Antimicrobial Efficacy of Different Irrigant Solutions Using a Novel Biofilm Model: An In Vitro Confocal Laser Scanning Microscopy Experiment.

AIM: To determine the ability of different irrigation solutions to biomechanically remove Enterococcus faecalis biofilm from a novel artificial root canal model during chemomechanical preparation. METHODS: High resolution micro-computer-tomography scans of a mandibular molar's mesial root were used to produce 50 identical 3D-printed resin root canal models. These were cultured with E.faecalis over seven days to generate biofilm and subjected to chemomechanical preparation using: saline; 17% ethylenediaminetetraacetic acid (EDTA) or 2% sodium hypochlorite (NaOCl) alongside positive/negative controls (n = 10). Canals were prepared to 40/.06 taper, with 1 mL irrigation between instruments, followed by 5 mL penultimate rinse, 30 s ultrasonic activation and 5 mL final rinse. Residual biofilm volume (pixels) was determined following immunofluorescent staining and confocal-laser-scanning-microscopy imaging. Statistical comparisons were made using Kruskal-Wallis with post-hoc Dunn's tests (α ⟨0.05). RESULTS: In all canal thirds, the greatest biofilm removal was observed with NaOCl, followed by EDTA and saline. The latter had significantly higher E.faecalis counts than NaOCl and EDTA (P ⟨0.01). However, no statistical differences were found between EDTA and NaOCl or saline and positive controls (P ⟩0.05). CONCLUSIONS: Within limitations of this model, 17% EDTA was found to be as effective as 2% NaOCl at eradicating E.faecalis biofilm following chemomechanical preparation. Further investigations with multi-species biofilms are encouraged.

Potential application of immunotherapy for modulation of pulp inflammation: opportunities for vital pulp treatment.

Caries results in the demineralization and destruction of enamel and dentine, and as the disease progresses, irreversible pulpitis can occur. Vital pulp therapy (VPT) is directed towards pulp preservation and the prevention of the progression of inflammation. The outcomes of VPT are not always predictable, and there is often a poor correlation between clinical signs and symptoms, and the events occurring at a molecular level. The inflamed pulp expresses increased levels of cytokines, including tumour necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1ß, IL-4, IL-6, IL-8, IL-17 and IL-23, which recruit and drive a complex cellular immune response. Chronic inflammation and sustained cytokine release can result in irreversible pulp damage and a decreased capacity for tissue healing. Other chronic inflammatory diseases, such as psoriasis, inflammatory bowel diseases and rheumatoid arthritis, are also characterized by an dysregulated immune response composed of relatively high cytokine levels and increased numbers of immune cells along with microbial and hard-soft tissue destructive pathologies. Whilst anti-cytokine therapies have been successfully applied in the treatment of these diseases, this approach is yet to be attempted in cases of pulp inflammation. This review therefore focuses on the similarities in the aetiology between chronic inflammatory diseases and pulpitis, and explores how anti-cytokine therapies could be applied to manage an inflamed pulp and facilitate healing. Further proof-of-concept studies and clinical trials are justified to determine the effectiveness of these treatments to enable more predictable outcomes in VPT.

Investigation of microbial profile, levels of endotoxin and lipoteichoic acid in teeth with symptomatic irreversible pulpitis: a clinical study.

