Macrophages polarize to the pro-inflammatory phenotype and delay neutrophil efferocytosis to augment Aggregatibacter actinomycetemcomitance JP2 clearance.

OBJECTIVE: The study examines how neutrophils cross-talk with macrophages during JP2 Aggregatibacter actinomycetemcomitance infection and factors that are involved in inflammatory resolution and efferocytosis. BACKGROUND: Although sub-gingival bacteria constitute the primary initiating factor in the pathogenesis of molar-incisor pattern periodontitis (MIPP), the non-resolved host response has a major role in tissue destruction. While evidence links neutrophils to MIPP pathogenesis, their clearance during inflammatory resolution, governed by macrophages, is poorly understood. METHODS: Human neutrophils (differentiated from HL60 cells) and macrophages (differentiated from THP1 cells) were inoculated with JP2. The supernatants were collected and exposed to naïve neutrophils or macrophages with or without exposure to JP2. Reactive oxygen species (ROS) were measured with 2'-7'-dichlorofluorescein-diacetate and a fluorescent plate reader. Immunofluorescence labeling of CD47 and cell vitality were examined using flow cytometry. Macrophage polarization was tested by immunofluorescence staining for CD163 and CD68 and a fluorescent microscope, and TNFα and IL-10 secretion was tested using ELISA and RT-PCR. Efferocytosis was examined by pHrodo and carboxyfluorescein succinimidyl ester staining and fluorescent microscopy. In vivo, macrophages were depleted from C57Bl/6 mice and neutrophil CD47 levels were tested using the subcutaneous chamber model. RESULTS: Neutrophils exposed to macrophage supernatant show increased ROS, mainly extracellularly, that increased during JP2 infection. Macrophages showed pro-inflammatory M1 phenotype polarization during JP2 infection, and their supernatants prolonged neutrophil survival by inhibiting CD47 down-expression and reducing neutrophil necrosis and apoptosis. Also, the macrophages delay neutrophil efferocytosis during JP2 infection which, in turn, enhanced JP2 clearance. Depletion of macrophages in mice mildly prevented neutrophils CD47 reduction and reduced JP2 clearance. The JP2 infection in mice also led to macrophage M1 polarization similar to the in vitro results. CONCLUSIONS: As shown in this study, neutrophil efferocytosis potentially may be reduced during JP2 infection, promoting JP2 clearance, which may contribute to the inflammatory-mediated periodontal tissue damage.

Mechanical force application and inflammation induce osteoclastogenesis by independent pathways.

OBJECTIVE: To investigate the functional changes of PDL fibroblasts in the presence of mechanical force, inflammation, or a combination of force and inflammation. MATERIALS AND METHODS: Inflammatory supernatants were prepared by inoculating human neutrophils with Porphyromonas gingivalis. Primary human PDL fibroblasts (PDLF), gingival fibroblasts (GFs), and osteoblasts (Saos2) were then exposed to the inflammatory supernatants. Orthodontic force on the PDLFs was simulated by centrifugation. Analyses included cell proliferation, cell viability, cell cycle, and collagen expression, as well as osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL) expression. RESULTS: Mechanical force did not affect PDLF viability, but it increased the metabolic rate compared to resting cells. Force application shifted the PDLF cell cycle to the G0/G1 phase, arresting cell proliferation and leading to elevated collagen production, mild OPG level elevation, and robust RANKL level elevation. Including an inflammatory supernatant in the presence of force did not affect PDLF viability, proliferation, or cytokine expression. By contrast, the inflammatory supernatant increased RANKL expression in GFs, but not in Saos2 cells. CONCLUSION: Applying mechanical force significantly affects PDLF function. Although inflammation had no effect on PDLF or Saos2 cells, it promoted RANKL expression in GF cells. Within the limitations of the in vitro model, the results suggest that periodontal inflammation and mechanical forces could affect bone catabolism through effects on different cell types, which may culminate in synergistic bone resorption.

