Inflammatory and bone remodelling related biomarkers following periodontal transplantation of the tissue engineered biocomplex.

OBJECTIVES: To assess gingival crevicular fluid (GCF) levels of inflammatory and bone remodelling related biomarkers following transplantation of a tissue-engineered biocomplex into intrabony defects at several time-points over 12-months. MATERIALS AND METHODS: Group-A (n = 9) received the Minimal Access Flap (MAF) surgical technique combined with a biocomplex of autologous clinical-grade alveolar bone-marrow mesenchymal stem cells in collagen scaffolds enriched with an autologous fibrin/platelet lysate (aFPL). Group-B (n = 10) received the MAF surgery, with collagen scaffolds enriched with aFPL and Group-C (n = 8) received the MAF surgery alone. GCF was collected from the osseous defects of subjects via paper strips/30 sec at baseline, 6-weeks, 3-, 6-, 9-, 12-months post-surgery. Levels of inflammatory and bone remodelling-related biomarkers in GCF were determined by ELISA. RESULTS: Group-A demonstrated significantly higher GCF levels of BMP-7 at 6-9 months than baseline, with gradually decreasing levels of pro-inflammatory and pro-osteoclastogenic markers (TNF-α, RANKL) over the study-period; and an overall decrease in the RANKL/OPG ratio at 9-12 months than baseline (all p < 0.001). In comparison, only modest interim changes were observed in Groups-B and -C. CONCLUSIONS: At the protein level, the approach of MAF and biocomplex transplantation provided greater tissue regeneration potential as cell-based therapy appeared to modulate inflammation and bone remodelling in residual periodontal defects. CLINICAL RELEVANCE: Transplantation of a tissue engineered construct into periodontal intrabony defects demonstrated a biochemical pattern for inflammatory control and tissue regeneration over 12-months compared to the control treatments. Understanding the biological healing events of stem cell transplantation may facilitate the design of novel treatment strategies. CLINICAL DATABASE REGISTRATION: ClinicalTrials.gov ID: NCT02449005.

Calcium-sensing receptor regulates Kv7 channels via Gi/o protein signalling and modulates excitability of human induced pluripotent stem cell-derived nociceptive-like neurons.

BACKGROUND AND PURPOSE: Neuropathic pain, a debilitating condition with unmet medical needs, can be characterised as hyperexcitability of nociceptive neurons caused by dysfunction of ion channels. Voltage-gated potassium channels type 7 (Kv7), responsible for maintaining neuronal resting membrane potential and thus excitability, reside under tight control of G protein-coupled receptors (GPCRs). Calcium-sensing receptor (CaSR) is a GPCR that regulates the activity of numerous ion channels, but whether CaSR can control Kv7 channel function has been unexplored until now. EXPERIMENTAL APPROACH: Experiments were conducted in recombinant cell models, mouse dorsal root ganglia (DRG) neurons and human induced pluripotent stem cell (hiPSC)-derived nociceptive-like neurons using patch-clamp electrophysiology and molecular biology techniques. KEY RESULTS: Our results demonstrate that CaSR is expressed in recombinant cell models, hiPSC-derived nociceptive-like neurons and mouse DRG neurons, and its activation induced depolarisation via Kv7.2/7.3 channel inhibition. The CaSR-Kv7.2/7.3 channel crosslink was mediated via the Gi/o protein-adenylate cyclase-cyclicAMP-protein kinase A signalling cascade. Suppression of CaSR function demonstrated a potential to rescue hiPSC-derived nociceptive-like neurons from algogenic cocktail-induced hyperexcitability. CONCLUSION AND IMPLICATIONS: This study demonstrates that the CaSR-Kv7.2/7.3 channel crosslink, via a Gi/o protein signalling pathway, effectively regulates neuronal excitability, providing a feasible pharmacological target for neuronal hyperexcitability management in neuropathic pain.

Limitations of using 16S rRNA microbiome sequencing to predict oral squamous cell carcinoma.