AIM: To investigate the microbial profile, and levels of endotoxin (LPS) and lipoteichoic acid (LTA), in infected dentine (ID) and root canals (RC) at different phases of root canal treatment in teeth with symptomatic irreversible pulpitis. METHODOLOGY: Ten volunteers were included, and samples were collected from infected dentine (ID) and the root canal lumen (RC) using sterile excavators and paper points, respectively. RC samples were taken before (S1) and after (S2) chemo-mechanical canal preparation (CMP), and after intracanal medication (ICM; S3). Checkerboard DNA-DNA hybridization was used for microbial analysis. The levels of LPS and LTA were evaluated using the limulus amebocyte lysate assay and ELISA, respectively. Shapiro-Wilk's test was used to verify data normality. Friedman's test was used to evaluate statistical differences using checkerboard DNA-DNA hybridization in the ID and RC at the different phases of the RC treatment. Post hoc Dunn's multiple comparison test was used to verify significant differences recorded at the different time-points. The levels of LPS and LTA were analysed statistically by using repeated measures anova and Tukey's post hoc test to evaluate differences in both sites. The significance level was set at 5% (P < 0.05). RESULTS: A total of 40 DNA probes were used for microbial investigation of ID and RC samples using checkerboard DNA-DNA hybridization. The levels and complexity of bacteria were similar in the ID and initial RC samples. The levels of LPS and LTA in ID were significantly higher than the initial RC samples (S1; P < 0.05). Canal preparation was effective in significantly decreasing the levels of bacteria, LPS and LTA (P < 0.05). ICM did not provide additional reduction in the levels of bacteria and LPS (P > 0.05). However, a significant reduction in the levels of LTA was observed after ICM (P < 0.05). CONCLUSION: The microbial profile of infected dentine and root canals of teeth with irreversible pulpitis was complex, harbouring different species including Gram-positive and Gram-negative, cocci and bacilli, and facultative and strict anaerobes. Root canal preparation was effective in reducing the levels of bacteria, LPS and LTA from the root canals of teeth with pulpitis.

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.

The influence of irrigant activation, concentration and contact time on sodium hypochlorite penetration into root dentine: an ex vivo experiment.

AIM: To establish whether irrigant activation techniques, namely manual dynamic activation (MDA), passive ultrasonic irrigation (PUI) and sonic irrigation (SI), improve the tubular penetration of sodium hypochlorite (NaOCl) into root dentine when compared with conventional needle irrigation (CNI). Secondly, investigate if increasing NaOCl concentration and/or contact time improves the performance of these techniques. METHODOLOGY: A total of 83 extracted human maxillary permanent canines were decoronated to 15 mm, and root canals prepared to a size 40, .10 taper. Root dentine was stained with crystal violet for 72 h and embedded in silicone. Eighty specimens were randomly distributed into 16 groups (n = 5) according to the irrigant activation technique, NaOCl concentration (2%; 5.25%) and irrigant contact time (10 min; 20 min). All activation techniques were used for 60 s in the last minute of irrigation. Additionally, three teeth were not exposed to NaOCl to confirm adequate dentine staining had occurred (i.e. negative control). All specimens were subsequently dissected, observed under a light microscope and NaOCl penetration depth (µm) determined by measuring the average width of bleached dentine using ImageJ software. Statistical comparisons were made with paired and unpaired t-tests, anovas followed by post hoc Tukey's and Dunnett's tests, and a general linear model (α < 0.05). RESULTS: Overall, NaOCl penetration ranged from 38.8 to 411.0 µm with MDA, PUI and SI consistently resulting in significantly greater tubular infiltration than CNI (P < 0.05). The deepest measurements in the coronal, middle and apical segments were all recorded in the MDA; 5.25%; 20 min group and the least in the CNI; 2%; 10 min group. Increasing either irrigant concentration or contact time resulted in significantly greater NaOCl penetration depths for all techniques and segments of the canal (P < 0.05). However, when irrigant concentration and contact time were increased together, a significant interaction effect between these two independent variables was observed on overall NaOCl penetration (P < 0.05). CONCLUSIONS: Agitating irrigants with MDA, PUI or SI, as well as using greater irrigant concentrations or contact times, potentiated NaOCl penetration into root dentine. However, longer durations of NaOCl exposure at lower concentrations resulted in similar depths of tubular penetration as those achieved at higher concentrations.

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.

Dissecting dentine-pulp injury and wound healing responses: consequences for regenerative endodontics.