Enterococcus faecalis sustained infection induces macrophage pro-resolution polarization.

AIM: To investigate macrophage function in the presence of sustained infection with Enterococcus faecalis, a prevalent root canal resident in asymptomatic apical periodontitis. METHODOLOGY: The human monocyte cell line (THP-1) was differentiated into macrophages by exposure to phorbol myristate acetate (PMA), and the cultures were inoculated with E. faecalis for up to 48 h. At three time-points 90 min, 24 and 48 h after inoculation, the macrophages and their supernatants were examined. Assays included macrophage phagocytosis rate and vitality, bacterial survival, reactive oxygen species (ROS) production, mitochondrial activity, cytokine production and the expression of pro/anti-inflammatory M1/M2 markers. Also, periapical tissue from apicectomy samples of human endodontically treated teeth were collected for histological and immunofluorescent analysis. Statistical differences were analysed with RM ANOVA. RESULTS: E. faecalis were phagocytized, and subsequently, most of the macrophages underwent apoptosis and necrosis. The small population of macrophages that remained vital after 48 h post-inoculation harboured surviving bacteria. Despite a reduction in the number of macrophages over time, the mitochondrial activity of the surviving macrophages remained constant and external ROS decreased, whereas internal ROS increased. During the infection, a shift to a M2 macrophage population at 48 h post-infection was observed; the results were similar to those obtained in periapical human tissue biopsies (p < .05). CONCLUSIONS: The study portrays a continuous non-resolved infection with E. faecalis and activation of macrophages that are polarized to the M2 pro-resolution phenotype.

Salivary inflammatory cytokines echo the low inflammatory burden in liver-transplanted children.

OBJECTIVES: The aims of this study were to compare the salivary cytokine profile, as a potential replacement for blood tests, in liver-transplanted children to that of a control group of healthy children, and to correlate the values of commonly tested laboratory blood tests to those of published blood values. METHODS: Liver-transplanted children, and a control group of healthy children of the same sex and age distribution, were recruited for the study. Saliva was collected at the same appointment for routine blood tests for the liver-transplanted children. Saliva was also collected from a control group of healthy children with similar age and sex distributions. Normal healthy blood values were extracted from the literature, for comparison. Cytokine levels in the saliva were quantified with ELISA. The analysis compared serum and saliva values between liver-transplanted and healthy children. In the serum, the values of albumin, GIT, GPT, GGT, CRP, WBC, neutrophils, and lymphocytes were examined, while the levels of IL-6, CXCL1, IL-1b, and IL-10 were measured in the saliva. RESULTS: Thirty liver-transplanted children and 30 healthy children were included in the study. Compared with published data for healthy children, the liver-transplanted group showed similar hepatic serum levels, yet reduced levels of serum inflammatory markers. Compared with the control group, in the transplanted group, the mean value of IL-6 was lower and the mean value of CXCL1 was similar. Interestingly, the anti-inflammatory IL-10 cytokine was lower in the transplanted group, while the pro-inflammatory IL-1ß cytokine was higher. CONCLUSION: The salivary inflammatory markers examined showed a similar pattern to the serum inflammatory values, though different markers were examined in the serum and saliva. CLINICAL RELEVANCE: The current study stresses the potential of oral fluids as an accessible biofluid, for use as a diagnostic substrate for systemic and oral diseases. TRIAL REGISTRATION: 0136-16-RMC, Registered on 01 March 2018.

Diabetes as a risk factor for periodontal disease-plausible mechanisms.

The present narrative review examines the scientific evidence of the biological mechanisms that may link periodontitis and diabetes, as a source of comorbidity. Publications regarding periodontitis and diabetes, in human, animals, and in vitro were screened for their relevance. Periodontal microbiome studies indicate a possible association between altered glucose metabolism in prediabetes and diabetes and changes in the periodontal microbiome. Coinciding with this, hyperglycemia enhances expression of pathogen receptors, which enhance host response to the dysbiotic microbiome. Hyperglycemia also promotes pro-inflammatory response independently or via the advanced glycation end product/receptor for advanced glycation end product pathway. These processes excite cellular tissue destruction functions, which further enhance pro-inflammatory cytokines expression and alteration in the RANKL/osteoprotegerin ratio, promoting formation and activation of osteoclasts. The evidence supports the role of several pathogenic mechanisms in the path of true causal comorbidity between poorly controlled diabetes and periodontitis. However, further research is needed to better understand these mechanisms and to explore other mechanisms.

