The relationship between sphingosine-1-phosphate receptor 2 and epidermal growth factor in migration and invasion of oral squamous cell carcinoma.

Sphingosine-1-phosphate (S1P) is a lipid mediator and its binding to the S1P receptor 2 (S1PR2) is reported to regulate cytoskeletal organization. Epidermal growth factor (EGF) has been shown to induce migration and invasion in tumour cells. Since binding of S1P to S1PR2 and EGF to the EGF receptors exhibit some overlapping functionality, this study aimed to determine whether S1PR2 was involved in EGF-induced migration and invasion of oral squamous cell carcinoma (OSCC) lines and to identify any potential crosstalk between the two pathways. Migration was investigated using the scratch wound assay while invasion was studied using the transwell invasion and multicellular tumour spheroid (MCTS) assays. Activity of Rac1, a RhoGTPase, was measured using G-LISA (small GTPase activation assays) while S1P production was indirectly measured via the expression of sphingosine kinase (Sphk). S1PR2 inhibition with 10 µM JTE013 reduced EGF-induced migration, invasion and Rac1 activity, however, stimulation of S1PR2 with 10 µM CYM5478 did not enhance the effect of EGF on migration, invasion or Rac1 activity. The data demonstrated a crosstalk between EGF/EGFR and S1P/S1PR2 pathways at the metabolic level. S1PR2 was not involved in EGF production, but EGF promoted S1P production through the upregulation of Sphk1. In conclusion, OSCC lines could not migrate and invade without S1PR2 regulation, even with EGF stimulation. EGF also activated S1PR2 by stimulating S1P production via Sphk1. The potential for S1PR2 to control cellular motility may lead to promising treatments for OSCC patients and potentially prevent or reduce metastasis.

Salivary E-cadherin as a biomarker for diagnosis and predicting grade of periodontitis.

OBJECTIVES: To determine the abilities of salivary E-cadherin to differentiate between periodontal health and periodontitis and to discriminate grades of periodontitis. BACKGROUND: E-cadherin is the main protein responsible for maintaining the integrity of epithelial-barrier function. Disintegration of this protein is one of the events associated with the destructive forms of periodontal disease leading to increase concentration of E-cadherin in the oral biofluids. MATERIALS AND METHODS: A total of 63 patients with periodontitis (case) and 35 periodontally healthy subjects (control) were included. For each patient, periodontal parameters including bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level (CAL) were recorded. Concentration of salivary E-cadherin was determined by ELISA. Receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to determine the diagnostic potentials of E-cadherin. RESULTS: Level of salivary E-cadherin was significantly higher in periodontitis cases than controls. The ROC analysis showed that salivary E-cadherin exhibits excellent sensitivity and specificity (AUC 1.000) to differentiate periodontal health from periodontitis with a cutoff concentration equal to 1.325 ng/mL. The AUCs of E-cadherin to differentiate grade A from grade B and C periodontitis were 0.731 (cutoff point = 1.754 ng/mL) and 0.746 (cutoff point = 1.722 ng/mL), respectively. However, the AUC of salivary E-cadherin to differentiate grade B from grade C periodontitis was lower (0.541). Additionally, BOP and PPD were significantly and positively correlated with the concentration of salivary E-cadherin. CONCLUSION: Salivary E-cadherin exhibited excellent sensitivity and specificity to differentiate periodontitis from a healthy periodontium. The level of accuracy of E-cadherin was also sufficient to recognize grade A periodontitis from grade B and C periodontitis.

Gingival tissue samples from periodontitis patients demonstrate epithelial-mesenchymal transition phenotype.

