Systemic antibiotics in the surgical treatment of peri-implantitis: A randomized placebo-controlled trial.

AIM: To study the clinical, radiographic and microbiological outcomes after surgical treatment of peri-implantitis, with or without adjunctive systemic antibiotics. MATERIALS AND METHODS: Eighty-four patients (113 implants) with peri-implantitis were randomized into three groups (A, amoxicillin and metronidazole; B, phenoxymethylpenicillin and metronidazole; or C, placebo). Treatment included resective surgery and implant surface decontamination with adjunctive antibiotics or placebo. Primary outcomes were probing pocket depth (PPD) reduction and marginal bone level (MBL) stability. Secondary outcomes were treatment success (defined as PPD ≤ 5 mm, bleeding on probing [BOP] ≤ 1site, absence of suppuration on probing [SOP] and absence of progressive bone loss of >0.5 mm), changes in BOP/SOP, mucosal recession (REC), clinical attachment level (CAL), bacterial levels and adverse events. Outcomes were evaluated for up to 12 months. The impact of potential prognostic indicators on treatment success was evaluated using multilevel logistic regression analysis. RESULTS: A total of 76 patients (104 implants) completed the study. All groups showed clinical and radiological improvements over time. Statistically significant differences were observed between groups for MBL stability (A = 97%, B = 89%, C = 76%), treatment success (A = 68%, B = 66%, C = 28%) and bacterial levels of Aggregatibacter actinomycetemcomitans and Tannerella forsythia, favouring antibiotics compared to placebo. Multiple regression identified antibiotic use as potential prognostic indicator for treatment success. Gastrointestinal disorders were the most reported adverse events in the antibiotic groups. CONCLUSIONS: Adjunctive systemic antibiotics resulted in additional improvements in MBL stability. However, the potential clinical benefits of antibiotics need to be carefully balanced against the risk of adverse events and possible antibiotic resistance.

Bacteriome and mycobiome dysbiosis in oral mucosal dysplasia and oral cancer.

It has long been considered that the oral microbiome is tightly connected to oral health and that dysbiotic changes can be detrimental to the occurrence and progression of dysplastic oral mucosal lesions or oral cancer. Improved understanding of the concepts of microbial dysbiosis together with advances in high-throughput molecular sequencing of these pathologies have charted in greater microbiological detail the nature of their clinical state. This review discusses the bacteriome and mycobiome associated with oral mucosal lesions, oral candidiasis, and oral squamous cell carcinoma, aiming to delineate the information available to date in pursuit of advancing diagnostic and prognostic utilities for oral medicine.

Cytokine profiles and the dynamic of gingivitis development in humans.

AIM: To investigate the relationship between cytokine profiles and "fast" and "slow" patterns of gingival inflammation development. MATERIALS AND METHODS: Forty-two adults participated in an experimental gingivitis study, comprising a 2-week hygiene phase (clinical examination and professional cleaning); a 3-week induction phase (absence of oral hygiene); and a 2-week resolution phase (re-establishment of oral hygiene). Plaque and gingival inflammation scores were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumour necrosis factor-alpha (TNF-α) from gingival crevicular fluid were collected and measured by multiplex ELISA. Group-based-trajectory-modelling (GBTM) was used to model cytokine profiles over the induction phase. The effect of gingival inflammation on cytokine levels over time was estimated with mixed-effects modelling. RESULTS: GBTM analysis revealed two cytokine profiles, "non-organized response" (IL-4, IL-6, IL-8, IL-12, and IL-13) and "organized response" (IL-2, IL-10, and TNF-α). Among the "slow" responders, neither cytokine profile was associated with gingivitis. In contrast, a "fast" response was associated with a higher "non-organized response" factor (coef. 0.14) and a lower "organized response" factor (coef. -0.03). CONCLUSION: A "fast" gingivitis development was associated with a higher "non-organized response" and a lower "organized response", which may elucidate the role of individual variability in gingivitis susceptibility.