The efficacy of pocket elimination/reduction compared to access flap surgery: A systematic review and meta-analysis.

AIM: To assess the efficacy and adverse effects of resective surgery compared to access flap in patients with periodontitis. METHODS: Randomized controlled trials with a follow-up ≥6 months were identified in ten databases. Screening, data extraction, and quality assessment were conducted by two reviewers. The primary outcome was probing pocket depth, and the main secondary outcome was clinical attachment level. Data on adverse events were collected. Meta-analysis was used to synthesize the findings of trials. RESULTS: A total of 880 publications were identified. Fourteen publications from nine clinical trials met the inclusion criteria and were included for analysis. Meta-analysis was carried out using all available results. The results indicated superior pocket depth reduction following resective surgery compared to access flap after 6-12 months of follow-up (weighted mean difference 0.47 mm; confidence interval 0.7-0.24; p = .010). After 36-60 months of follow-up, no differences were found between the two treatments in pocket depth and attachment level. The prevalence of adverse effects was not different between the groups. Post-operative recession tended to be more severe for the resective approaches. CONCLUSION: Resective surgical approach was superior to access flap in reducing pocket depth 6-12 months post-surgery, while no differences between the two modalities were found at 36-60 months of follow-up.

High-resolution novel method for tracking bacteria in a multi-species biofilm.

The aim of this study is to establish a novel high resolution tracking ability of a specific bacterium in multispecies biofilm. A periodontal multispecies biofilm was constructed with Streptococcus sanguis, Actinomyces naeslundii, Porphyromonas gingivalis and Fusobacterium nucleatum. A single species was stained with fluorescein isothiocyanate (FITC). The mature biofilm was stained for viability (propidium iodide) and analysis was performed with flow cytometry. The sensitivity of the assay was compared with colony forming units (CFU) counts. A single cell suspension of P. gingivalis was grown in broth and biofilm to identify the location of these events on side scatter and forward scatter. The sensitivity of the assay was comparable to that of the CFU counts. The assay allows quantification of the ratio of a single bacterium within the biofilm, and its viable proportion. The described method is reproducible and of high resolution, and allows the examination of microbes' composition and viability within a biofilm structure.

Myd88 plays a major role in the keratinocyte response to infection with Porphyromonas gingivalis.

AIM: To explore the role of keratinocyte myeloid differentiation primary response 88 (MyD88) expression in the adhesion of Porphyromonas gingivalis to the cells and its subsequent invasion and intracellular survival. MATERIALS AND METHODS: Primary mouse keratinocytes from wild-type (WT) or Myd88-/- mice were infected with P gingivalis alone or co-infected with Fusobacterium nucleatum. Bacterial adhesion and invasion were measured using fluorescent microscopy and flow cytometry, and intracellular survival in keratinocytes was quantified by an antibiotic protection assay. Keratinocyte expression of antimicrobial peptides was measured by real-time PCR. RESULTS: In the absence of MyD88, P gingivalis adherence, invasion, and intracellular survival were enhanced compared with WT keratinocytes. The presence of F nucleatum during infection increased the adhesion of P gingivalis to WT keratinocytes but reduced the adhesion to Myd88-/- keratinocytes. Fusobacterium nucleatum improved mildly the invasion and survival of P gingivalis in both cell types. Baseline expression of beta-defensin 2, 3, 4 and RegIII-γ was elevated in Myd88-/- keratinocytes compared to WT cells; however, following infection beta-defensin expression was strongly induced in WT cells but decreased dramatically in the MyD88 deficient cells. CONCLUSION: In the absence of MyD88 expression, P gingivalis adhesion to keratinocytes is improved, and invasion and intracellular survival are increased. Furthermore, keratinocyte infection by P gingivalis induces antimicrobial peptide expression in a MyD88-dependent manner. Thus, MyD88 plays a key role in the interaction between P gingivalis and keratinocytes.