OBJECTIVE: To determine the expression of key epithelial-mesenchymal transition (EMT) markers in gingival tissue samples collected from patients with periodontitis. BACKGROUND: Epithelial-mesenchymal transition is a process responsible for shifting epithelial-phenotype to mesenchymal-phenotype leading to loss of epithelial-barrier function. Thus, EMT could be involved as a pathogenic mechanism in periodontitis as both conditions share common promoters and signalling pathways. MATERIALS AND METHODS: Gingival tissue samples were collected from patients with periodontitis (case) and healthy periodontium (control). Periodontal parameters including bleeding on probing, probing pocket depth (PPD), and clinical attachment loss were recorded. Paraffinized tissue samples were processed and immunohistochemically stained to determine the expression of key EMT markers which included E-cadherin, ß-catenin, Snail1 and vimentin. RESULTS: The majority of cases (n = 65, 72.2%) were diagnosed with periodontitis stage 3 or 4, grade b or c vs 25 (27.8%) subjects with intact healthy periodontium. Discontinuity of epithelium was detected in up to 80.9% of periodontitis cases associated with reduced number of epithelial layers as compared to controls. Immunohistochemical expression of epithelial markers (E-cadherin and ß-catenin) was significantly downregulated in periodontitis patients as compared with controls. Periodontitis cases exhibited significant upregulation of Snail1 expression. Furthermore, cytoplasmic vimentin (66.2%) and nuclear ß-catenin (27.7%) were solely expressed in periodontally diseased tissues compared with control. Epithelial markers, E-cadherin and ß-catenin, were significantly negatively correlated with increasing PPD, while vimentin showed positive correlation with this parameter. CONCLUSION: There were marked downregulation of epithelial molecules and upregulation of mesenchymal markers in gingival tissues derived from periodontitis patients, suggesting expression of the EMT phenotype in the pathological epithelial lining of periodontal pockets.

Inflammasome dysregulation in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: Uncontrolled production of Interleukin-1ß (IL-1ß), a major proinflammatory cytokine, is associated with tissue destruction in periodontal disease. IL-1ß production is controlled by inflammasomes which are multiprotein regulatory complexes. The current study aimed to elucidate potential regulatory pathways by monitoring the effects of periodontal pathogens Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) on inflammasomes and their regulators in human gingival fibroblasts (HGFs) in vitro. METHODS: HGFs were exposed to Fn and Pg alone or in combination for 24 hr at a multiplicity of infection of 100, ±30 min exposure with 5 mM adenosine triphosphate (ATP) incubation. Gene expression of NLRP3 and AIM2, inflammasome regulatory proteins POP1, CARD16 and TRIM16, and inflammasome components ASC and CASPASE 1, and IL-1ß, were evaluated by RT-PCR. Pro- and mature IL-1ß levels were monitored intracellularly by immunocytochemistry and extracellularly by ELISA. RESULTS: Fn + ATP significantly upregulated NLRP3, AIM2, IL-1ß, ASC, and CASPASE 1; however, it downregulated POP1 and TRIM16. Pg + ATP downregulated NLRP3, ASC, POP1, but upregulated IL-1ß and CARD16. Pg + Fn+ATP significantly upregulated AIM2, IL-1ß and CARD16, and downregulated POP1, TRIM16, and CASPASE 1. Pg + ATP exposure significantly increased pro- and mature IL-1ß production. CONCLUSION: Bacterial exposure with ATP may deregulate IL-1ß by dysregulating inflammasomes and their regulators in HGFs.

