Immunological isolation and characterization of neuronal progenitors from human dental pulp: A laboratory-based investigation.

AIMS: Dental pulp stem cells (DPSCs) contain a population of stem cells with a broad range of differentiation potentials, as well as more lineage-committed progenitors. Such heterogeneity is a significant obstacle to experimental and clinical applications. The aim of this study is to isolate and characterize a homogenous neuronal progenitor cell population from human DPSCs. METHODOLOGY: Polysialylated-neural cell adhesion molecule (PSA-NCAM+) neural progenitors were isolated from the dental pulp of three independent donors using magnetic-activated cell sorting (MACS) technology. Immunofluorescent staining with a panel of neural and non-neural markers was used to characterize the magnetically isolated PSA-NCAM+ fraction. PSA-NCAM+ cells were then cultured in Neurobasal A supplemented with neurotrophic factors: dibutyryl cyclic-AMP, neurotrophin-3, B27 and N2 supplements to induce neuronal differentiation. Both PSA-NCAM+ and differentiated PSA-NCAM+ cells were used in Ca2+ imaging studies to assess the functionality of P2X3 receptors as well as membrane depolarization. RESULTS: PSA-NCAM+ neural progenitors were isolated from a heterogeneous population of hDPSCs using magnetic-activated cell sorting and anti-PSA-NCAM MicroBeads. Flow cytometry analysis demonstrated that immunomagnetic sorting significantly increased the purity of PSA-NCAM+ cells. Immunofluorescent staining revealed expression of pan-neuronal and mature neuronal markers, PGP9.5 and MAP2, respectively, as well as weak expression of the mature sensory markers, peripherin and islet1. ATP-induced response was mediated predominately by P2X3 receptors in both undifferentiated and differentiated cells, with a greater magnitude observed in the latter. In addition, membrane depolarizations were also detected in cells before and after differentiation when loaded with fast-voltage-responding fluorescent molecule, FluoVolt™ in response to potassium chloride. Interestingly, only differentiated PSA-NCAM+ cells were capable of spontaneous membrane oscillations. CONCLUSIONS: In summary, DPSCs contain a population of neuronal progenitors with enhanced neural differentiation and functional neural-like properties that can be effectively isolated with magnetic-activated cell sorting (MACS).

In Vivo Regulation of Active Matrix Metalloproteinase-8 (aMMP-8) in Periodontitis: From Transcriptomics to Real-Time Online Diagnostics and Treatment Monitoring.

BACKGROUND: This study investigated in vivo regulation and levels of active matrix metalloproteinase-8 (aMMP-8), a major collagenolytic protease, in periodontitis. METHODS: Twenty-seven adults with chronic periodontitis (CP) and 30 periodontally healthy controls (HC) were enrolled in immunohistochemistry and transcriptomics analytics in order to assess Treponema denticola (Td) dentilisin and MMP-8 immunoexpression, mRNA expression of MMP-8 and its regulators (IL-1ß, MMP-2, MMP-7, TIMP-1). Furthermore, the periodontal anti-infective treatment effect was monitored by four different MMP-8 assays (aMMP-8-IFMA, aMMP-8-Oralyzer, MMP-8-activity [RFU/minute], and total MMP-8 by ELISA) among 12 CP (compared to 25 HC). RESULTS: Immunohistochemistry revealed significantly more Td-dentilisin and MMP-8 immunoreactivities in CP vs. HC. Transcriptomics revealed significantly elevated IL-1ß and MMP-7 RNA expressions, and MMP-2 RNA was slightly reduced. No significant differences were recorded in the relatively low or barely detectable levels of MMP-8 mRNAs. Periodontal treatment significantly decreased all MMP-8 assay levels accompanied by the assessed clinical indices (periodontal probing depths, bleeding-on-probing, and visual plaque levels). However, active but not total MMP-8 levels persisted higher in CP than in periodontally healthy controls. CONCLUSION: In periodontal health, there are low aMMP-8 levels. The presence of Td-dentilisin in CP gingivae is associated with elevated aMMP-8 levels, potentially contributing to a higher risk of active periodontal tissue collagenolysis and progression of periodontitis. This can be detected by aMMP-8-specific assays and online/real-time aMMP-8 chair-side testing.