Nupharidine enhances Aggregatibacter actinomycetemcomitans clearance by priming neutrophils and augmenting their effector functions.

OBJECTIVES: Nupharidine (6,6'-Dihydroxythiobinupharidine), purified from the aquatic plant Nuphar lutea leaves (Water lily) prompts antimicrobial activity of immune cells. The aim of the study was to test the effect of Nupharidine on neutrophil function against Aggregatibacter actinomycetemcomitans, JP2 clone (Aa-JP2). METHODS: Neutrophils derived from the human cell line HL60 and human peripheral blood derived from aggressive periodontitis and periodontally healthy subjects were incubated with Nupharidine or vehicle and inoculated with JP2. Bacterial survival was tested using viable counts on blood agar (CFU's). Neutrophils' necrosis/apoptosis, reactive oxygen species (ROS) production, phagocytosis and neutrophil extracellular traps (NET) production following infection were tested, as well as markers of neutrophil priming. RESULTS: Nupharidine had no direct bactericidal effect on JP2, but it enhanced Aa-JP2 clearance by neutrophils. Nupharidine enhanced neutrophil phagocytosis, ROS production and NET formation during JP2 infection. Furthermore, Nupharidine enhanced the expression of certain markers of neutrophils priming, specifically iCAM1, DECTIN-2 and intracellular IL-1ß. CONCLUSION: Nupharidine was shown to promote neutrophil effector bactericidal functions, boosting Aa-JP2 clearance. The results point to the potential of Nupharidine as an adjunctive agent in the treatment of Aa-JP2 periodontitis, but this should be tested initially using pre-clinical and clinical studies.

Microbial accumulation on different suture materials following oral surgery: a randomized controlled study.

BACKGROUND: The aim of the study was to compare bacterial accumulation on different suture materials following oral surgery. METHODS: Patients scheduled for implant or periodontal surgery were included in the study. Upon flap closure, four different sutures were placed in a randomized sequence-silk, coated polyglactin, nylon, and polyester. Ten days following surgery, the sutures were removed and incubated in aerobic as well as anaerobic conditions for 7 days and colony-forming units (CFUs) were calculated. Association between bacterial accumulation and periodontal diagnosis, type of surgery, and antibiotic treatment were also tested. RESULTS: All sutures in all patients were found to contain bacteria. Overall, nylon sutures showed significantly lower CFU levels compared to silk, coated polyglactin, and polyester sutures. The type of surgery (implant vs. periodontal surgery) did not significantly influence bacterial accumulation. Also, periodontal diagnosis had little impact on CFU counts. Interestingly, post-surgical antibiotic treatment also had only a minor effect on bacterial accumulation on the various sutures. DISCUSSION: The results indicate that the monofilamentous nylon sutures showed less microbial accumulation than the other tested materials that were all braided. This effect may be due to material qualities as well as suture macrostructure. Type of surgery, periodontal diagnosis, and antibiotic consumption have little effect on bacterial accumulation of sutures. CLINICAL RELEVANCE: The study provides the microbial profile of commonly used sutures and may assist in suture selection during clinical procedures.

An update on the evidence for pathogenic mechanisms that may link periodontitis and diabetes.