Photobiomodulation of mineralisation in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) and photobiomodulation (PBM) both offer significant therapeutic potential in regenerative medicine. MSCs have the ability to self-renew and differentiate; giving rise to multiple cellular and tissue lineages that are utilised in repair and regeneration of damaged tissues. PBM utilises light energy delivered at a range of wavelengths to promote wound healing. The positive effects of light on MSC proliferation are well documented; and recently, several studies have determined the outcomes of PBM on mineralised tissue differentiation in MSC populations. As PBM effects are biphasic, it is important to understand the underlying cellular regulatory mechanisms, as well as, provide accurate details of the irradiation conditions, to optimise and standardise outcomes. This review article focuses on the use of red, near-infra-red (R/NIR) and blue wavelengths to promote the mineralisation potential of MSCs; and also reports on the possible molecular mechanisms which underpin transduction of these effects. A variety of potential photon absorbers have been identified which are reported to mediate the signalling mechanisms, including respiratory chain enzymes, flavins, and cryptochromes. Studies report that R/NIR and blue light stimulate MSC differentiation by enhancing respiratory chain activity and increasing reactive oxygen species levels; however, currently, there are considerable variations between irradiation parameters reported. We conclude that due to its non-invasive properties, PBM may, following optimisation, provide an efficient therapeutic approach to clinically support MSC-mediated hard tissue repair. However, to optimise application, further studies are required to identify appropriate light delivery parameters, as well as elucidate the photo-signalling mechanisms involved.

Inflammasomes and their regulation in periodontal disease: A review.

Interleukin-1ß (IL-1ß), which is secreted by host tissues leading to periodontal tissue inflammation, is a major pro-inflammatory cytokine in the pathogenesis of periodontal disease. The conversion of pro-IL-1ß into its biologically active form is controlled by multiprotein complexes named as inflammasomes, which are key regulator of host defense mechanisms and inflammasome involved diseases, including the periodontal diseases. Inflammasomes are regulated by different proteins and processes, including pyrin domain (PYD)-only proteins (POPs), CARD-only proteins (COPs), tripartite motif family proteins (TRIMs), autophagy, and interferons. A review of in vitro, in vivo, and clinical data from these publications revealed that several inflammasomes including (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) have been found to be involved in periodontal disease pathogenesis. To the best of our knowledge, the current article provides the first review of the literature focusing on studies that evaluated both inflammasomes and their regulators in periodontal disease. An upregulation for inflammasomes and a downregulation of inflammasome regulator proteins including POPs, COPs, and TRIMs have been reported in periodontal disease. Although interferons (types I and II) and autophagy have been found to be involved in periodontal disease, their possible role in inflammasome activation has not evaluated yet. Modulating the excessive inflammatory response by the use of inflammasome regulators may have potential in the management of periodontal disease.

Under the spotlight: mechanisms of photobiomodulation concentrating on blue and green light.

Photobiomodulation (PBM) describes the application of light at wavelengths ranging from 400-1100 nm to promote tissue healing, reduce inflammation and promote analgesia. Traditionally, red and near-infra red (NIR) light have been used therapeutically, however recent studies indicate that other wavelengths within the visible spectrum could prove beneficial including blue and green light. This review aims to evaluate the literature surrounding the potential therapeutic effects of PBM with particular emphasis on the effects of blue and green light. In particular focus is on the possible primary and secondary molecular mechanisms of PBM and also evaluation of the potential effective parameters for application both in vitro and in vivo. Studies have reported that PBM affects an array of molecular targets, including chromophores such as signalling molecules containing flavins and porphyrins as well as components of the electron transport chain. However, secondary mechanisms tend to converge on pathways induced by increases in reactive oxygen species (ROS) production. Systematic evaluation of the literature indicated 72% of publications reported beneficial effects of blue light and 75% reported therapeutic effects of green light. However, of the publications evaluating the effects of green light, reporting of treatment parameters was uneven with 41% failing to report irradiance (mW cm-2) and 44% failing to report radiant exposure (J cm-2). This review highlights the potential of PBM to exert broad effects on a range of different chromophores within the body, dependent upon the wavelength of light applied. Emphasis still remains on the need to report exposure and treatment parameters, as this will enable direct comparison between different studies and hence enable the determination of the full potential of PBM.

Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria.

Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii, stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated.

Periodontitis prevalence and serum antibody reactivity to periodontal bacteria in primary Sjögren's syndrome: a pilot study.