A new era of next-generation sequencing has changed our perception of the oral microbiome in health and disease, and with this there is a growing understanding that the oral microbiome is a contributing factor to oral squamous cell carcinoma (OSCC), a malignancy of the oral cavity. This study aimed to analyse the trends and relevant literature based on the 16S rRNA oral microbiome in head and neck cancer using next-generation sequencing technologies, and to conduct a meta-analysis of the studies with OSCC cases and healthy controls. A literature search using the databases Web of Science and PubMed was conducted in a scoping-like review to collect information based on the study design, and plots were generated using RStudio. We selected case-control studies using 16S rRNA oral microbiome sequencing analysis in OSCC cases versus healthy controls for re-analysis. Statistical analyses were conducted using R. Out of 916 original articles, we filtered and selected 58 studies for review, and 11 studies for meta-analysis. Differences between sampling type, DNA extraction methods, next-generation sequencing technology and region of the 16S rRNA were identified. No significant differences in the α- and ß-diversity between health and oral squamous cell carcinoma were observed (p < 0.05). Random Forest classification marginally improved predictability of four studies (training set) when split 80/20. We found an increase in Selenomonas, Leptotrichia and Prevotella species to be indicative of disease. A number of technological advances have been accomplished to study oral microbial dysbiosis in oral squamous cell carcinoma. There is a clear need for standardization of study design and methodology to ensure 16S rRNA outputs are comparable across the discipline in the hope of identifying 'biomarker' organisms for designing screening or diagnostic tools.

Acetylcholine Receptors in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their regenerative potential. Even though the ability of MSCs to proliferate and differentiate has been studied extensively, there remains much to learn about the signaling mechanisms and pathways that control proliferation and influence the differentiation phenotype. In recent years, there has been growing evidence for the utility of non-neuronal cholinergic signaling systems and that acetylcholine (ACh) plays an important ubiquitous role in cell-to-cell communication. Indeed, cholinergic signaling is hypothesized to occur in stem cells and ACh synthesis, as well as in ACh receptor (AChR) expression, has been identified in several stem cell populations, including MSCs. Furthermore, AChRs have been found to influence MSC regenerative potential. In humans, there are two major classes of AChRs, muscarinic AChRs and nicotinic AChRs, with each class possessing several subtypes or subunits. In this review, the expression and function of AChRs in different types of MSC are summarized with the aim of highlighting how AChRs play a pivotal role in regulating MSC regenerative function.

Assessing the inflammatory response to in vitro polymicrobial wound biofilms in a skin epidermis model.

Wounds can commonly become infected with polymicrobial biofilms containing bacterial and fungal microorganisms. Microbial colonization of the wound can interfere with sufficient healing and repair, leading to high rates of chronicity in certain individuals, which can have a huge socioeconomic burden worldwide. One route for alleviating biofilm formation in chronic wounds is sufficient treatment of the infected area with topical wound washes and ointments. Thus, the primary aim here was to create a complex in vitro biofilm model containing a range of microorganisms commonly isolated from the infected wound milieu. These polymicrobial biofilms were treated with three conventional anti-biofilm wound washes, chlorhexidine (CHX), povidone-iodine (PVP-I), and hydrogen peroxide (H2O2), and efficacy against the microorganisms assessed using live/dead qPCR. All treatments reduced the viability of the biofilms, although H2O2 was found to be the most effective treatment modality. These biofilms were then co-cultured with 3D skin epidermis to assess the inflammatory profile within the tissue. A detailed transcriptional and proteomic profile of the epidermis was gathered following biofilm stimulation. At the transcriptional level, all treatments reduced the expression of inflammatory markers back to baseline (untreated tissue controls). Olink technology revealed a unique proteomic response in the tissue following stimulation with untreated and CHX-treated biofilms. This highlights treatment choice for clinicians could be dictated by how the tissue responds to such biofilm treatment, and not merely how effective the treatment is in killing the biofilm.