AIM: To provide an update of the review by Taylor (Journal of Clinical Periodontology, 2013, 40, S113) regarding the scientific evidence of the biological association between periodontitis and diabetes. METHODS: Literature searches were performed using MeSH terms, keywords and title words and were published between 2012 and November 2016. All publications were screened for their relevance. The data from the articles were extracted and summarized in tables and a narrative review. RESULTS: Small-scale molecular periodontal microbiome studies indicate a possible association between altered glucose metabolism in pre-diabetes and diabetes and changes in the periodontal microbiome, with no evidence for casual relationships. Clinical and animal studies found elevated gingival levels of IL1-ß, TNF-α, IL-6, RANKL/OPG and oxygen metabolites in poorly controlled diabetes. In addition, individuals with diabetes and periodontitis exhibit high levels of circulating TNF-α, CRP and mediators of oxidative stress, and successful periodontal treatment reduces their levels. CONCLUSIONS: The elevated pro-inflammatory factors in the gingiva of patients with poorly controlled diabetes suggest a biological pathway that may aggravate periodontitis. Some evidence suggests that the systemic inflammatory burden in periodontitis has the potential to affect diabetes control, but no studies addressed the impact of successful periodontal therapy on the pathophysiological mechanisms involved in systemic complications of diabetes.

Antibacterial Orthodontic Adhesive Incorporating Polyethyleneimine Nanoparticles.

PURPOSE: To evaluate the antibacterial, mechanical and biocompatibility characteristics of an orthodontic adhesive that contains quaternary ammonium polyethyleneimine (QPEI) nanoparticles. MATERIALS AND METHODS: QPEI nanoparticles were added to an orthodontic adhesive at 0%, 1% and 1.5% wt/wt. Antibacterial activity was tested after aging for 14 days using the direct contact test (DCT). The degree of monomer conversion (DC) was measured using Fourier transform infrared (FTIR) spectroscopy. Shear bond atrength (SBS) was tested on the etched enamel of extracted human teeth. Biocompatibility was tested using keratinocyte and neutrophil cell lines in the XTT assay. RESULTS: The DCT results showed significant bacterial growth inhibition in the test group incorporating 1.5% wt/wt QPEI nanoparticles (p < 0.05). The DC of the 0%, 1%, and 1.5% wt/wt samples measured immediately and after 10 min was 62.2-71.0%, 59.1-68.7%, and 52.9-58.6%, respectively, and the average SBS were 9.25 MPa, 11.57 MPa, and 9.10 MPa, respectively. Keratinocyte and neutrophil viability did not change following the addition of QPEI to the orthodontic adhesives. CONCLUSIONS: The incorporation of QPEI nanoparticles into orthodontic cement provides long-lasting antibacterial activity against Streptococcus mutans without reducing the strength of adhesion to enamel, the degree of double bond conversion during the polymerisation, or the biocompatibility of the orthodontic cement.

Are anti-inflammatory agents effective in treating gingivitis as solo or adjunct therapies? A systematic review.

OBJECTIVE: Systematically review the scientific evidence for efficiency of anti-inflammatory agents against gingivitis, either as solo treatments or adjunctive therapies. METHODS: A protocol was developed aimed to answer the following focused question: "Are anti-inflammatory agents effective in treating gingivitis as solo or adjunct therapies?" RCTs and cohort studies on anti-inflammatory agents against gingivitis studies were searched electronically. Screening, data extraction and quality assessment were conducted. The primary outcome measures were indices of gingival inflammation. A sub-analysis was performed dividing the active agents into anti-inflammatory and other drugs. RESULTS: The search identified 3188 studies, of which 14 RCTs met the inclusion criteria. The use of anti-inflammatory or other agents, in general showed a higher reduction in the test than in the control in terms of gingival indexes and bleeding scores. Only two RCTs on inflammatory drugs could be meta-analysed, showing a statistically significant reduction in the GI in the experimental group [WMD = -0.090; 95% CI (-0.105; -0.074); p = 0.000]. However, the contribution of both studies to the global result was unbalanced (% weight: 99.88 and 0.12 respectively). CONCLUSIONS: Most of the tested material showed beneficial effect as anti-inflammatory agents against gingivitis, either as a single treatment modality or as an adjunctive therapy.

Primary prevention of periodontitis: managing gingivitis.