Dysbiosis of the Human Oral Microbiome During the Menstrual Cycle and Vulnerability to the External Exposures of Smoking and Dietary Sugar.

Physiological hormonal fluctuations exert endogenous pressures on the structure and function of the human microbiome. As such, the menstrual cycle may selectively disrupt the homeostasis of the resident oral microbiome, thus compromising oral health. Hence, the aim of the present study was to structurally and functionally profile the salivary microbiome of 103 women in reproductive age with regular menstrual cycle, while evaluating the modifying influences of hormonal contraceptives, sex hormones, diet, and smoking. Whole saliva was sampled during the menstrual, follicular, and luteal phases (n = 309) of the cycle, and the participants reported questionnaire-based data concerning their life habits and oral or systemic health. No significant differences in alpha-diversity or phase-specific clustering of the overall microbiome were observed. Nevertheless, the salivary abundances of genera Campylobacter, Haemophilus, Prevotella, and Oribacterium varied throughout the cycle, and a higher species-richness was observed during the luteal phase. While the overall community structure maintained relatively intact, its functional properties were drastically affected. In particular, 11 functional modules were differentially abundant throughout the menstrual cycle, including pentose phosphate metabolism, and biosynthesis of cobalamin and neurotransmitter gamma-aminobutyric acid. The menstrual cycle phase, but not oral contraceptive usage, was accountable for greater variations in the metabolic pathways of the salivary microbiome. Further co-risk factor analysis demonstrated that Prevotella and Veillonella were increased in current smokers, whereas high dietary sugar consumption modified the richness and diversity of the microbiome during the cycle. This is the first large study to systematically address dysbiotic variations of the oral microbiome during the course of menstrual cycle, and document the additive effect of smoking and sugar consumption as environmental risk factors. It reveals the structural resilience and functional adaptability of the oral microbiome to the endogenous hormonal pressures of the menstrual cycle, while revealing its vulnerability to the exogenous exposures of diet and smoking.

Dysbiosis of the Oral Ecosystem in Severe Congenital Neutropenia Patients.

PURPOSE: To decipher the underlying immunological mechanisms in predisposition to oral microbial dysbiosis in severe congenital neutropenia (SCN) patients. EXPERIMENTAL DESIGN: Ten SCN patients (5-23 years old) and 12 healthy controls (5-22 years old) are periodontally examined and provided saliva, subgingival plaque, and gingival crevicular fluid (GCF) samples. The SCN patients received oral hygiene therapy and are re-evaluated after 6 months. Antimicrobial peptides HPN1-3 and LL-37 are assessed in saliva by ELISA. Concentration of 30 cytokines is measured in saliva and GCF by human 30-plex panel, while bacterial profiles of saliva and subgingival plaque are assessed using 16S rDNA amplicon sequencing. RESULTS: There is no significant difference in salivary HPN1-3 and LL-37 concentration between the SCN patients and controls. At baseline, clinical, immunological, and microbiological parameters of the patients are indicative of oral ecological dysbiosis. The SCN patients have significantly higher bleeding on probing (BOP)%, GCF volume, and cytokine levels, high bacterial load with low bacterial diversity in saliva. The associations between the microbiome and immunological parameters in the SCN patients differ from those in the healthy individuals. CONCLUSIONS AND CLINICAL RELEVANCE: SCN patients have a dysregulated immune response toward commensal oral microbiota, which could be responsible for the observed clinical and microbiological signs of dysbiosis.

Root caries: the intersection between periodontal disease and dental caries in the course of ageing.

