through an endothelial monolayer. Ex vivo-isolated mDCs from the blood of chronic periodontitis subjects, but not healthy controls, expressed a similar migratory profile; moreover, sera from chronic periodontitis subjects expressed elevated levels of CXCL12. Overall, we conclude that P. gingivalis actively "commandeers" DCs by reprogramming the chemokine receptor profile, thus disrupting SLO homing, while driving migration toward inflammatory vascular sites.

Human periodontal ligament fibroblast responses to compression in chronic periodontitis.

AIM: Test whether human periodontal ligament fibroblasts (PDLFs) retain homeostatic responses to a physiological compressive force during chronic periodontitis. MATERIAL AND METHODS: Six cell lines were established from periodontally healthy individuals (H-PDLFs) and another six were cultured from patients diagnosed with chronic periodontitis (D-PDLFs). Compressive force at 150 psi was applied to H-and D-PDLFs for 3 h on 2 consecutive days. After compression, comparisons between H-and D-PDLFs were performed by gene expression analysis of IL-6, proteases and 84 inflammation-related targets using real-time PCR. RESULTS: Compression of H-PDLFs resulted in a significant increase only in MMP-1 mRNA. In contrast, the same compressive force on D-PDLFs produced significant increases in the expression of MMPs-1,-7,-9 and -16. Moreover, compression of H-PDLFs resulted in down-regulation of IL-6, while IL-6 was significantly up-regulated in compressed D-PDLFs. Compression of H-PDLFs slightly up-regulated 3 and significantly down-regulated 15 inflammation-related genes, while the same treatment strongly up-regulated 21 inflammation-related genes in D-PDLFs. CONCLUSION: These results suggest a fundamental difference in the inflammatory response of healthy versus diseased PDLFs under physiological compression. Maintenance of these characteristics in vitro suggests that these cells may be at least partly responsible for the persistence of inflammation and localized susceptibility in chronic periodontitis.