UNLABELLED: Periodontitis is a ubiquitous and irreversible inflammatory condition and represents a significant public health burden. Severe periodontitis affects over 11% of adults, is a major cause of tooth loss impacting negatively upon speech, nutrition, quality of life and self-esteem, and has systemic inflammatory consequences. Periodontitis is preventable and treatment leads to reduced rates of tooth loss and improved quality of life. However, successful treatment necessitates behaviour change in patients to address lifestyle risk factors (e.g. smoking) and, most importantly, to attain and sustain high standards of daily plaque removal, lifelong. While mechanical plaque removal remains the bedrock of successful periodontal disease management, in high-risk patients it appears that the critical threshold for plaque accumulation to trigger periodontitis is low, and such patients may benefit from adjunctive agents for primary prevention of periodontitis. AIM: The aims of this working group were to systematically review the evidence for primary prevention of periodontitis by preventing gingivitis via four approaches: 1) the efficacy of mechanical self-administered plaque control regimes; 2) the efficacy of self-administered inter-dental mechanical plaque control; 3) the efficacy of adjunctive chemical plaque control; and 4) anti-inflammatory (sole or adjunctive) approaches. METHODS: Two meta-reviews (mechanical plaque removal) and two traditional systematic reviews (chemical plaque control/anti-inflammatory agents) formed the basis of this consensus. RESULTS: Data support the belief that professionally administered plaque control significantly improves gingival inflammation and lowers plaque scores, with some evidence that reinforcement of oral hygiene provides further benefit. Re-chargeable power toothbrushes provide small but statistically significant additional reductions in gingival inflammation and plaque levels. Flossing cannot be recommended other than for sites of gingival and periodontal health, where inter-dental brushes (IDBs) will not pass through the interproximal area without trauma. Otherwise, IDBs are the device of choice for interproximal plaque removal. Use of local or systemic anti-inflammatory agents in the management of gingivitis has no robust evidence base. We support the almost universal recommendations that all people should brush their teeth twice a day for at least 2 min. with fluoridated dentifrice. Expert opinion is that for periodontitis patients 2 min. is likely to be insufficient, especially when considering the need for additional use of inter-dental cleaning devices. In patients with gingivitis once daily inter-dental cleaning is recommended and the adjunctive use of chemical plaque control agents offers advantages in this group.

Direct recognition of Fusobacterium nucleatum by the NK cell natural cytotoxicity receptor NKp46 aggravates periodontal disease.

Periodontitis is a common human chronic inflammatory disease that results in the destruction of the tooth attachment apparatus and tooth loss. Although infections with periopathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum) are essential for inducing periodontitis, the nature and magnitude of the disease is determined by the host's immune response. Here, we investigate the role played by the NK killer receptor NKp46 (NCR1 in mice), in the pathogenesis of periodontitis. Using an oral infection periodontitis model we demonstrate that following F. nucleatum infection no alveolar bone loss is observed in mice deficient for NCR1 expression, whereas around 20% bone loss is observed in wild type mice and in mice infected with P. gingivalis. By using subcutaneous chambers inoculated with F. nucleatum we demonstrate that immune cells, including NK cells, rapidly accumulate in the chambers and that this leads to a fast and transient, NCR1-dependant TNF-α secretion. We further show that both the mouse NCR1 and the human NKp46 bind directly to F. nucleatum and we demonstrate that this binding is sensitive to heat, to proteinase K and to pronase treatments. Finally, we show in vitro that the interaction of NK cells with F. nucleatum leads to an NCR1-dependent secretion of TNF-α. Thus, the present study provides the first evidence that NCR1 and NKp46 directly recognize a periodontal pathogen and that this interaction influences the outcome of F. nucleatum-mediated periodontitis.

Development and Application of Reversible and Irreversible Covalent Probes for Human and Mouse Cathepsin-K Activity Detection, Revealing Nuclear Activity.