A thorough understanding of the biology of the dentine-pulp complex is essential to underpin new treatment approaches and maximize clinical impact for regenerative endodontics and minimally invasive vital pulp treatment (VPT) strategies. Following traumatic and carious injury to dentine-pulp, a complex interplay between infection, inflammation and the host defence responses will occur, which is critical to tissue outcomes. Diagnostic procedures aim to inform treatment planning; however, these remain clinically subjective and have considerable limitations. As a consequence, significant effort has focussed on identification of diagnostic biomarkers, although these are also problematic due to difficulties in identifying appropriate diagnostic fluid sources and selecting reproducible biomarkers. This is further compounded by the link between inflammation and repair as many of the molecules involved exhibit significant multifunctionality. The tertiary dentine formed in response to dental injury has been purposefully termed reactionary and reparative dentine to enable focus on associated biological processes. Whilst reactionary dentine produced in response to milder injury is generated from surviving primary odontoblasts, reparative dentine, in response to more intense injury, requires the differentiation of new odontoblast-like cells derived from progenitor/stem cells recruited to the injury site. These two diverse processes result in very different outcomes in terms of the tertiary dentine produced and reflect the intensity rather than specific nature (nonexposure versus exposure) of the injury. The subsequent identification of the odontoblast-like cell phenotype remains challenging due to lack of unique molecular or morphological markers. Furthermore, the cells ultimately lining the newly deposited dentine provide only a snapshot of events. The specific source and plasticity of the progenitor cells giving rise to the odontoblast-like cell phenotype are also of significant debate. It is likely that improved characterization of tertiary dentine may better clarify the influence of cell derivation for odontoblast-like cells and their diversity. The field of regenerative endodontics offers exciting new treatment opportunities, and to maximize outcomes, we propose that the term regenerative endodontics should embrace the repair, replacement and regeneration of dentine-pulp.

A systematic review of methods used to sample and analyse periradicular tissue fluid during root canal treatment.

AIM: The primary aim was to identify techniques used to sample and analyse periradicular tissue fluid (PTF) in permanent teeth diagnosed with apical disease during root canal treatment. Secondly, to identify the types of inflammatory mediators studied using this approach. METHODOLOGY: Data Sources: PubMed, EMBASE, Cochrane Library, Science Direct, Web of Science and OpenGrey. Eligibility Criteria: Clinical studies published until 1 June 2018 which utilized orthograde techniques to sample and analyse PTF were included. Cell culture, laboratory or animal studies and those concerned with investigating inflammatory mediator activity from within healthy or diseased pulp tissue, and not periradicular tissues, were excluded. Study appraisal and methods: In accordance with PRISMA guidelines, data were extracted on study characteristics, target mediators, sampling and assay techniques and the parameters associated with the PTF sampling and eluting protocol. A qualitative synthesis was conducted, and studies were critically appraised using a modified version of the Cochrane risk of bias tool. RESULTS: Study Characteristics: From 251 studies, 33 were eligible for inclusion. Sampling techniques included the use of paper points (n = 27), fine needle aspiration (n = 4) and filter strips (n = 2). Assay techniques included enzyme-linked immunosorbent assay (n = 18), quantitative polymerase chain reaction (n = 9), radioimmunoassay (n = 4), colorimetric assay (n = 2), immunofluorometric assay (n = 1) and cytometric bead array (n = 1). Forty-five different inflammatory mediators were targeted at the proteomic/metabolomic (n = 25) or transcriptomic level (n = 9). LIMITATIONS: Significant heterogeneity exists within the methodology, and only 5 studies disclosed unambiguous information about their PTF sampling and eluting protocols. CONCLUSIONS: Paper points and proteomic/metabolomic analysis are currently the preferred methods for studying and analysing PTF during root canal treatment. The most studied analytes were IL-1ß and TNF-α. IMPLICATIONS: Further research is required to develop an optimized PTF sampling and eluting protocol to overcome methodological heterogeneity, and future studies are advised to follow a standardized approach to reporting their methodology.

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.

Cleaning lateral morphological features of the root canal: the role of streaming and cavitation.