AIMS: The aims of this study were as follows: (i) To assess the prevalence of periodontitis among patients with primary Sjögren's syndrome (pSS) and comparator groups of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). (ii) To perform a pilot study to compare serum antibody responses to 10 oral/periodontal bacteria in these patient groups and a historical comparator group of patients with periodontitis. MATERIALS AND METHODS: Standard clinical periodontal assessments were performed on 39 pSS, 36 RA and 23 OA patients and "In-house" antibody ELISAs for serum antibodies against 10 oral/periodontal bacteria were performed in these groups. RESULTS: Forty-six percent of the pSS group, 64% of the RA group and 48% of the OA group had moderate/severe periodontitis. These frequencies did not reach statistical significance between groups. Raised antibody levels to Prevotella denticola were found in the pSS, RA and periodontitis groups compared to the OA group. Significant between group differences were seen for Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Campylobacter showae. None of these differences were specifically associated with pSS. CONCLUSION: This study showed no increase in periodontitis in pSS patients. Although the P. denticola data are of interest, identifying bacterial triggering factors for pSS will likely require alternative strategies including modern techniques such as microbiome analysis.

The dark art of light measurement: accurate radiometry for low-level light therapy.

Lasers and light-emitting diodes are used for a range of biomedical applications with many studies reporting their beneficial effects. However, three main concerns exist regarding much of the low-level light therapy (LLLT) or photobiomodulation literature; (1) incomplete, inaccurate and unverified irradiation parameters, (2) miscalculation of 'dose,' and (3) the misuse of appropriate light property terminology. The aim of this systematic review was to assess where, and to what extent, these inadequacies exist and to provide an overview of 'best practice' in light measurement methods and importance of correct light measurement. A review of recent relevant literature was performed in PubMed using the terms LLLT and photobiomodulation (March 2014-March 2015) to investigate the contemporary information available in LLLT and photobiomodulation literature in terms of reporting light properties and irradiation parameters. A total of 74 articles formed the basis of this systematic review. Although most articles reported beneficial effects following LLLT, the majority contained no information in terms of how light was measured (73%) and relied on manufacturer-stated values. For all papers reviewed, missing information for specific light parameters included wavelength (3%), light source type (8%), power (41%), pulse frequency (52%), beam area (40%), irradiance (43%), exposure time (16%), radiant energy (74%) and fluence (16%). Frequent use of incorrect terminology was also observed within the reviewed literature. A poor understanding of photophysics is evident as a significant number of papers neglected to report or misreported important radiometric data. These errors affect repeatability and reliability of studies shared between scientists, manufacturers and clinicians and could degrade efficacy of patient treatments. Researchers need a physicist or appropriately skilled engineer on the team, and manuscript reviewers should reject papers that do not report beam measurement methods and all ten key parameters: wavelength, power, irradiation time, beam area (at the skin or culture surface; this is not necessarily the same size as the aperture), radiant energy, radiant exposure, pulse parameters, number of treatments, interval between treatments and anatomical location. Inclusion of these parameters will improve the information available to compare and contrast study outcomes and improve repeatability, reliability of studies.

Evaluation of the role of mitofusin-1 and mitofusin-2 in periodontal disease.

BACKGROUND: Mitochondria and endoplasmic reticulum are key cellular organelles and create contact sites (mitochondria-endoplasmic reticulum contact [MERC]), which plays a major role in calcium metabolism, apoptotic processes, and inflammation. Previously, proteins that have been associated with these MERC contact sites mitofusin-1 (MFN1) and mitofusin-2 (MFN2) have been found to be downregulated in periodontal disease in vitro. Therefore, the aim of the current study was to evaluate MFN1 and MFN2 in gingival crevicular fluid (GCF) of patients with periodontal disease compared with healthy controls clinically. METHODS: A total of 48 participants were divided into three groups including periodontally healthy (n = 16), patients with gingivitis (n = 16), and patients with stage 3 grade B periodontitis (n = 16). GCF levels of MFN1, MFN2, calcium (Ca), caspase-1, and tumor necrosis factor-alpha (TNF-α) were determined via enzyme-linked immunosorbent assay (ELISA). Results were calculated as total amount and concentration. RESULTS: MFN1 levels (total amount) were significantly higher in patients with periodontitis and gingivitis when compared with healthy controls (p < 0.05). However, concentration levels of MFN1, MFN2, Ca, caspase-1, TNF-α significantly decreased in periodontal disease groups compared with healthy controls (p < 0.05). A positive correlation was detected among all evaluated markers (p < 0.05). CONCLUSION: The MERC protein MFN1 may have a role in the pathogenesis of periodontal disease due to its increase in GCF of patients with periodontitis and gingivitis.