Caries and periodontitis are the primary non-communicable oral diseases among elderly individuals. The burden of the disease increases with ageing, particularly as the elderly are tending to retain more teeth due to improvement of oral health measures and increased life expectancy. Root caries represents itself as an overlapping pathology, but not necessarily a summation of the two diseases. This narrative commentary discusses the cross-boundary nature of root caries, a periodontal-cariological condition, taking into account the multi-morbidities of ageing. The evidence includes epidemiological and pathophysiological features of root caries, and specific influencing factors of ageing, such as xerostomia, polypharmacy, functional and cognitive impairment and oral ecological alterations. Active or previous history of periodontal disease poses a risk for root caries, whereas the systemic co-morbidities of ageing may also increase the susceptibility to this pathology. It is plausible that root caries is the net outcome of coexisting risk for these conditions. There exists no standardised system for risk assessment and diagnosis that takes into account the interactive effect of caries, periodontitis and the constellation of age-specific influencing factors. As restorative treatment is challenging, cost-effective prevention and diagnosis methods are needed for vulnerable elderly populations. These may include improved clinical registration methods and establishment of individualised prevention and treatment protocols.

Advances in Oral Mucosal Immunity and the Microbiome.

The 1st International Conference on Oral Mucosal Immunity and the Microbiome (OMIM) took place at the Avra Imperial Hotel, Chania, Crete, Greece, between 26th and 30th September 2018, under the auspices of the Aegean Conferences. This was the first Aegean Conference of its kind in thematic oral research, and a unique blend of immunological and microbiological perspectives, which attracted leading scientists from around the world to discuss the latest advances in the field. The Conference was divided into eight sessions that spanned across 4 days and included the following topics: (a) mucosal barrier immunity; (b) host response and inflammation; (c) microbiome in homeostasis and dysbiosis; (d) fungal and viral pathogenesis; (e) oral microbiome and proteome; (f) microbial virulence and biofilms; (g) microbiome, cancer, and systemic disease; and (h) microbiota and inflammation. There was substantial thematic overlap among all sessions, which promoted constant involvement of the participating scientists. An important hallmark was the active debate between oral microbiologists and oral immunologists, who explored new ideas and potential research collaborations, a crucial aspect for bridging our understanding of oral diseases in the context of the whole body. Key findings are highlighted and thematically presented in the following sections.

Change of saliva composition with radiotherapy.

OBJECTIVE: The aim of this study was to analyze the physiological and microbiological changes of saliva from patients with head and neck cancer during and after intensity-modulated radiotherapy (IMRT). DESIGN: In this prospective clinical trial saliva samples and oral candida swabs were collected from patients receiving IMRT due to head and neck cancer (examination group). The first measurement was scheduled before radiotherapy, the other measurements during and after radiotherapy up to a one year follow-up. Additionally samples from healthy controls were collected over six weeks. Salivary flow rate and pH were measured. Microbiological analysis of cariogenic and periodontopathogenic taxa was performed by fluorescence in situ hybridization and oral Candida spp occurrence was evaluated by swab tests. RESULTS: 11 patients and 19 controls were included. The salivary flow rate and the unstimulated pH of the examination group were significantly reduced during radiotherapy compared with the measurement before radiotherapy and to the control group. Total bacteria, streptococci and lactobacilli numbers slightly increased after radiotherapy, resuming baseline levels after one year. Mutans streptococci, Porphyromonas gingivalis and Treponema denticola were barely detectable, whereas Tannerella forsythia slightly increased following radiotherapy. No differences in Candida levels were observed in the study. CONCLUSIONS: Salivary changes in quantitative, qualitative and microbial composition occur during and after radiotherapy, with resumption of the measurements towards baseline levels after one year. While low levels of cariogenic and periodontopathogenic species were detected, the lower pH and salivary flow combined with increased numbers of aciduric and acidogenic lactobacilli corroborates a higher risk for caries, necessitating prevention.

Cytokine, chemokine, and growth factor levels in peri-implant sulcus during wound healing and osseointegration after piezosurgical versus conventional implant site preparation: Randomized, controlled, split-mouth trial.

