Minimally invasive management of vital teeth requiring root canal therapy.

The present study aimed to investigate the possible use of a non-instrumentation technique including blue light irradiation for root canal cleaning. Extracted human single rooted teeth were selected. Nine different groups included distilled water, NaOCl, intra-canal heated NaOCl, and NaOCl + EDTA irrigation after either instrumentation or non-instrumentation, and a laser application group following non-instrumentation technique. The chemical assessment of the root canal dentine was evaluated using energy dispersive spectroscopy (EDS) and Fourier transform infrared (FT-IR) spectroscopy. Surface microstructural analyses were performed by using scanning electron microscopy (SEM). The antimicrobial efficacy of different preparation techniques was evaluated using microbial tests. Light application didn't change the calcium/phosphorus, carbonate/phosphate and amide I/phosphate ratios of the root canal dentin. The root canal dentin preserved its original chemistry and microstructure after light application. The instrumentation decreased the carbonate/phosphate and amide I/phosphate ratios of the root canal dentin regardless of the irrigation solution or technique (p < 0.05). The application of light could not provide antibacterial efficacy to match the NaOCl irrigation. The NaOCl irrigation both in the non-instrumentation and instrumentation groups significantly reduced the number of bacteria (p < 0.05). The use of minimally invasive root canal preparation techniques where the root canal is not instrumented and is disinfected by light followed by obturation with a hydraulic cement sealer reduced the microbial load and preserved the dentin thus may be an attractive treatment option for management of vital teeth needing root canal therapy.

Photobiomodulation of oral fibroblasts stimulated with periodontal pathogens.

Photobiomodulation (PBM) utilises light energy to treat oral disease, periodontitis. However, there remains inconsistency in the reporting of treatment parameters and a lack of knowledge as to how PBM elicits its molecular effects in vitro. Therefore, this study aimed to establish the potential immunomodulatory effects of blue and near infra-red light irradiation on gingival fibroblasts (GFs), a key cell involved in the pathogenesis of periodontitis. GFs were seeded in 96-well plates in media + / - Escherichia coli lipopolysaccharide (LPS 1 µg/ml), or heat-killed Fusobacterium nucleatum (F. nucleatum, 100:1MOI) or Porphyromonas gingivalis (P. gingivalis, 500:1MOI). Cultures were incubated overnight and subsequently irradiated using a bespoke radiometrically calibrated LED array (400-830 nm, irradiance: 24 mW/cm2 dose: 5.76 J/cm2). Effects of PBM on mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays, total reactive oxygen species production (ROS assay) and pro-inflammatory/cytokine response (interleukin-8 (IL-8) and tumour growth factor-ß1 (TGFß1)) were assessed 24 h post-irradiation. Data were analysed using one-way ANOVA followed by the Tukey test. Irradiation of untreated (no inflammatory stimulus) cultures at 400 nm induced 15%, 27% and 13% increases in MTT, ROS and IL-8 levels, respectively (p < 0.05). Exposure with 450 nm light following application of P. gingivalis, F. nucleatum or LPS induced significant decreases in TGFß1 secretion relative to their bacterially stimulated controls (p < 0.001). Following stimulation with P. gingivalis, 400 nm irradiation induced 14% increases in MTT, respectively, relative to bacteria-stimulated controls (p < 0.05). These findings could identify important irradiation parameters to enable management of the hyper-inflammatory response characteristic of periodontitis.

Violet-Blue Light Arrays at 405 Nanometers Exert Enhanced Antimicrobial Activity for Photodisinfection of Monomicrobial Nosocomial Biofilms.