Cathepsin-K (CTSK) is an osteoclast-secreted cysteine protease that efficiently cleaves extracellular matrices and promotes bone homeostasis and remodeling, making it an excellent therapeutic target. Detection of CTSK activity in complex biological samples using tailored tools such as activity-based probes (ABPs) will aid tremendously in drug development. Here, potent and selective CTSK probes are designed and created, comparing irreversible and reversible covalent ABPs with improved recognition components and electrophiles. The newly developed CTSK ABPs precisely detect active CTSK in mouse and human cells and tissues, from diseased and healthy states such as inflamed tooth implants, osteoclasts, and lung samples, indicating changes in CTSK's activity in the pathological samples. These probes are used to study how acidic pH stimulates mature CTSK activation, specifically, its transition from pro-form to mature form. Furthermore, this study reveals for the first time, why intact cells and cell lysate exhibit diverse CTSK activity while having equal levels of mature CTSK enzyme. Interestingly, these tools enabled the discovery of active CTSK in human osteoclast nuclei and in the nucleoli. Altogether, these novel probes are excellent research tools and can be applied in vivo to examine CTSK activity and inhibition in diverse diseases without immunogenicity hazards.

The role of RgpA in the pathogenicity of Porphyromonas gingivalis in the murine periodontitis model.

AIM: To investigate the in vivo role of gingipains in Porphyromonas gingivalis' virulence, and suggest a possible host mechanisms through which the bacteria cause alveolar bone loss. MATERIALS AND METHODS: Mice were orally infected with P. gingivalis wild type, or the gingipains mutants (RgpA⁻, Kgp⁻, RgpA⁻/Kgp⁻). Mice were analysed for alveolar bone loss using micro-computed tomography. The molecular effects of the proteases were evaluated using the subcutaneous chamber model. Mice were infected with P. gingivalis wild type or mutants. Exudates were analysed for cytokine and leukocytes levels, in vivo phagocytosis, P. gingivalis survival and serum anti-P. gingivalis IgG titres. RESULTS: Only RgpA-expressing bacteria induced significantly alveolar bone loss, and suppressed phagocytosis resulting in increased survival of P. gingivalis in the chamber exudates. In addition, RgpA-expressing bacteria induced higher levels of leukocytes and cytokines 2 h post-infection, and reduced levels of serum anti-P. gingivalis IgG titres 7 days post-infection. CONCLUSIONS: Our findings showed that elimination of RgpA from P. gingivalis diminished inflammation, but augmented phagocytosis and antibody titres, coincidental with reduced alveolar bone loss. These findings support the hypothesis that RgpA is a critical virulence factor in the pathogenesis of experimental periodontitis in mice.

Biofilm formation, its removal, and consequent effect on the osteoblast response to titanium surfaces: A model for re-osseointegration.

BACKGROUND: The aim of the study was to characterize pathogenic biofilm formation on titanium surfaces, the ability to remove the biofilm and the osteoblast response to infected and cleaned titanium surfaces as a model for re-osseointegration. METHODS: Multispecies biofilm composed of Pseudomonas. gingivalis, F. nucleatum, S. sanguis, and A. naeslundii were grown on smooth, acid-etched, and acid-etched-aluminum-sprayed titanium surfaces. Bacterial viability was determined with live/dead staining. The biofilm was removed mechanically or together with adjunctive antibiotics. The osteoblast (Saos2) response to previously infected, treated and non-infected titanium surfaces were measured according to 4'-6-diamidino-2-phenylindole staining. Alkaline phosphatase levels and receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin expression were measured with enzyme-linked immunosorbent assay and immunofluorescence staining, respectively The inflammatory environment was established by using differentiated HL-60 cells (neutrophils) pre-inoculated onto the biofilm clusters that were more prominent and less scattered on infected titanium surfaces before osteoblast attachment. RESULTS: Biofilm formed on all the tested surfaces, with an increased thickness on rough surfaces and no differences in bacterial viability. All the treatments reduced the amount of biofilm, but none led to bacteria-free surfaces. The treated surfaces showed reduced osteoblast attachment and reduced alkaline phosphatase activity compared with non-infected surfaces. Additionally, treated surfaces showed an osteoblast shift to a pro-osteoclastic-induction phenotype, compared with non-infected surfaces. The presence of experimental inflammation before osteoblast attachment reduced the levels of osteoblast attachment compared with that of the non-inflamed control. CONCLUSIONS: Biofilm removal from titanium surfaces is incomplete when hand instruments are used alone or in combination with antibiotics. The treated surfaces showed impaired osteoblast attachment and function, particularly in the presence of inflammation, which may prevent or decrease the ability for re-osseointegration.