BACKGROUND: The aim of this trial was to evaluate the cytokine, chemokine, and growth factor levels in peri-implant sulcular fluid (PISF) during healing and osseointegration at osteotomy sites prepared either with piezosurgery (PS) or drills. METHODS: Fourteen patients having contralateral partial edentulism in the posterior maxilla were enrolled and 38 osteotomies were prepared. Implants were placed with one-stage surgery. Insertion torque, early healing index, probing depth and modified gingival and plaque indices and crestal bone loss (CBL) were measured. PISF was collected from each implant at weeks 2, 4, 8, 12, and 24 and were analyzed by a 30-Plex immunoassay. Data analysis employed Brunner-Langer method. RESULTS: CBL values did not depend on osteotomy modality (P > 0.05). Eighteen molecules (interleukine (IL)-1ß, granulocyte colony stimulating factor (G.CSF), IL-13, IL-6, IL-12, interferon (IFN)-γ, IFN-α, IL-2, IL-2 R, IL-8, macrophage inflammatory protein (MIP)-1α, MIP-1ß, monocyte chemoattractant protein (MCP)-1, interferon gamma-induced protein (IP)-10, monokine induced by IFN-γ (MIG), epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) showed time-dependent decrease (P < 0.05), but they were not treatment-dependent (P > 0.05). When values of weeks 4, 8, 12, and 24 were compared to week 2, it was found that all were highest at week 2 and decreased thereafter (P < 0.05). The decrease was significant at weeks 4 or 8 for multitude of molecules and was mostly sustained throughout the follow-up. Week 8 regulated on activation, normal T cell expressed and secreted (RANTES) values in PS group were lower in PS group compared to drill group (P < 0.05). CONCLUSIONS: Implants placed into osteotomies created with PS and drills are similar in terms of PISF biomarker changes during the osseointegration and wound healing period. When clinical and CBL parameters were taken into account together with the PISF molecular data it can be speculated that PS and conventional drill osteotomy have similar effects on peri-implant tissues on the biochemical, clinical and radiological levels.

Targeted Proteomics Guided by Label-free Quantitative Proteome Analysis in Saliva Reveal Transition Signatures from Health to Periodontal Disease.

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. Here we carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n = 67, including individuals with health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n = 82). The LFQ platform led to the discovery of 119 proteins with at least 2-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 functionally related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases with maximum area under the receiver operating curve >0.97 (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1). This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum leap brought into the field of periodontal diagnostics by this study is the application of the biomarker discovery-through-verification pipeline, which can be used for validation in further cohorts.

The epigenetic architecture at gene promoters determines cell type-specific LPS tolerance.

Synovial fibroblasts (SF) drive inflammation and joint destruction in chronic arthritis. Here we show that SF possess a distinct type of LPS tolerance compared to macrophages and other types of fibroblasts. In SF and dermal fibroblasts, genes that were non-tolerizable after repeated LPS stimulation included pro-inflammatory cytokines, chemokines and matrix metalloproteinases, whereas anti-viral genes were tolerizable. In macrophages, all measured genes were tolerizable, whereas in gingival and foreskin fibroblasts these genes were non-tolerizable. Repeated stimulation of SF with LPS resulted in loss of activating histone marks only in promoters of tolerizable genes. The epigenetic landscape at promoters of tolerizable genes was similar in unstimulated SF and monocytes, whereas the basal configuration of histone marks profoundly differed in genes that were non-tolerizable in SF only. Our data suggest that the epigenetic configuration at gene promoters regulates cell-specific LPS-induced responses and primes SF to sustain their inflammatory response in chronic arthritis.

Gingival Inflammation and Salivary or Serum Granulocyte-Secreted Enzymes in Patients With Polycystic Ovary Syndrome.