AIM: To investigate the effects of ultrasonic activation file type, lateral canal location and irrigant on the removal of a biofilm-mimicking hydrogel from a fabricated lateral canal. Additionally, the amount of cavitation and streaming was quantified for these parameters. METHODOLOGY: An intracanal sonochemical dosimetry method was used to quantify the cavitation generated by an IrriSafe 25 mm length, size 25 file inside a root canal model filled with filtered degassed/saturated water or three different concentrations of NaOCl. Removal of a hydrogel, demonstrated previously to be an appropriate biofilm mimic, was recorded to measure the lateral canal cleaning rate from two different instruments (IrriSafe 25 mm length, size 25 and K 21 mm length, size 15) activated with a P5 Suprasson (Satelec) at power P8.5 in degassed/saturated water or NaOCl. Removal rates were compared for significant differences using nonparametric Kruskal-Wallis and/or Mann-Whitney U-tests. Streaming was measured using high-speed particle imaging velocimetry at 250 kfps, analysing both the oscillatory and steady flow inside the lateral canals. RESULTS: There was no significant difference in amount of cavitation between tap water and oversaturated water (P = 0.538), although more cavitation was observed than in degassed water. The highest cavitation signal was generated with NaOCl solutions (1.0%, 4.5%, 9.0%) (P < 0.007) and increased with concentration (P < 0.014). The IrriSafe file outperformed significantly the K-file in removing hydrogel (P < 0.05). Up to 64% of the total hydrogel volume was removed after 20 s. The IrriSafe file typically outperformed the K-file in generating streaming. The oscillatory velocities were higher inside the lateral canal 3 mm compared to 6 mm from WL and were higher for NaOCl than for saturated water, which in turn was higher than for degassed water. CONCLUSIONS: Measurements of cavitation and acoustic streaming have provided insight into their contribution to cleaning. Significant differences in cleaning, cavitation and streaming were found depending on the file type and size, lateral canal location and irrigant used. In general, the IrriSafe file outperformed the K-file, and NaOCl performed better than the other irrigants tested. The cavitation and streaming measurements revealed that both contributed to hydrogel removal and both play a significant role in root canal cleaning.

Growth factor release from dentine matrix by pulp-capping agents promotes pulp tissue repair-associated events.

AIM: To characterize growth factor release from dentine by pulp-capping agents and to determine the effects of liberated dentine extracellular matrix (dECM) components on pulp cells in the key wound healing processes of migration and cell growth. METHODOLOGY: Powdered human dentine was exposed to solutions of calcium hydroxide, white and grey mineral trioxide aggregate (MTA) (ProRoot, (Dentsply Tulsa, Tulsa, OK, USA) over 14 days. The solubilized dECM components were dialysed and lyophilized and characterized using multiplex quantitative ELISA. Following dECM component extraction dentine was analysed using Fourier-transformed infrared spectroscopy (FTIR). Primary rat dental pulp cells (RDPCs) were exposed to dECM components (0.1-100 µg mL-1 ) released by calcium hydroxide, white and grey MTA, and cell growth and chemotactic responses were assessed. Statistical differences between the experimental and control groups were determined using one-way anova. RESULTS: A broad range of growth factors, many not previously reported in dentine, were liberated by these pulp-capping agents, including SCF, M-CSF, GM-CSF, IGFBP-1, NGF and GDNF. White and grey MTA liberated more growth factors than calcium hydroxide. FTIR analysis of dentine exposed to pulp-capping agents showed partial depletion of amide bands I, II and III, with little alteration in phosphate peaks compared to untreated dentine. dECM components released by white and grey MTA induced significantly more cell growth at low-to-moderate concentrations (P â‰¦ 0.05) examined in this study and significantly enhanced cell chemotaxis at all concentrations compared with controls (P â‰¦ 0.05). CONCLUSIONS: White and grey MTA solubilize a broad range of bioactive molecules from dentine, which can induce proliferation and chemotaxis in pulp cells.

Release of bio-active dentine extracellular matrix components by histone deacetylase inhibitors (HDACi).