Exposure to blue light reduces antimicrobial resistant Pseudomonas aeruginosa isolated from dog ear infections.

Introduction: Pseudomonas aeruginosa is a leading cause of canine otitis externa. Enrofloxacin is often applied topically to treat this condition, although recalcitrant and recurring infections are common. There is evidence that exposure to blue light (400-470 nm) has a bactericidal effect on P. aeruginosa and other microorganisms. Methods: In the present study, we tested the biocidal effect of blue light (375-450 nm), alone or in combination with enrofloxacin, against six isolates of P. aeruginosa from dogs with otitis externa (5 of which were resistant to enrofloxacin). Results: Treatment of planktonic cell cultures with blue light resulted in significant (p < 0.5) reductions in Colony Forming Units (CFU) for all seven strains tested, in some cases below the limit of detection. The greatest bactericidal effect was observed following exposure to light at 405 nm wavelength (p < 0.05). Exposure to blue light for 20 min usually resulted in a greater reduction in Pseudomonas aeruginosa than enrofloxacin treatment, and combination treatment typically resulted in the largest reductions in CFU. Analysis of the genome sequences of these strains established that enrofloxacin resistance was likely the result of a S466F substitution in GyrB. However, there was no clear association between genotype and susceptibility to blue light treatment. Discussion: These results suggest that blue light treatment, particularly at 405 nm wavelength, and especially in combination with enrofloxacin therapy, could be an effective treatment for otherwise recalcitrant canine otitis externa caused by Pseudomonas aeruginosa. It may also provide a way of extending the usefulness of enrofloxacin therapy which would otherwise be ineffective as a sole therapeutic agent.

Ceramic conversion treated titanium implant abutments with gold for enhanced antimicrobial activity.

INTRODUCTION: Peri-implantitis is an inflammatory process around dental implants that is characterised by bone loss that may jeopardize the long-term survival of osseo integrated dental implants. The aim of this study was to create a surface coating on titanium abutments that possesses cellular adhesion and anti-microbial properties as a post-implant placement strategy for patients at risk of peri-implantitis. MATERIALS AND METHODSMETHODS: Titanium alloy Grade V stubs were coated with gold particles and then subjected to ceramic conversion treatment (CCT) at 620 °C for 3, 8 and 80 h. The surface characteristics and chemistry were assessed using scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and X-ray diffraction (XRD) analysis. The leaching profile was investigated by inductively coupled plasma mass spectroscopy (ICP-MS) for all groups after 7, 14 and 28 days in contact with distilled water. A scratch test was conducted to assess the adhesion of the gold coating to the underlying titanium discs. Two bacterial species (Staphylococcus aureus (SA) & Fusobacterium nucleatum (FN)) were used to assess the antibacterial behaviour of the coated discs using a direct attachment assay test. The potential changes in surface chemistry by the bacterial species were investigated by grazing angle XRD. RESULTS: The gold pre-coated titanium discs exhibited good stability of the coating especially after immersion in distilled water and after bacterial colonisation as evident by XRD analysis. Good surface adhesion of the coating was demonstrated for gold treated discs after scratch test analysis, especially titanium, following a 3-hour (3 H) ceramic conversion treatment. All coated discs exhibited significantly improved antimicrobial properties against both tested bacterial species compared to untreated titanium discs. CONCLUSIONS: Ceramic conversion treated titanium with a pre-deposited gold layer showed improved antimicrobial properties against both SA and FN species than untreated Ti-C discs. Scratch test analysis showed good adherence properties of the coated discs the oxide layer formed is firmly adherent to the underlying titanium substrate, suggesting that this approach may have clinical efficacy for coating implant abutments.