BACKGROUND: The objective of this cross-sectional study is to investigate levels of salivary and serum matrix metalloproteinase (MMP)-9, myeloperoxidase (MPO), neutrophil elastase (NE), and MMP-9/tissue inhibitor of MMP-1 (TIMP)-1 ratio in patients with polycystic ovary syndrome (PCOS) and systemically healthy controls in the presence or absence of gingivitis. METHODS: Serum and salivary levels of these biomarkers were evaluated in the following: 1) periodontally healthy women with PCOS (n = 45); 2) women with PCOS and gingivitis (n = 35); 3) systemically and periodontally healthy women (n = 25); and 4) systemically healthy women with gingivitis (n = 20). Enzyme-linked immunosorbent assay was used to determine levels of these biomarkers. A full-mouth clinical periodontal evaluation was performed for each patient. RESULTS: Salivary MMP-9 and NE levels, as well as MMP-9/TIMP-1 ratios, were higher in the systemically healthy women with gingivitis compared with periodontally healthy women with PCOS (P <0.001; P <0.01; and P <0.0001, respectively). Serum MMP-9 and MPO levels were higher in women with PCOS and gingivitis compared with periodontally healthy women with PCOS (P <0.05). Serum MMP-9 levels were lower in healthy women with gingivitis than systemically and periodontally healthy women or women with PCOS and gingivitis (P <0.05). PCOS groups exhibited a positive correlation among clinical periodontal parameters and serum MMP-9 levels or salivary MPO, NE levels, and MMP-9/MMP-1 ratio. Correlation was negative among clinical periodontal parameters and serum MMP-9 levels and MMP-9/TIMP-1 ratio in systemically healthy patients (P <0.05). CONCLUSIONS: The present findings emphasize that PCOS and gingival inflammation are associated with each other, as evidenced by salivary and serum levels of neutrophilic enzymes. This interaction may contribute to the perturbation of ovarian remodeling in PCOS.

Impact of implant-abutment connection on osteoimmunological and microbiological parameters in short implants: a randomized controlled clinical trial.

OBJECTIVES: The study aimed to determine the levels of soluble receptor activator of nuclear factor-кB ligand (sRANKL) and osteoprotegerin (OPG) as well as their relative calculated ratio in peri-implant crevicular fluid (PICF) obtained around two different types of implant-abutment connection on short implants following a 12-month monitoring period. Moreover, the levels of a number of oral bacterial species were investigated in the corresponding submucosal biofilm samples. MATERIALS AND METHODS: Thirty short implants were randomly placed in posterior maxillary edentulous sites using a split-mouth design in 15 periodontally healthy subjects. Tapered interference fit (TIF) and taper-integrated screwed-in (TIS) types of implant-abutment connections were selected for investigation. PICF and submucosal biofilm samples were collected 1 month after surgery and repeated 12 months after prosthetic loading. Clinical parameters, including probing depth, dichotomous presence of bleeding on probing, and plaque index, were recorded and digital periapical radiographs were taken at each time point. sRANKL and OPG levels in PICF were analyzed using an enzyme-linked immunosorbent assay. Total bacterial levels, as well as levels of Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia, and Streptococcus oralis, were analyzed in the corresponding submucosal biofilm samples using quantitative real-time polymerase chain reaction. RESULTS: The total amount of sRANKL in TIF implants was 2.64-fold lower than that in TIS implants at baseline (P < 0.001), whereas similar levels were found after 12 months (P > 0.05). Accordingly, OPG and RANKL/OPG ratio were similar between the groups at each time point (P > 0.05). Microbiological results were similar in both groups at each time point (P > 0.05). CONCLUSION: The results of this longitudinal study suggested that sRANKL and OPG in PICF, as well as microbiological parameters in submucosal biofilms, were similar between TIF and TIS implants, after a 12-month monitoring period, despite early differences in the former. Therefore, the type of implant-abutment connection does not appear to influence longitudinally the levels of osteoimmunological and microbiological markers in the peri-implant tissues of short implants.

The effect of piezoelectric surgery implant osteotomy on radiological and molecular parameters of peri-implant crestal bone loss: a randomized, controlled, split-mouth trial.