AIM: To characterize dentine matrix component (DMC) release and smear layer removal by histone deacetylase inhibitors (HDACis). METHODOLOGY: DMCs were extracted from powdered human dentine over 14 days using three HDACis, valproic acid (VPA), trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) and compared with a control extractant, 10% (w/v) EDTA. Protein compositions of the resultant extracts were analysed by 1D-polyacrylamide gel electrophoresis (1D-PAGE), TGF-ß-1 and MMP-9 ELISAs and a high-throughput growth factor antibody array. Dentine discs with a standardized smear layer were prepared from human molars and treated with EDTA (17% w/v), polyacrylic acid (PA) (20% v/v) and the experimental HDACis prior to analysis by scanning electron microscopy. Parametric ELISA data were analysed using one-way anova and Tukey's post hoc test, whilst nonparametric smear layer data were analysed by Kruskal-Wallis test and Mann-Whitney U-test (P < 0.05). RESULTS: HDACis did not remove smear layer in the presence or absence of PA pre-treatment (P ≥ 0.478). 1D-PAGE analysis demonstrated different protein profiles for EDTA and HDACi extracts. All HDACi solutions released TGF-ß-1 although less effectively than EDTA (P < 0.001), whilst MMP-9 was extracted in significantly higher concentration by EDTA and VPA compared with TSA (P < 0.012). Antibody array analysis demonstrated the ability of HDACis to extract a complex cocktail of established/novel growth factors from dentine, albeit significantly less efficiently than EDTA for certain cytokines (TGF-ß-1, PDGF-AA, VEGF-A) and significantly more effectively for others (GDF-15, IGF-1, EGRF-1, NGFR, BDNF, SCF-R). CONCLUSIONS: HDACi release a range of bioactive DMCs that could promote dentine repair processes in vivo; however, they are ineffective at removing smear layer.

Epigenetic modulation of dental pulp stem cells: implications for regenerative endodontics.

Dental pulp stem cells (DPSCs) offer significant potential for use in regenerative endodontics, and therefore, identifying cellular regulators that control stem cell fate is critical to devising novel treatment strategies. Stem cell lineage commitment and differentiation are regulated by an intricate range of host and environmental factors of which epigenetic influence is considered vital. Epigenetic modification of DNA and DNA-associated histone proteins has been demonstrated to control cell phenotype and regulate the renewal and pluripotency of stem cell populations. The activities of the nuclear enzymes, histone deacetylases, are increasingly being recognized as potential targets for pharmacologically inducing stem cell differentiation and dedifferentiation. Depending on cell maturity and niche in vitro, low concentration histone deacetylase inhibitor (HDACi) application can promote dedifferentiation of several post-natal and mouse embryonic stem cell populations and conversely increase differentiation and accelerate mineralization in DPSC populations, whilst animal studies have shown an HDACi-induced increase in stem cell marker expression during organ regeneration. Notably, both HDAC and DNA methyltransferase inhibitors have also been demonstrated to dramatically increase the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) for use in regenerative therapeutic procedures. As the regulation of cell fate will likely remain the subject of intense future research activity, this review aims to describe the current knowledge relating to stem cell epigenetic modification, focusing on the role of HDACi on alteration of DPSC phenotype, whilst presenting the potential for therapeutic application as part of regenerative endodontic regimens.

A comparison of the in vitro mineralisation and dentinogenic potential of mesenchymal stem cells derived from adipose tissue, bone marrow and dental pulp.

Stem-cell-based therapies provide a biological basis for the regeneration of mineralised tissues. Stem cells isolated from adipose tissue (ADSCs), bone marrow (BMSCs) and dental pulp (DPSCs) have the capacity to form mineralised tissue. However, studies comparing the capacity of ADSCs with BMSCs and DPSCs for mineralised tissue engineering are lacking, and their ability to regenerate dental tissues has not been fully explored. Characterisation of the cells using fluorescence-activated cell sorting and semi-quantitative reverse transcription PCR for MSC markers indicated that they were immunophenotypically similar. Alizarin red (AR) staining and micro-computed tomography (µCT) analyses demonstrated that the osteogenic potential of DPSCs was significantly greater than that of BMSCs and ADSCs. Scanning electron microscopy and AR staining showed that the pattern of mineralisation in DPSC cultures differed from ADSCs and BMSCs, with DPSC cultures lacking defined mineralised nodules and instead forming a diffuse layer of low-density mineral. Dentine matrix components (DMCs) were used to promote dentinogenic differentiation. Their addition to cultures resulted in increased amounts of mineral deposited in all three cultures and significantly increased the density of mineral deposited in BMSC cultures, as determined by µCT analysis. Addition of DMCs also increased the relative gene expression levels of the dentinogenic markers dentine sialophosphoprotein and dentine matrix protein 1 in ADSC and BMSC cultures. In conclusion, DPSCs show the greatest potential to produce a comparatively high volume of mineralised matrix; however, both dentinogenesis and mineral volume was enhanced in ADSC and BMSC cultures by DMCs, suggesting that these cells show promise for regenerative dental therapies.