The role of coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum on the host response to mixed infection.

AIM: To evaluate the role of coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum on the virulence of the mixed infection in mice. MATERIALS AND METHODS: Inhibition of coaggregation was carried out using lactose. In vitro, inhibition of coaggregation was verified using a coaggregation assay. In vivo, the virulence of the mixed infection, with and without coaggregation, was examined in a model of experimental periodontitis in mice. The local host response to the mixed infection, with or without coaggregation, was examined using the subcutaneous chamber model of infection. RESULTS: Lactose inhibited the coaggregation between P. gingivalis and F. nucleatum at all the tested concentrations (1-0.0625 M). Surprisingly, the addition of lactose to the mixed infection increased the severity of experimental periodontitis (as measured by alveolar bone loss) compared with mixed infection with coaggregating bacteria. The addition of lactose to the mixed infection resulted in mild attenuation of TNFα and IL-1ß levels. In addition, inhibition of coaggregation resulted in inhibition of the phagocytosis of F. nucleatum and augmentation of the phagocytosis of P. gingivalis. CONCLUSIONS: The ability of P. gingivalis and F. nucleatum to coaggregate may limit their ability to induce experimental periodontitis in a mixed infection model. Moreover, there is a shift in the phagocytosis pattern of the bacteria with the annulment of coaggregeaiton, with a reduction in F. nucleatum phagocytosis and amplification of P. gingivalis phagocytosis. The increased virulence of the mixed infection without coaggregation may surprisingly lay in the sustention of F. nucleatum in the infected sites.

The in vitro efficacy of biofilm removal from titanium surfaces using Er:YAG laser: Comparison of treatment protocols and ablation parameters.

BACKGROUND: The aims of the present study were to compare the antibacterial effect of Er:YAG laser with other acceptable decontamination methods and to single out the optimal laser device parameters for effective bacterial elimination. METHODS: A multispecies biofilm which was composed of Streptococcus sanguis, Actinomyces naeslundii, Porphyromonas gingivalis, and Fusobacterium nucleatum was grown on sandblasted and acid-etched (SLA, homogeneous moderately microrough, and nanosmooth surface) titanium disks. The biofilm was removed from the coated disks by hand curets, ultrasonic device, nylon brush (dental polishing prophy cup), or Er:YAG. Additionally, different parameter combinations of the laser machine were examined to reach an optimal lasing power for bacterial elimination/reduction. Residual biofilm samples were stained with bacterial live/dead staining and quantified using a fluorescent microscope. RESULTS: A multispecies biofilm was accumulated on the SLA titanium surfaces exhibiting cluster distribution next to bacteria-poor areas. Hand curets, nylon brushes, and the ultrasonic device showed limited capability to effectively remove the biofilm from the SLA surfaces as opposed to the Er:YAG which displayed a superior ability to remove the biofilm. All Er:YAG parameter combinations that were evaluated as well as the tested "tip to target" distances showed similar excellent anti-biofilm effects. Furthermore, we observed that the Er:YAG capability of biofilm removal is not only due to its light emission, but depends on its water irrigation as well. CONCLUSIONS: Er:YAG laser has an excellent biofilm removal capability compared with hand curets, ultrasonic devices, or nylon brushes even when low energy parameters and low power settings are used. Additionally, an excellent antibacterial effect can be reached using a non-contact mode of 1 to 5 mm "tip to target" distance.