AIM: To evaluate the effect of piezoelectric surgery (PS) implant osteotomy on biochemical and radiological parameters of crestal bone (CB) loss. MATERIAL AND METHODS: In this randomized, controlled, clinical study, 38 osteotomies were prepared with PS and drilling in the posterior maxilla in a split-mouth design. Implants were placed and left for non-submerged healing. Osteotomy time, insertion torque, pain perception, probing depth, and modified gingival and plaque indices were recorded. Peri-implant sulcular fluid (PISF) was collected from four sites of each implant at 2, 4, 8, 12, and 24 weeks. PISF samples were analyzed by ELISA for receptor activator of nuclear factor kappa-B-ligand (RANKL) and osteoprotegerin. CB loss was assessed on periapical radiographs at the 12th and on cone beam computed tomography (CBCT) at the 24th weeks. The influence of time and osteotomy method on biochemical and radiological parameters of CB loss employed statistical method of Brunner-Langer. RESULTS: Osteotomy time for PS group was significantly longer than the drill group (P < 0.05). Pain perception that was lower in the PS than in the drill group depended on osteotomy method (P < 0.05). PS group had lower RANKL total amount than the drill group (P < 0.05). Mean CB loss on periapical radiographs at the 12th week for PS and drill groups were 0.11 and 0.18 mm, respectively (P > 0.05). At the 24th week, PS and drill groups showed 0.11 and 0.12 mm CB losses on CBCT, respectively (P > 0.05). However, CB loss values did not depend on osteotomy modality (P > 0.05). CONCLUSION: PS may modify and reduce bone-destructive inflammatory response during implant osseointegration. Therefore, on the molecular level, it might be a less traumatic osteotomy modality than drilling although this was not reflected by CB loss values in the present study.

Microbiome of peri-implant infections: lessons from conventional, molecular and metagenomic analyses.

Osseointegrated dental implants are now a well-established treatment option in the armament of restorative dentistry. These technologically advanced devices are designed to functionally and esthetically replace missing teeth. Despite the revolutionary advances that implants have incurred, they have also provided the oral cavity with new artificial surfaces prone to the formation of oral biofilms, similarly to the hard tissue surfaces of natural teeth. Biofilm formation on the implant surface can trigger the inflammatory destruction of the peri-implant tissue, in what is known as peri-implantitis. The mixed microbial flora of peri-implant infections resembles that of periodontal infections, with some notable differences. These are likely to expand with the ever increasing application of metagenomics and metatrascriptomics in the analysis of oral ecology. This review presents the wealth of knowledge we have gained from microbiological methods used in the characterization of peri-implant microflora and sheds light over potential new benefits, as well as limitations, of the new sequencing technology in our understanding of peri-implant disease pathogenesis.

Elevated matrix metalloproteinase-8 in saliva and serum in polycystic ovary syndrome and association with gingival inflammation.

This study aimed to investigate the levels of matrix metalloproteinase-8 (MMP-8) and tissue inhibitors of MMP-1 (TIMP-1) in saliva and serum samples of women with polycystic ovary syndrome (PCOS; n = 80) and matched systemically healthy controls (n = 45), with varying degrees of gingival inflammation. Salivary levels of MMP-8 and the MMP-8/TIMP-1 ratio were significantly elevated in women with PCOS, who also exhibited more gingivitis than systemically healthy women. No major changes were observed in salivary TIMP-1 levels with regard to PCOS. Serum levels of MMP-8 and the MMP-8/TIMP-1 ratio were significantly higher in women with PCOS, irrespective of the presence of gingivitis, while there were no differences in TIMP-1 levels. A positive correlation was indicated between probing depth, bleeding on probing, plaque index and salivary or serum MMP-8 levels or MMP-8/TIMP-1 ratio in the case of PCOS, while a negative such correlation was revealed for TIMP-1 in systemically healthy women. Increased levels of MMP-8 in saliva and serum seem to be more pronounced in women with PCOS, and potentiated in the presence of gingival inflammation. Alterations in MMP/TIMP system triggered by local and systemic inflammation may be implicated in the pathogenesis of PCOS, or the deterioration of its clinical presentation.

Establishment of an oral infection model resembling the periodontal pocket in a perfusion bioreactor system.