Recurrent Vulvovaginal Candidiasis: a Dynamic Interkingdom Biofilm Disease of Candida and Lactobacillus.

Despite the strikingly high worldwide prevalence of vulvovaginal candidiasis (VVC), treatment options for recurrent VVC (RVVC) remain limited, with many women experiencing failed clinical treatment with frontline azoles. Further, the cause of onset and recurrence of disease is largely unknown, with few studies identifying potential mechanisms of treatment failure. This study aimed to assess a panel of clinical samples from healthy women and those with RVVC to investigate the influence of Candida, the vaginal microbiome, and how their interaction influences disease pathology. 16S rRNA sequencing characterized disease by a reduction in specific health-associated Lactobacillus species, such as Lactobacillus crispatus, coupled with an increase in Lactobacillus iners. In vitro analysis showed that Candida albicans clinical isolates are capable of heterogeneous biofilm formation, and we found the presence of hyphae and C. albicans aggregates in vaginal lavage fluid. Additionally, the ability of Lactobacillus to inhibit C. albicans biofilm formation and biofilm-related gene expression was demonstrated. Using RNA sequencing technology, we were able to identify a possible mechanism by which L. crispatus may contribute to re-establishing a healthy vaginal environment through amino acid acquisition from C. albicans. This study highlights the potential formation and impact of Candida biofilms in RVVC. Additionally, it suggests that RVVC is not entirely due to an arbitrary switch in C. albicans from commensal to pathogen and that understanding interactions between this yeast and vaginal Lactobacillus species may be crucial to elucidating the cause of RVVC and developing appropriate therapies. IMPORTANCE RVVC is a significant burden, both economically and for women's health, but its prevalence is poorly documented globally due to the levels of self-treatment. Identifying triggers for development and recurrence of VVC and the pathogenesis of the microbes involved could considerably improve prevention and treatment options for women with recurrent, azole-resistant cases. This study therefore aimed to examine the interkingdom dynamics from healthy women and those with RVVC using next-generation sequencing techniques and to further investigate the molecular interactions between C. albicans and L. crispatus in a relevant biofilm coculture system.

Exploring the roles of neuropeptides in trigeminal neuropathic pain: A systematic review and narrative synthesis of animal studies.

OBJECTIVE: This systematic review aims to explore the changes in expression of neuropeptides and/or their receptors following experimental trigeminal neuropathic pain in animals. DESIGN: MEDLINE, Embase, and Scopus were searched for publications up to 31st March 2021. Study selection and data extraction were completed by two independent reviewers based on the eligibility criteria. The quality of articles was judged based on the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) risk-of-bias tool. RESULTS: A total of 19 studies satisfied the eligibility criteria and were included for narrative synthesis. Methods of trigeminal neuropathic pain induction were nerve ligation, nerve compression/crush, nerve transection and dental pulp injury. Animal behaviours used for pain verification were evoked responses to mechanical and thermal stimuli. Non-evoked behaviours, including vertical exploration, grooming and food consumption, were also employed in some studies. Calcitonin gene-related peptide (CGRP) and substance P were the most frequently reported neuropeptides. Overall, unclear to high risk of bias was identified in the included studies. CONCLUSIONS: Limited evidence has suggested the pro-nociceptive role of CGRP in trigeminal neuropathic pain. In order to further translational pain research, animal models of trigeminal neuropathic pain and pain validation methods need to be optimised. Complete reporting of future studies based on available guidelines to improve confidence in research is encouraged.

Microbiome analysis of feline odontoclastic resorptive lesion (FORL) and feline oral health.