Neutrophil superoxide production in the presence of cigarette smoke extract, nicotine and cotinine.

AIM: To determine the effect of cigarette smoke extract, nicotine and cotinine on lucigenin-detectable neutrophil superoxide production. MATERIALS & METHODS: Neutrophils from periodontally healthy individuals were treated with aqueous smoke extract, nicotine and cotinine, prior to stimulation or at the same time as stimulation with Fusobacterium nucleatum, IgG-opsonized Staphylococcus aureus and Escherichia coli Lipopolysaccharide (LPS). Superoxide generation was determined by lucigenin chemiluminescence. RESULTS: Smoke extract induced superoxide release from neutrophils (p <0.0001) in a dose-dependent manner. By contrast, superoxide generation by neutrophils in response to pathologically relevant stimuli was inhibited by pre-treatment with smoke extract (p <0.01). This inhibition did not require the continued presence of the extract. A similar reduction in stimulated superoxide production by smoke extract was detected when neutrophils were simultaneously exposed to the extract and stimuli. Nicotine and cotinine (0-10 µg/ml) had no effect on superoxide release from unstimulated or stimulated neutrophils. CONCLUSIONS: Stable water-soluble components of cigarette smoke directly induce superoxide generation by otherwise unstimulated neutrophils, but reduce superoxide responses of cells to pathologically relevant stimuli. These data suggest potential neutrophil-mediated mechanisms by which smoking may initiate and maintain oxidative stress at periodontally healthy sites and participate in disease progression, by reducing innate immune responses.

Adjunctive daily supplementation with encapsulated fruit, vegetable and berry juice powder concentrates and clinical periodontal outcomes: a double-blind RCT.

AIM: A double-blind randomized controlled trial to determine whether dietary supplementation with fruit/vegetable/berry juice powder concentrates, simultaneously with non-surgical periodontal therapy, improved 2-month treatment outcomes. METHODS: Volunteers with chronic periodontitis were randomly assigned to one of three groups: fruit/vegetable (FV), fruit/vegetable/berry (FVB) or placebo. Supplements were taken daily during non-surgical debridement and maintenance and outcomes assessed at 2, 5 and 8 months after completion. Primary outcomes were mean probing pocket depth (PPD), clinical attachment gain, % sites bleeding on probing (% BOP) at 2 months. Adherence and plasma ß-carotene were determined. RESULTS: Sixty-one nutritionally replete (by serum biochemistry) volunteers enrolled and 60 (n = 20 per arm) completed the 2-month review. Clinical outcomes improved in all groups at 2 months, with additional improvement in PPD versus placebo for FV (p < 0.03). Gingival crevicular fluid volumes diminished more in supplement groups than placebo (FVB; p < 0.05) at 2 months, but not at later times. The % BOP (5 months) and cumulative plaque scores (8 months) were lowered more in the FV group (p < 0.05). CONCLUSIONS: Adjunctive juice powder concentrates appear to improve initial pocket depth reductions in nutritionally replete patients, where plasma micronutrient bioavailability is attainable. Definitive multicentre studies in untreated and treated patients are required to ascertain the clinical significance of such changes.

Pathogenesis of periodontitis - A potential role for epithelial-mesenchymal transition.