Light-emitting diodes (LEDs) demonstrate therapeutic effects for a range of biomedical applications, including photodisinfection. Bands of specific wavelengths (centered at 405 nm) are reported to be the most antimicrobial; however, there remains no consensus on the most effective irradiation parameters for optimal photodisinfection. The aim of this study was to assess decontamination efficiency by direct photodisinfection of monomicrobial biofilms using a violet-blue light (VBL) single-wavelength array (SWA) and multiwavelength array (MWA). Mature biofilms of nosocomial bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus) were grown on 96-well polypropylene PCR plates. The biofilms were then exposed to VBL for 2,700 s (SWA) and 1,170 s (MWA) to deliver 0 to 670 J/cm2, and the antibacterial activity of VBL was assessed by comparing the seeding of the irradiated and the nonirradiated biofilms. Nonirradiated groups were used as controls. The VBL arrays were characterized optically (spectral irradiance and beam profile) and thermally. The SWA delivered 401-nm VBL and the MWA delivered between 379-nm and 452-nm VBL, albeit at different irradiances and with different beam profiles. In both arrays, the irradiated groups were exposed to increased temperatures compared to the nonirradiated controls. All bacterial isolates were susceptible to VBL and demonstrated reductions in the seeding of exposed biofilms compared with the nonirradiated controls. VBL at 405 nm exerted the most antimicrobial activity, exhibiting reductions in seeding of up to 94%. Decontamination efficiency is dependent on the irradiation parameters, bacterial species and strain, and experimental conditions. Controlled experiments that ameliorate the heating effects and improve the optical properties are required to optimize the dosing parameters to advance the successful clinical translation of this technology.IMPORTANCE This study reports the efficacy of VBL and blue light (BL) and their antimicrobial activity against mature biofilms of a range of important nosocomial pathogens. While this study investigated the antibacterial activity of a range of wavelengths of between 375 and 450 nm and identified a specific wavelength region (∼405 nm) with increased antibacterial activity, decontamination was dependent on the bacterial species, strain, irradiation parameters, and experimental conditions. Further research with controlled experiments that ameliorate the heating effects and improve the optical properties are required to optimize the dosing parameters to advance the successful clinical translation of this technology.

Periodontal diagnosis in the context of the 2017 classification system of periodontal diseases and conditions: presentation of a patient with periodontitis localised to the molar teeth.

The objective of this case report is to illustrate the diagnosis and classification of periodontitis, according to the 2017 classification system, as recommended in the British Society of Periodontology (BSP) implementation plan. A 37-year-old female was diagnosed with periodontitis (molar-incisor pattern), stage III, grade C, currently unstable. Several issues pertinent to the diagnosis of localised forms of periodontitis in young patients are discussed in relation to the current and previous classification systems. Periodontitis can be limited to a few sites and this case highlights the importance of the careful application of the basic periodontal examination (BPE).

Periodontal diagnosis in the context of the 2017 classification system of periodontal diseases and conditions - implementation in clinical practice.

The 2017 World Workshop Classification system for periodontal and peri-implant diseases and conditions was developed in order to accommodate advances in knowledge derived from both biological and clinical research, that have emerged since the 1999 International Classification of Periodontal Diseases. Importantly, it defines clinical health for the first time, and distinguishes an intact and a reduced periodontium throughout. The term 'aggressive periodontitis' was removed, creating a staging and grading system for periodontitis that is based primarily upon attachment and bone loss and classifies the disease into four stages based on severity (I, II, III or IV) and three grades based on disease susceptibility (A, B or C). The British Society of Periodontology (BSP) convened an implementation group to develop guidance on how the new classification system should be implemented in clinical practice. A particular focus was to describe how the new classification system integrates with established diagnostic parameters and pathways, such as the basic periodontal examination (BPE). This implementation plan focuses on clinical practice; for research, readers are advised to follow the international classification system. In this paper we describe a diagnostic pathway for plaque-induced periodontal diseases that is consistent with established guidance and accommodates the novel 2017 classification system, as recommended by the BSP implementation group. Subsequent case reports will provide examples of the application of this guidance in clinical practice.

Periodontal diagnosis in the context of the BSP implementation plan for the 2017 classification system of periodontal diseases and conditions: presentation of a pair of young siblings with periodontitis.

The objective of this case report is to illustrate the diagnosis and classification of periodontitis according to the 2017 classification system as recommended in the British Society of Periodontology (BSP) implementation plan. We describe two cases in the form of a pair of siblings, who developed periodontitis very early in life. A 19-year-old female was diagnosed with 'generalised periodontitis; stage III/grade C; currently unstable'. Her 14-year-old sister was diagnosed with 'localised periodontitis; stage II, grade C; currently unstable'. The present case report presents an example for the application of the new classification system and illustrates the importance of a periodontal check for children and adolescents and/or their relatives.

Periodontal diagnosis in the context of the 2017 classification system of periodontal diseases and conditions: Presentation of a middle-aged patient with localised periodontitis.