Introduction. Feline odontoclastic resorptive lesion (FORL) is one of the most common and painful oral diseases of the cat. It is characterised by tooth resorption due to destructive activity of odontoclasts. FORL can result in tooth loss. While the aetiology of FORL is not clearly understood, it is thought to be multifactorial and bacteria are likely to play a major role.Hypothesis. Dysbiosis of the normal feline oral microbiota leads to an alteration in commensal bacteria populations, which results in the development of FORL.Aim. The purpose of the current study was to determine the composition of the microbiomes associated with feline oral health and FORL.Methodology. Supragingival plaque was collected from 25 cats with a healthy oral cavity and 40 cats with FORL. DNA was extracted from each sample, the V4 region of the 16S rRNA gene amplified by polymerase chain reaction and amplicons sequenced. Diversity and species richness analyses were performed, principal component analysis was used to explore differences between the oral microbiomes of healthy cats and those with FORL, and linear discriminant analysis effect size was used to assess differences between the groups.Results. The six most abundant bacterial genera identified were Bergeyella, Capnocytophaga, Lampropedia, Morexella, Porphyromonas and Treponema. Two-step cluster analysis of the data identified two FORL sub-groups (FORL-1, FORL-2). The FORL-2 sub-group was very similar to the healthy group, whilst the FORL-1 sub-group was clearly different from both the FORL-2 sub-group and the healthy groups. In this analysis, Capnocytophaga (P <0.001) and Lampropedia (P <0.01) were found at significantly lower levels and Porphyromonas at a slightly higher level in the FORL-1 sub-group compared to the healthy and FORL-2 sub-groups. Microbial diversity was found to be less in the FORL-1 sub-group than in the healthy group. Lampropedia sp., a phosphate-accumulating oral commensal species, was significantly lower in the FORL-1 sub-group.Conclusion. The oral microbiota associated with the FORL-1 sub-group is distinct from that found in the healthy group and FORL-2 sub-group. Lampropedia species may influence the local calcium-phosphate ratio, which could be a factor in tooth and bone resorption observed in FORL.

Evaluating aerosol and splatter following dental procedures: Addressing new challenges for oral health care and rehabilitation.

BACKGROUND: Dental procedures often produce aerosol and splatter which have the potential to transmit pathogens such as SARS-CoV-2. The existing literature is limited. OBJECTIVE(S): To develop a robust, reliable and valid methodology to evaluate distribution and persistence of dental aerosol and splatter, including the evaluation of clinical procedures. METHODS: Fluorescein was introduced into the irrigation reservoirs of a high-speed air-turbine, ultrasonic scaler and 3-in-1 spray, and procedures were performed on a mannequin in triplicate. Filter papers were placed in the immediate environment. The impact of dental suction and assistant presence were also evaluated. Samples were analysed using photographic image analysis and spectrofluorometric analysis. Descriptive statistics were calculated and Pearson's correlation for comparison of analytic methods. RESULTS: All procedures were aerosol and splatter generating. Contamination was highest closest to the source, remaining high to 1-1.5 m. Contamination was detectable at the maximum distance measured (4 m) for high-speed air-turbine with maximum relative fluorescence units (RFU) being: 46,091 at 0.5 m, 3,541 at 1.0 m and 1,695 at 4 m. There was uneven spatial distribution with highest levels of contamination opposite the operator. Very low levels of contamination (≤0.1% of original) were detected at 30 and 60 minutes post-procedure. Suction reduced contamination by 67-75% at 0.5-1.5 m. Mannequin and operator were heavily contaminated. The two analytic methods showed good correlation (r = 0.930, n = 244, P < .001). CONCLUSION: Dental procedures have potential to deposit aerosol and splatter at some distance from the source, being effectively cleared by 30 minutes in our setting.

Detection, treatment and prevention of endodontic biofilm infections: what's new in 2020?

Endodontic disease, a biofilm infection of the root canal space, is a significant cause of dental morbidity worldwide. Endodontic treatment, or root canal treatment, as it is commonly known is founded on the ability to eradicate microbial biofilm infection and prevent re-infection of the highly complex root canal space. Despite many "advances" in clinical endodontics we have seen little improvement in outcomes. The aim of this critical review paper is to provide a contemporary view of endodontic microbiology and biofilm polymicrobiality, provide an understanding of the host response, and how together these impact upon clinical treatment. Ultimately, it is intended to provide insight into novel opportunities and strategies for the future diagnostics, treatment, and prevention of endodontic disease.