The effects of cryopreservation on cells isolated from adipose, bone marrow and dental pulp tissues.

The effects of cryopreservation on mesenchymal stem cell (MSC) phenotype are not well documented; however this process is of increasing importance for regenerative therapies. This study examined the effect of cryopreservation (10% dimethyl-sulfoxide) on the morphology, viability, gene-expression and relative proportion of MSC surface-markers on cells derived from rat adipose, bone marrow and dental pulp. Cryopreservation significantly reduced the number of viable cells in bone marrow and dental pulp cell populations but had no observable effect on adipose cells. Flow cytometry analysis demonstrated significant increases in the relative expression of MSC surface-markers, CD90 and CD29/CD90 following cryopreservation. sqRT-PCR analysis of MSC gene-expression demonstrated increases in pluripotent markers for adipose and dental pulp, together with significant tissue-specific increases in CD44, CD73-CD105 following cryopreservation. Cells isolated from different tissue sources did not respond equally to cryopreservation with adipose tissue representing a more robust source of MSCs.

A novel methodology providing insights into removal of biofilm-mimicking hydrogel from lateral morphological features of the root canal during irrigation procedures.

AIM: To introduce and characterize a reproducible hydrogel as a suitable biofilm mimic in endodontic research. To monitor and visualize the removal of hydrogel from a simulated lateral canal and isthmus for the following: I) Ultrasonic-Activated Irrigation (UAI) with water, ii) UAI with NaOCl and iii) NaOCl without UAI. METHODOLOGY: A rheometer was used to characterize the viscoelastic properties and cohesive strength of the hydrogel for suitability as a biofilm mimic. The removal rate of the hydrogel from a simulated lateral canal or isthmus was measured by high-speed imaging operating at frame rates from 50 to 30,000 fps. RESULTS: The hydrogel demonstrated viscoelastic behaviour with mechanical properties comparable to real biofilms. UAI enhanced the cleaning effect of NaOCl in isthmi (P < 0.001) and both NaOCl and water in lateral canals (P < 0.001). A greater depth of cleaning was achieved from an isthmus (P = 0.009) than from a lateral canal with UAI and also at a faster rate for the first 20 s. NaOCl without UAI resulted in a greater depth of hydrogel removal from a lateral canal than an isthmus (P < 0.001). The effect of UAI was reduced when stable bubbles were formed and trapped in the lateral canal. Different removal characteristics were observed in the isthmus and the lateral canal, with initial highly unstable behaviour followed by slower viscous removal inside the isthmus. CONCLUSIONS: The biofilm-mimicking hydrogel is reproducible, homogenous and can be easily applied and modified. Visualization of its removal from lateral canal anatomy provides insights into the cleaning mechanisms of UAI for a biofilm-like material. Initial results showed that UAI improves hydrogel removal from the accessory canal anatomy, but the creation of stable bubbles on the hydrogel-liquid interface may reduce the cleaning rate.

Preparedness and competency of New Zealand graduates for general dental practice - perceptions from the workforce.

BACKGROUND: Dental graduates need to demonstrate clinical competency. This mixed-methods study explored the perceptions of clinicians who employ or work with new graduates from the University of Otago, New Zealand, and identified themes reflecting graduates' preparedness for independent practice. METHODS: An online survey using a semantic differential scale and open-ended questions collected opinions and experiences from the workforce. Quantitative data were analysed using SPSS software, and qualitative data were analysed thematically. RESULTS: A representative sample of the workforce was obtained with a response rate of 35% (N = 83). Most clinicians engage new graduates to support the profession and/or rural communities. They perceived that graduates were well prepared in most areas, could translate theory to clinical practice and demonstrate professionalism. Graduates were reportedly stronger in basic dentistry, communication, ethics, and record keeping however were less strong in complex treatment planning, molar endodontics, fixed prosthodontics and exodontia. Clinical exposure during dental training was perceived as more limited, and mentoring and guidance in the transition to practice were deemed to be important. CONCLUSIONS: New Zealand dental graduates appear prepared for independent practice; however, maximising clinical opportunities during training, mentoring and early professional development in advanced areas of practice is essential to enhance competency and confidence.