Epithelial mesenchymal transition (EMT) is a process comprising cellular and molecular events which result in cells shifting from an epithelial to a mesenchymal phenotype. Periodontitis is a destructive chronic disease of the periodontium initiated in response to a dysbiotic microbiome, and dominated by Gram-negative bacteria in the subgingival niches accompanied by an aberrant immune response in susceptible subjects. Both EMT and periodontitis share common risk factors and drivers, including Gram-negative bacteria, excess inflammatory cytokine production, smoking, oxidative stress and diabetes mellitus. In addition, periodontitis is characterized by down-regulation of key epithelial markers such as E-cadherin together with up-regulation of transcriptional factors and mesenchymal proteins, including Snail1, vimentin and N-cadherin, which also occur in the EMT program. Clinically, these phenotypic changes may be reflected by increases in microulceration of the pocket epithelial lining, granulation tissue formation, and fibrosis. Both in vitro and in vivo data now support the potential involvement of EMT as a pathogenic mechanism in periodontal diseases which may facilitate bacterial invasion into the underlying gingival tissues and propagation of inflammation. This review surveys the available literature and provides evidence linking EMT to periodontitis pathogenesis.

A 3D Printed Device for In Vitro Generation of Stratified Epithelia at the Air-Liquid Interface.

Air-liquid interface (ALI) cultures are used to produce stratified epithelial tissues in vitro, notably for the production of oral mucosal equivalents. Currently, there are few purpose-built devices, which aim to enhance the ease and reproducibility of generating such tissue. Most ALI cultures utilize stainless steel grids or cell culture inserts to elevate the matrix or scaffold to the surface of the culture media. In this study, a novel buoyant epithelial culture device (BECD) was designed to both contain a fibroblast-seeded collagen hydrogel and float in culture media, thereby automatically maintaining the ALI without further user intervention. BECDs aim to mitigate several issues associated with ALI culture; reducing the chance of media flooding the epithelial layer from physical disturbance, reducing technique sensitivity for less-experienced users, and improving the reproducibility of the epithelia generated. H400 oral squamous cell carcinoma cells cultured in BECDs for 7, 14, and 21 days showed continuous increase in epithelial tissue thickness with expected localization of epithelial differentiation markers: cytokeratin 5, involucrin, and E-cadherin. Fused filament fabrication three-dimensional printing with polypropylene used in BECD production allows for rapid turnover and design iteration, presenting a versatile, adaptable, and useful tool for application in in vitro cell culture.

Effect of nicotine, cotinine and cigarette smoke extract on the neutrophil respiratory burst.

AIMS: To determine the effect of nicotine, cotinine and cigarette smoke extract (CSE) on the neutrophil respiratory burst and their effect on activation of the nuclear factor-κB (NFκB) pathway in oral epithelium. MATERIALS AND METHODS: Neutrophils from periodontally healthy individuals were treated with nicotine, cotinine and CSE before stimulation with Fusobacterium nucleatum, IgG-opsonized Staphylococcus aureus and Escherichia coli lipopolysaccharide. Total and extracellular reactive oxygen species (ROS) generation was determined by luminol/isoluminol chemiluminescence. Activation of NFκB in oral epithelial cells was determined by immunocytochemistry. RESULTS: Smoke extract alone caused increased neutrophil extracellular isoluminol-dependent chemiluminescence, not detectable with luminol. However, pre-treatment with smoke extract reduced both total and extracellular ROS generation in response to all stimuli. Nicotine and cotinine had no effect on the neutrophil respiratory burst. Smoke extract, nicotine and cotinine did not induce oral epithelial cell NFκB activation. CONCLUSIONS: These data demonstrate that smoke extract reduces the ability of neutrophils to generate ROS after stimulation with F. nucleatum and IgG-opsonized S. aureus but, at high concentrations, stimulates extracellular ROS generation. During periodontitis, cigarette smoking may differentially affect neutrophil function, generally preventing elimination of periodontal pathogens but, in heavy smokers, also stimulating ROS release and oxidative stress mediated tissue damage.

Gene expression profiles of mitochondria-endoplasmic reticulum tethering in human gingival fibroblasts in response to periodontal pathogens.