Effects of smoking on non-surgical periodontal therapy in patients with periodontitis Stage III or IV, and Grade C.

BACKGROUND: To evaluate possible effects of smoking on clinical, biochemical, and microbiological outcomes of non-surgical periodontal treatment in patients with periodontitis Stage III or IV and Grade C. METHODS: Conventional quadrant-wise non-surgical periodontal treatment was performed and whole-mouth periodontal measurements were recorded at baseline, 1, 3, and 6 months after completion of treatment. Saliva, gingival crevicular fluid, subgingival plaque, and blood samples were obtained at the same time points. Inflammatory cytokine levels, presence, and quantities of 11 different bacterial species were determined. Smoking status was validated by cotinine assay. RESULTS: Fourteen smoker and 13 non-smoker patients completed the study protocol and revealed similar clinical findings except for the higher plaque scores in the non-smokers at 6 months (P <0.01). Significant differences were found between the study groups in biofluid cytokine levels at 1 and 3 months (P <0.01). Gram-negative bacteria were more abundant in the smokers at baseline and so were Gram-positive bacteria in the non-smokers (P <0.01). Gram-negative bacteria repopulated in the smokers faster than in the non-smokers (P <0.01). CONCLUSIONS: The present findings suggest that smoker patients with periodontitis Stage III and IV, Grade C respond well to the non-surgical periodontal treatment during the 6-month follow-up. However, smokers exhibit faster repopulation of Gram-negative bacteria.

Candida albicans Biofilm Heterogeneity and Tolerance of Clinical Isolates: Implications for Secondary Endodontic Infections.

AIM: Endodontic infections are caused by the invasion of various microorganisms into the root canal system. Candida albicans is a biofilm forming yeast and the most prevalent eukaryotic microorganism in endodontic infections. In this study we investigated the ability of C. albicans to tolerate treatment with standard endodontic irrigants NaOCl (sodium hypochlorite), ethylenediaminetetraacetic acid (EDTA) and a combination thereof. We hypothesized that biofilm formed from a panel of clinical isolates differentially tolerate disinfectant regimens, and this may have implications for secondary endodontic infections. METHODOLOGY: Mature C. albicans biofilms were formed from 30 laboratory and oral clinical isolates and treated with either 3% NaOCl, 17% EDTA or a sequential treatment of 3% NaOCl followed by 17% EDTA for 5 min. Biofilms were then washed, media replenished and cells reincubated for an additional 24, 48 and 72 h at 37 °C. Regrowth was quantified using metabolic reduction, electrical impedance, biofilm biomass and microscopy at 0, 24, 48 and 72 h. RESULTS: Microscopic analysis and viability readings revealed a significant initial killing effect by NaOCl, followed by a time dependent significant regrowth of C. albicans, but with inter-strain variability. In contrast to NaOCl, there was a continuous reduction in viability after EDTA treatment. Moreover, EDTA significantly inhibited regrowth after NaOCl treatment, though viable cells were still observed. CONCLUSIONS: Our results indicate that different C. albicans biofilm phenotypes grown in a non-complex surface topography have the potential to differentially tolerate standard endodontic irrigation protocols. This is the first study to report a strain dependent impact on efficacy of endodontic irrigants. Its suggested that within the complex topography of the root canal, a more difficult antimicrobial challenge, that existing endodontic irrigant regimens permit cells to regrow and drive secondary infections.

Interkingdom interactions on the denture surface: Implications for oral hygiene.