The objective of this case report is to illustrate the diagnosis and classification of periodontitis according to the 2017 classification system as recommended in the British Society of Periodontology (BSP) implementation plan. We describe a case of a patient who was diagnosed with 'localised periodontitis; stage II, grade B; currently unstable'. The present case report presents an example for the application of the new classification system and illustrates how the new classification system captures disease severity, extent and disease susceptibility by staging and grading periodontitis.

Automated noninvasive epithelial cell counting in phase contrast microscopy images with automated parameter selection.

Cell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a 'destructive' process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy-based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal. The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.

Potential role of periodontal pathogens in compromising epithelial barrier function by inducing epithelial-mesenchymal transition.

BACKGROUND AND OBJECTIVE: Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal-like phenotype and this may be induced by exposure to gram-negative bacteria. It has been proposed that EMT is responsible for compromising epithelial barrier function in the pathogenesis of several diseases. However, the possible role of EMT in the pathogenesis of periodontitis has not previously been investigated. The aim of this study therefore was to investigate whether gram-negative, anaerobic periodontal pathogens could trigger EMT in primary oral keratinocytes in vitro. MATERIAL AND METHODS: Primary oral keratinocytes were harvested from labial mandibular mucosa of Wistar Han rats. Cells were exposed to heat-killed Fusobacterium nucleatum and Porphyromonas gingivalis (100 bacteria/epithelial cell) and to 20 µg/mL of Escherichia coli lipopolysaccharide over an 8-day period. Exposure to bacteria did not significantly change epithelial cell number or vitality in comparison with unstimulated controls at the majority of time-points examined. Expression of EMT marker genes was determined by semiquantitative RT-PCR at 1, 5, and 8 days following stimulation. The expression of EMT markers was also assessed by immunofluorescence (E-cadherin and vimentin) and using immunocytochemistry to determine Snail activation. The loss of epithelial monolayer coherence, in response to bacterial challenge, was determined by measuring trans-epithelial electrical resistance. The induction of a migratory phenotype was investigated using scratch-wound and transwell migration assays. RESULTS: Exposure of primary epithelial cell cultures to periodontal pathogens was associated with a significant decrease in transcription (~3-fold) of E-cadherin and the upregulation of N-cadherin, vimentin, Snail, matrix metalloproteinase-2 (~3-5 fold) and toll-like receptor 4. Bacterial stimulation (for 8 days) also resulted in an increased percentage of vimentin-positive cells (an increase of 20% after stimulation with P. gingivalis and an increase of 30% after stimulation with F. nucleatum, compared with controls). Furthermore, periodontal pathogens significantly increased the activation of Snail (60%) and cultures exhibited a decrease in electrical impedance (P < .001) in comparison with unexposed controls. The migratory ability of the cells increased significantly in response to bacterial stimulation, as shown by both the number of migrated cells and scratch-wound closure rates. CONCLUSION: Prolonged exposure of primary rat oral keratinocyte cultures to periodontal pathogens generated EMT-like features, which introduces the possibility that this process may be involved in loss of epithelial integrity during periodontitis.

Cigarette smoke modifies neutrophil chemotaxis, neutrophil extracellular trap formation and inflammatory response-related gene expression.

BACKGROUND AND OBJECTIVE: Cigarette smoking is a major risk factor for periodontitis, and smoking perturbs neutrophil reactive oxygen species production. This study tested the hypothesis that cigarette smoke extract (CSE) and its components/metabolites nicotine, cotinine and thiocyanate (SCN-), may influence neutrophil functions. MATERIAL AND METHODS: Chemotaxis was assessed in neutrophils pre-treated with CSE using real-time video microscopy. Neutrophil extracellular trap (NET) release in response to CSE, nicotine, cotinine, SCN- as well as to phorbol 12-myristate-13-acetate and hypochlorous acid following pre-treatment with CSE, nicotine, cotinine or SCN- was assessed using fluorescence-based assays. The impact of CSE and SCN- treatment on neutrophil respiratory burst- and inflammation-related gene expression (NFKBIE, DNAJB1, CXCL8, NCF1, NCF2, CYBB) was determined by real-time polymerase chain reaction. RESULTS: Both CSE and SCN- pre-treatment inhibited phorbol 12-myristate-13-acetate-stimulated NET release. Additionally, SCN- inhibited hypochlorous acid-stimulated NET formation, while SCN- alone stimulated NET release. Overall, neutrophils pre-treated with CSE exhibited reduced speed, velocity and directionality relative to untreated neutrophils. Although CSE and SCN- promoted DNAJB1 expression, increased redox-related gene expression was only detected in response to SCN-. CONCLUSION: These results suggest that CSE can alter ex vivo neutrophil activation by mechanisms independent of SCN- and nicotine, and SCN- may contribute to the perturbed innate immune responses observed in smokers.