OBJECTIVE: The current study aimed to elucidate the potential involvement of mitochondria-endoplasmic reticulum contact genes in the pathogenesis of periodontal disease by monitoring levels of contact associated genes including Mitofusion 1 (MFN1) and MFN2, inositol 1,4,5-trisphosphate receptor (IP3R), chaperone glucose-regulated protein 75 (GRP75), sigma non-opioid intracellular receptor 1 (SIGMAR1) and phosphate and tensin homolog induced putative kinase 1 (PINK1) in human gingival fibroblasts in response to periodontal pathogens Fusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) in vitro. DESIGN: Primary human gingival fibroblasts were exposed to live cultures of P. gingivalis (W83; ATCC BAA-308) and F. nucleatum (subsp. Polymorphum; ATCC 10953) alone or in combination for 4 h at a 50 or 200 multiplicity of infection. Escherichia coli lipopolysaccharide (10 µg/mL) exposure was used as a positive control. Gene expression levels of contact genes (MFN1, MFN2, IP3R, GRP75, SIGMAR1 and PINK1) as well as a proinflammatory cytokine, Tumor necrosis factor-α (TNF-α), and the apoptosis associated gene, Immediate early response 3 (IER3), were evaluated by reverse transcription polymerase chain reaction analysis. RESULTS: MFN1, GRP75, IP3R and PINK1 were significantly upregulated by P. gingivalis with or without F. nucleatum. Only P. gingivalis with F. nucleatum caused a significant upregulation of SIGMAR1. TNF-α and IER3 gene expression positively correlated with the contact-associated gene expression changes. CONCLUSION: F. nucleatum and P. gingivalis alone or in combination may differentially dysregulate the gene expression levels of contact-associated genes in human gingival fibroblasts. These host-microbiome interactions may mechanistically be important in the pathogenesis of periodontal disease.

Potential for direct application of blue light for photo-disinfection of dentine.

The direct application of light for photo-disinfection potentially provides a safe and novel modality to inhibit or eliminate cariogenic bacteria residing upon and within dentine. This study aimed to both; characterize the pattern of transmission of 405 nm light through molar dentine at different tooth locations, as well as, determine the irradiation parameters that are antibacterial for Streptococcus mutans under various growth conditions, including lawns, planktonic cultures, and biofilms. To determine the amount of light (405 nm) transmitted at different anatomical tooth locations; irradiance values were recorded after blue light (470-4054 mW/cm2) had traversed through occlusal, oblique, and buccal dentine sections; and three thicknesses - 1, 2 and 3 mm were investigated. To determine tubular density; scanning electron micrographs from 2 mm outer (dentine-enamel junction) and inner (pulp) dentine sections were analysed. For photo-disinfection studies; S. mutans was irradiated using the same 405 nm wavelength light at a range of doses (110-1254 J/cm2) in both biofilm and planktonic cultures. The inhibitory effect of the irradiation on bacterial lawns was compared by measuring zones of inhibition; and for planktonic cultures both spectrophotometric and colony forming unit (CFU) assays were performed. A live/dead staining assay was utilised to determine the effect of irradiation on bacterial viability in mature biofilms. Data indicated that increasing dentine thickness decreased light transmission significantly irrespective of its orientation. Occlusal and oblique samples exhibited higher transmission compared with buccal dentine. Oblique dentine 405 nm light transmission was comparable with that of occlusal dentine independent of section thickness. An increased tubule density directly positively correlated with light transmission. Irradiation at 405 nm inhibited S. mutans growth in both biofilm and planktonic cultures and a dose response relationship was observed. Irradiation at doses of 340 and 831 J/cm2 led to significant reductions in bacterial growth and viability; as determined by CFU counting and live/dead staining. Data suggests that phototherapy approaches utilising a 405 nm wavelength have therapeutic potential to limit cariogenic bacterial infections both at the surface and within dentine.