BACKGROUND: Evidence to support the role of Candida species in oral disease is limited. Often considered a commensal, this opportunistic yeast has been shown to play a role in denture related disease, though whether it is an active participant or innocent bystander remains to be determined. This study sought to understand the role of Candida species alongside the bacterial microbiome in a denture patient cohort, exploring how the microbiology of the denture was affected by oral hygiene practices. MATERIALS AND METHODS: In vitro denture cleansing studies were performed on a complex 9-species interkingdom denture biofilm model, with quantitative assessment of retained bacterial and fungal viable bioburdens. Patient hygiene measures were also collected from 131 patients, including OHIP, frequency of denture cleansing, oral hygiene measure and patient demographics. The bacterial microbiome was analysed from each patient, alongside quantitative PCR assessment of ITS (fungal) and 16S (bacterial) bioburden from denture, mucosa and intact dentition. RESULTS: It was shown that following in vitro denture cleansing C. albicans were unresponsive to treatment, whereas bacterial biofilms could repopulate 100-fold, but were susceptible to subsequent treatment. Within the patient cohort, oral hygiene did not impact candidal or bacterial composition, nor diversity. The levels of Candida did not significantly influence the bacterial microbiome, though an observed gradient was suggestive of a microbial composition change in response to Candida load, indicating interkingdom interaction rather than an oral hygiene effect. Indeed, correlation analysis was able to show significant correlations between Candida species and key genera (Lactobacillus, Scardovia, Fusobacterium). CONCLUSIONS: Overall, this study has shown that the denture microbiome/mycobiome is relatively resilient to oral hygiene challenges, but that Candida species have potential interactions with key oral genera. These interactions may have a bearing on shaping community structure and a shift from health to disease when the opportunity arises.

Clinical associations between acetylcholine levels and cholinesterase activity in saliva and gingival crevicular fluid and periodontal diseases.

AIM: The oral mucosa possesses a non-neuronal cholinergic system. This study aimed to determine clinical evidence for a role of cholinergic mechanisms in the pathogenesis of periodontal diseases. MATERIALS AND METHODS: Fifty healthy participants, 52 patients with gingivitis and 49 with periodontitis were recruited. Full periodontal parameters were recorded and saliva and gingival crevicular fluid (GCF) collected. Levels of acetylcholine and inflammatory mediators were quantified using commercially available assay kits. Acetylcholinesterase and butyrylcholinesterase activities were measured using a published biochemical assay. RESULTS: Acetylcholine levels are significantly elevated in saliva and GCF, whereas GCF levels of butyrylcholinesterase activity are significantly decreased, in patients with periodontal diseases. Acetylcholine levels in saliva and GCF correlated positively with clinical markers of disease severity and with increased levels of IL-17A and IL-17F. In contrast, butyrylcholinesterase activity levels in GCF showed significant negative correlations with clinical markers of disease severity and IL-17A and IL-17F levels. None of the findings were due to smoking. CONCLUSIONS: Elevated acetylcholine levels and reduced butyrylcholinesterase activity are clinically associated with periodontal diseases and elevated levels of IL-17A and IL-17F. Therefore, non-neuronal cholinergic mechanisms may influence IL-17 biology and the aetiopathogenesis of periodontal diseases and therefore are possible therapeutic targets.

Nanoimprinting of biomedical polymers reduces candidal physical adhesion.

Management of fungal biofilms represents a significant challenge to healthcare. As a preventive approach, minimizing adhesion between indwelling medical devices and microorganisms would be an important step forward. This study investigated the anti-fouling capacity of engineered nanoscale topographies to the pathogenic yeast Candida albicans. Highly ordered arrays of nano-pit topographies were shown to significantly reduce the physical adherence capacity of C. albicans. This study shows a potential of nanoscale patterns to inhibit and prevent pathogenic biofilm formation on biomedical substrates.

The Anti-Adhesive Effect of Curcumin on Candida albicans Biofilms on Denture Materials.