Periodontal pathogens promote epithelial-mesenchymal transition in oral squamous carcinoma cells in vitro.

Epithelial-mesenchymal transition (EMT) is potentially involved in increasing metastasis of oral squamous cell carcinoma (OSCC). Periodontal pathogens are well-known for their ability to induce intense immune responses and here we investigated whether they are involved in inducing EMT. Cultures of OSCC cell line (H400) were treated separately with heat-killed periodontal pathogens F. nucleatum, or P. gingivalis or E. coli LPS for 8 d. EMT-associated features were assayed using sq-PCR and PCR-arrays, for EMT-related markers, and ELISAs for TGF-ß1, TNF-α, and EGF. The migratory ability of cells was investigated using scratch and transwell migration assays. E-cadherin and vimentin expression was assessed using immunofluorescence while Snail activation was detected with immunocytochemistry. In addition, the integrity of the cultured epithelial layer was investigated using transepithelial electrical resistance (TEER). PCR data showed significant upregulation after 1, 5, and 8 d in transcription of mesenchymal markers and downregulation of epithelial ones compared with unstimulated controls, which were confirmed by immunofluorescence. Periodontal pathogens also caused a significant increase in level of all cytokines investigated which could be involved in EMT-induction and Snail activation. Exposure of cells to the bacteria increased migration and the rate of wound closure. Downregulation of epithelial markers also resulted in a significant decrease in impedance resistance of cell monolayers to passage of electrical current. These results suggested that EMT was likely induced in OSCC cells in response to stimulation by periodontal pathogens.

Neutrophil Extracellular Traps in Periodontitis: A Web of Intrigue.

Neutrophil extracellular traps (NETs) represent a novel paradigm in neutrophil-mediated immunity. NETs are believed to constitute a highly conserved antimicrobial strategy comprising decondensed nuclear DNA and associated histones that are extruded into the extracellular space. Associated with the web-like strands of DNA is an array of antimicrobial peptides (AMPs), which facilitate the extracellular destruction of microorganisms that become entrapped within the NETs. NETs can be released by cells that remain viable or following a unique form of programmed cell death known as NETosis, which is dependent on the production of reactive oxygen species (ROS) and the decondensing of the nuclear DNA catalyzed by peptidyl arginine deiminase-4. NETs are produced in response to a range of pathogens, including bacteria, viruses, fungi, and protozoa, as well as host-derived mediators. NET release is, however, not without cost, as the concomitant release of cytotoxic molecules can also cause host tissue damage. This is evidenced by a number of immune-mediated diseases, in which excess or dysfunctional NET production, bacterial NET evasion, and decreased NET removal are associated with disease pathogenesis. Periodontitis is the most prevalent infectious-inflammatory disease of humans, characterized by a dysregulated neutrophilic response to specific bacterial species within the subgingival plaque biofilm. Neutrophils are the predominant inflammatory cell involved in periodontitis and have previously been found to exhibit hyperactivity and hyperreactivity in terms of ROS production in chronic periodontitis patients. However, the contribution of ROS-dependent NET formation to periodontal health or disease remains unclear. In this focused review, we discuss the mechanisms, stimuli, and requirements for NET production; the ability of NET-DNA and NET-associated AMPs to entrap and kill pathogens; and the potential immunogenicity of NETs in disease. We also speculate on the potential role of NETs in the pathogenesis of periodontitis.

Effects of red light-emitting diode irradiation on dental pulp cells.

Light irradiation activates a range of cellular processes in a variety of cell types, including stem cells, and can promote tissue repair. This study investigated the effects of light-emitting diode (LED) exposure on dental pulp cells (DPCs). Dose response analysis at 20-second intervals up to 120 seconds demonstrated that a LED array emitting 653-nm red light stimulated significantly increased cell growth at 3 and 7 days post-irradiation with 40 (149 mJ/cm(2)) and 60 (224 mJ/cm(2)) seconds of radiant exposure. Double-dosing cells at days 1 and 4 of a 7-day culture period with 60-second (224 mJ/cm(2)) LED exposure significantly increased cell growth compared with a single dosing regime. BrdU analysis demonstrated significantly increased proliferation rates associated with significantly increased ATP, nitric oxide (NO), and mitochondrial metabolic activity. LED-stimulated NO levels were not reduced by inhibition of NO-synthase activity. Light exposure also rescued the inhibition of mitochondrial dysfunction and increased levels of in vitro mineralization compared with control. Media exchange experiments indicated that autocrine signaling was not likely responsible for red-light-induced DPC activity. In conclusion, data analysis indicated that 653-nm LED irradiation promoted DPC responses relevant to tissue repair, and this is likely mediated by increased mitochondrial activity.