Periodontal infection involves a complex interplay between oral biofilms, gingival tissues and cells of the immune system in a dynamic microenvironment. A humanized in vitro model that reduces the need for experimental animal models, while recapitulating key biological events in a periodontal pocket, would constitute a technical advancement in the study of periodontal disease. The aim of this study was to use a dynamic perfusion bioreactor in order to develop a gingival epithelial-fibroblast-monocyte organotypic co-culture on collagen sponges. An 11 species subgingival biofilm was used to challenge the generated tissue in the bioreactor for a period of 24 h. The histological and scanning electron microscopy analysis displayed an epithelial-like layer on the surface of the collagen sponge, supported by the underlying ingrowth of gingival fibroblasts, while monocytic cells were also found within the sponge mass. Bacterial quantification of the biofilm showed that in the presence of the organotypic tissue, the growth of selected biofilm species, especially Campylobacter rectus, Actinomyces oris, Streptococcus anginosus, Veillonella dispar, and Porphyromonas gingivalis, was suppressed, indicating a potential antimicrobial effect by the tissue. Multiplex immunoassay analysis of cytokine secretion showed that interleukin (IL)-1 ß, IL-2, IL-4, and tumor necrosis factor (TNF)-α levels in cell culture supernatants were significantly up-regulated in presence of the biofilm, indicating a positive inflammatory response of the organotypic tissue to the biofilm challenge. In conclusion, this novel host-biofilm interaction organotypic model might resemble the periodontal pocket and have an important impact on the study of periodontal infections, by minimizing the need for the use of experimental animal models.

Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation.

BACKGROUND: Periodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 "subgingival" bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition. RESULTS: Following 64 h of biofilm growth, the levels of all 10 species were quantified by fluorescence in situ hybridization or immunofluorescence. The wild-type and the two gingipain-deficient P. gingivalis strains exhibited similar growth in their corresponding biofilms. Among the remaining nine species, only the numbers of T. forsythia were significantly reduced, and only when the Lys-gingipain mutant was present in the biofilm. When evaluating the structure of the biofilm by confocal laser scanning microscopy, the most prominent observation was a shift in the spatial arrangement of T. denticola, in the presence of P. gingivalis Arg-gingipain mutant. CONCLUSIONS: The gingipains of P. gingivalis may qualitatively and quantitatively affect composition of polymicrobial biofilms. The present experimental model reveals interdependency between the gingipains of P. gingivalis and T. forsythia or T. denticola.

Association between polycystic ovary syndrome, oral microbiota and systemic antibody responses.

Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.

Periapical fluid RANKL and IL-8 are differentially regulated in pulpitis and apical periodontitis.

The dental pulp space can become infected due to a breach in the surrounding hard tissues. This leads to inflammation of the pulp (pulpitis), soft tissue breakdown, and finally to bone loss around the root apex (apical periodontitis). The succession of the molecular events leading to apical periodontitis is currently not known. The main inflammatory mediator associated with neutrophil chemotaxis is interleukin-8 (IL-8), and with bone resorption the dyad of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). The levels of RANKL, OPG and IL-8 were studied in periapical tissue fluid of human teeth (n = 48) diagnosed with symptomatic irreversible pulpitis (SIP) and asymptomatic apical periodontitis (AAP). SIP represents the starting point, and AAP an established steady state of the disease. Periapical tissue fluid samples were collected using paper points and then evaluated using enzyme-linked immunosorbent assays (ELISAs). Target protein levels per case were calibrated against the corresponding total protein content, as determined fluorometrically. RANKL was expressed at significantly higher levels in SIP compared to AAP (P < 0.05), whereas OPG was under the detection limit in most samples. In contrast, IL-8 levels were significantly lower in SIP compared to AAP (P < 0.05). Spearman's correlation analysis between RANKL and IL-8 revealed a significantly (P < 0.05) negative correlation between the two measures (rho = -.44). The results of this study suggest that, in the development of apical periodontitis, periapical bone resorption signaling, as determined by RANKL, occurs prior to inflammatory cell recruitment signaling, as determined by IL-8.