The use of natural compounds as an alternative source of antimicrobials has become a necessity given the growing concern over global antimicrobial resistance. Polyphenols, found in various edible plants, offers one potential solution to this. We aimed to investigate the possibility of using curcumin within the context of oral health as a way of inhibiting and preventing the harmful development of Candida albicans biofilms. We undertook a series of adsorption experiments with varying concentrations of curcumin, showing that 50 µg/ml could prevent adhesion. This effect could be further synergized by the curcumin pre-treatment of yeast cells to obtain significantly greater inhibition (>90%, p < 0.001). Investigation of the biological impact of curcumin showed that it preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that key adhesins were down-regulated (ALS1 and ALS3), whereas aggregation related genes (ALS5 and AAF1) were up-regulated. Collectively, these data demonstrated that curcumin elicits anti-adhesive effects and that induces transcription of genes integrally involved in the processes related to biofilm formation. Curcumin and associated polyphenols therefore have the capacity to be developed for use in oral healthcare to augment existing preventative strategies for candidal biofilms on the denture surface.

Biomarkers and Bacteria Around Implants and Natural Teeth in the Same Individuals.

BACKGROUND: This cross-sectional study assesses cytokine levels in peri-implant crevicular fluid (PICF)/gingival crevicular fluid (GCF) and a selection of subgingival/submucosal plaque bacteria from clinically healthy or diseased sites in the same individuals. METHODS: Samples from 97 implants/teeth (58 implants [19 healthy, 20 mucositis, 19 peri-implantitis] and 39 natural teeth [19 healthy, 12 gingivitis, eight periodontitis] in 15 systemically healthy patients were investigated by immunoassay and real-time polymerase chain reaction. Samples were obtained first, with probing depth, clinical attachment level, bleeding on probing, plaque index scores, and keratinized tissue width then recorded. Data were analyzed by Wilcoxon, Mann-Whitney U, and permutation tests on dependent, independent, and mixed dependent and independent samples and Spearman correlation. RESULTS: Interleukin (IL)-1ß levels were significantly higher in PICF samples of healthy implants than in GCF samples of healthy teeth (P = 0.003), and soluble receptor activator of nuclear factor-κB ligand (sRANKL) concentrations were significantly higher in the gingivitis than the mucositis group (P = 0.004). Biomarker levels were similar in peri-implantitis and periodontitis groups (P >0.05). Actinomyces naeslundi and Streptococcus oralis levels were significantly higher in the healthy implant group than in healthy teeth (P <0.05). Prevotella intermedia and Treponema denticola (Td) levels were lower in the mucositis group than the gingivitis group (P <0.05). Prevotella oralis and S. oralis levels were significantly higher in the periodontitis group (P <0.05), and Td levels were significantly higher in the peri-implantitis group (P <0.05). CONCLUSION: There were many similarities but, crucially, some differences in biomarker levels (IL-1ß and sRANKL) and bacterial species between peri-implant and periodontal sites in the same individuals, suggesting similar pathogenic mechanisms.

Candida albicans biofilm heterogeneity does not influence denture stomatitis but strongly influences denture cleansing capacity.

Approximately 20 % of the UK population wear some form of denture prosthesis, resulting in denture stomatitis in half of these individuals. Candida albicans is primarily attributed as the causative agent, due to its biofilm -forming ability. Recently, there has been increasing evidence of C. albicans biofilm heterogeneity and the negative impact it can have clinically; however, this phenomenon has yet to be studied in relation to denture isolates. The aims of this study were to evaluate C. albicans biofilm formation of clinical denture isolates in a denture environment and to assess antimicrobial activity of common denture cleansers against these tenacious communities. C. albicans isolated from dentures of healthy and diseased individuals was quantified using real-time PCR and biofilm biomass assessed using crystal violet. Biofilm development on the denture substratum poly(methyl methacrylate), Molloplast B and Ufi-gel was determined. Biofilm formation was assessed using metabolic and biomass stains, following treatment with denture hygiene products. Although C. albicans was detected in greater quantities in diseased individuals, it was not associated with increased biofilm biomass. Denture substrata were shown to influence biofilm biomass, with poly(methyl methacrylate) providing the most suitable environment for C. albicans to reside. Of all denture hygiene products tested, Milton had the most effective antimicrobial activity, reducing biofilm biomass and viability the greatest. Overall, our results highlight the complex nature of denture- related disease, and disease development cannot always be attributed to a sole cause. It is the distinct combination of various factors that ultimately determines the pathogenic outcome.