Differential activation of NF-kappaB and gene expression in oral epithelial cells by periodontal pathogens.

To investigate the molecular effects of the periodontopathogens Fusobacterium nucleatum (FN) and Porphyromonas gingivalis (PG) on the oral epithelium, the H400 oral epithelial cell line was cultured in the presence of non-viable bacteria. Following confirmation of the presence of transcripts for the bacterial pattern recognition receptors in H400 cells, Toll-like receptors -2, -4 and -9, and components of the NF-kappaB signalling pathway, immunocytochemical analyses were performed showing that NF-kappaB was activated within 1 h of exposure to both periodontopathogens. A significantly greater number of NF-kappaB nuclear translocations were apparent following H400 cell exposure to FN as compared with PG. Gene expression analyses indicated that transcripts known to be regulated by the NF-kappaB pathway, including cytokines/chemokines TNF-alpha, IL-1beta, IL-8, MCP-1/CCL2 and GM-CSF, were up-regulated following 4 and 24 h of exposure to both periodontopathogens. In addition, H400 periodontopathogen exposure resulted in differential regulation of transcripts for several cytokeratin gene family members. Consistent with the immunocytochemical data, microarray results indicated that FN induced a greater number of gene expression changes than PG following 24 h of exposure, 609 and 409 genes, respectively. Ninety-one genes were commonly differentially expressed by both periodontopathogens and represented biological processes commonly associated with periodontitis. Gene expression analyses by reserve transcriptase-polymerase chain reaction (RT-PCR) of molecules identified from the microarray data sets, including Heme oxygenase-1, lysyl oxidase, SOD2, CCL20 and calprotectin components, confirmed their differential expression profiles induced by the two periodontopathogens. FN and PG have clearly different molecular effects on oral epithelial cells, potentially highlighting the importance of the composition of the plaque biofilm in periodontitis pathogenesis.

Compromised GCF total antioxidant capacity in periodontitis: cause or effect?

BACKGROUND: Oxidative stress is implicated in the pathogenesis of periodontitis. The total antioxidant capacity (TAOC) of gingival crevicular fluid volume (GCF) and plasma appears compromised in periodontitis, but it is unclear whether this predisposes to, or results from the inflammatory process. AIM: To investigate longitudinal changes in GCF and plasma TAOC following reductions in periodontal inflammation with successful non-surgical therapy. MATERIALS AND METHODS: Two longitudinal studies were run in series on non-smokers with chronic periodontitis (CP). Study-1 (n=17) assessed index sites with mild disease; Study-2 (n=18) investigated deep sites. GCF sampling and clinical measures were performed at baseline and 3 months post-therapy. Plasma and GCF TAOC was determined by enhanced chemiluminescence and 32 age/sex-matched periodontally healthy controls were used. RESULTS: Therapy improved clinical outcomes consistent with the literature. There were no differences in plasma TAOC between periodontitis patients (507+/-92 microMTeq) and controls (520+/-100 microMTeq; p=0.57) at baseline, but GCF TAOC was lower (p<0.0001) in CP patients (680+/-371 microMTeq) than controls (1129+/-722 microMTeq). Successful periodontal therapy did not alter plasma TAOC (p=0.56), but GCF TAOC increased (by 449+/-722 microMTeq, p<0.001) to control subject levels (p=0.47) CONCLUSIONS: Local total antioxidant capacity in CP appears to reflect increased oxygen radical activity during periodontal inflammation and can be restored to control subject levels by successful non-surgical therapy.

Classification of periodontal diseases: where were we? Where are we now? Where are we going?

This paper discusses the past, present and possible future classification of periodontal diseases. It outlines the reasons for using a classification system from a clinical perspective and provides a critical appraisal of the latest classification. The major changes introduced in the 1999 system are discussed alongside the rationale behind the recommended nomenclature.