Viable Compositional Analysis of an Eleven Species Oral Polymicrobial Biofilm.

PURPOSE: Polymicrobial biofilms are abundant in clinical disease, particularly within the oral cavity. Creating complex biofilm models that recapitulate the polymicrobiality of oral disease are important in the development of new chemotherapeutic agents. In order to do this accurately we require the ability to undertake compositional analysis, in addition to determine individual cell viability, which is difficult using conventional microbiology. The aim of this study was to develop a defined multispecies denture biofilm model in vitro, and to assess viable compositional analysis following defined oral hygiene regimens. METHODS: An in vitro multispecies denture biofilm containing various oral commensal and pathogenic bacteria and yeast was created on poly (methyl methacrylate) (PMMA). Denture hygiene regimens tested against the biofilm model included brushing only, denture cleansing only and combinational brushing and denture cleansing. Biofilm composition and viability were assessed by culture (CFU) and molecular (qPCR) methodologies. Scanning electron microscopy and confocal laser scanning microscopy were also employed to visualize changes in denture biofilms following treatment. RESULTS: Combinational treatment of brushing and denture cleansing had the greatest impact on multispecies denture biofilms, reducing the number of live cells by more than 2 logs, and altering the overall composition in favor of streptococci. This was even more evident during the sequential testing, whereby daily sequential treatment reduced the total and live number of bacteria and yeast more than those treated intermittently. Bacteria and yeast remaining following treatment tended to aggregate in the pores of the PMMA, proving more difficult to fully eradicate the biofilm. CONCLUSIONS: Overall, we are the first to develop a method to enable viable compositional analysis of an 11 species denture biofilm following chemotherapeutic challenge. We were able to demonstrate viable cell reduction and changes in population dynamics following evaluation of various denture cleansing regimens. Specifically, it was demonstrated that daily combinational treatment of brushing and cleansing proved to be the most advantageous denture hygiene regimen, however, residual organisms still remained within the pores of PMMA surface, which could act as a reservoir for further biofilm regrowth. We have identified an industry need for denture cleansing agents with the capacity to penetrate these pores and disaggregate these complex biofilm consortia.

The Oral Microbiome of Denture Wearers Is Influenced by Levels of Natural Dentition.

OBJECTIVES: The composition of dental plaque has been well defined, whereas currently there is limited understanding of the composition of denture plaque and how it directly influences denture related stomatitis (DS). The aims of this study were to compare the microbiomes of denture wearers, and to understand the implications of these towards inter-kingdom and host-pathogen interactions within the oral cavity. METHODS: Swab samples were obtained from 123 participants wearing either a complete or partial denture; the bacterial composition of each sample was determined using bar-coded illumina MiSeq sequencing of the bacterial hypervariable V4 region of 16S rDNA. Sequencing data processing was undertaken using QIIME, clustered in Operational Taxonomic Units (OTUs) and assigned to taxonomy. The dentures were sonicated to remove the microbial flora residing on the prosthesis, sonicate was then cultured using diagnostic colorex Candida media. Samples of unstimulated saliva were obtained and antimicrobial peptides (AMP) levels were measured by ELISA. RESULTS: We have shown that dental and denture plaques are significantly distinct both in composition and diversity and that the oral microbiome composition of a denture wearer is variable and is influenced by the location within the mouth. Dentures and mucosa were predominantly made up of Bacilli and Actinobacteria. Moreover, the presence of natural teeth has a significant impact on the overall microbial composition, when compared to the fully edentulous. Furthermore, increasing levels of Candida spp. positively correlate with Lactobacillus spp. AMPs were quantified, though showed no specific correlations. CONCLUSIONS: This is the first study to provide a detailed understanding of the oral microbiome of denture wearers and has provided evidence that DS development is more complex than simply a candidal infection. Both fungal and bacterial kingdoms clearly play a role in defining the progression of DS, though we were unable to show a defined role for AMPs.