TRPV2 modulates mechanically Induced ATP Release from Human bronchial epithelial cells.

Repetitive bouts of coughing expose the large airways to significant cycles of shear stress. This leads to the release of alarmins and the tussive agent adenosine triphosphate (ATP) which may be modulated by the activity of ion channels present in the human airway. This study aimed to investigate the role of the transient receptor potential subfamily vanilloid member 2 (TRPV2) channel in mechanically induced ATP release from primary bronchial epithelial cells (PBECs).PBECs were obtained from individuals undergoing bronchoscopy. They were cultured in vitro and exposed to mechanical stress in the form of compressive and fluid shear stress (CFSS) or fluid shear stress (FSS) alone at various intensities. ATP release was measured using a luciferin-luciferase assay. Functional TRPV2 protein expression in human PBECs was investigated by confocal calcium imaging. The role of TRPV2 inhibition on FSS-induced ATP release was investigated using the TRPV2 inhibitor tranilast or siRNA knockdown of TRPV2. TRPV2 protein expression in human lung tissue was also determined by immunohistochemistry.ATP release was significantly increased in PBECs subjected to CFSS compared with control (unstimulated) PBECs (N = 3, ***P < 0.001). PBECs expressed functional TRPV2 channels. TRPV2 protein was also detected in fixed human lung tissue. ATP release from FFS stimulated PBECs was decreased by the TRPV2 inhibitor tranilast (N = 3, **P < 0.01) (vehicle: 159 ± 17.49 nM, tranilast: 25.08 ± 5.1 nM) or by TRPV2 siRNA knockdown (N = 3, *P < 0.05) (vehicle: 197 ± 24.52 nM, siRNA: 119 ± 26.85 nM).In conclusion, TRPV2 is expressed in the human airway and modulates ATP release from mechanically stimulated PBECs.

An international consensus study to identify "what" outcomes should be included in a core outcome set for endodontic treatments (COSET) for utilization in clinical practice and research.

BACKGROUND: Development of a standardized set of topic-specific outcomes known as a Core Outcome Set (COS) is important to address issues of heterogeneity in reporting research findings in order to streamline evidence synthesis and clinical decision making. AIM: The aim of the current international consensus study is to identify "what" outcomes to include in the Core Outcome Set for Endodontic Treatments (COSET). Outcomes of various endodontic treatments (non-surgical root canal treatment, surgical endodontics, vital pulp treatment and revitalization procedures) performed on permanent teeth were considered. METHODS: A standard validated methodology for COS development and reporting was adopted. The process involved identification of existing outcomes through four published scoping reviews. This enabled creation of a list of outcomes to be prioritized via semi-structured patient interviews, e-Delphi process and a consensus meeting with a range of relevant global stakeholders. Outcomes were prioritized using a 1-9 Likert scale, with outcomes rated 7-9 considered critical, 4-6 are important and 1-3 are less important. Outcomes rated 7-9 by ≥70% and 1-3 by <15% of participants were considered to achieve consensus for inclusion in the COS. The outcomes that did not achieve consensus in the first round were considered for further prioritization in the second Delphi round and consensus meeting. Final decisions about the outcomes to include in COSET were made by voting during the consensus panel meeting using the Zoom Poll function. RESULTS: A total of 95 participants including patients contributed to the COS development process. The consensus panel recommended, with strong consensus, eight outcomes shared across all treatment modalities for inclusion in COSET: pain; signs of infection (swelling, sinus tract); further intervention/exacerbation; tenderness to percussion/palpation; radiographic evidence of disease progression/healing; function; tooth survival; and patient satisfaction. Additional treatment specific outcomes were also recommended. DISCUSSION: Many of the outcomes included in COSET are patient reported. All should be included in future outcomes studies. CONCLUSION: COSET identified outcomes that are important for patients and clinicians and validated these using a rigorous methodology. Further work is ongoing to determine "how" and "when" these outcomes should be measured.

Modulation of fast sodium current in airway smooth muscle cells by exchange protein directly activated by cAMP.

Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by NaV1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, INa, was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 µM), a ß-adrenergic agonist. Forskolin (1 µM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 µM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 µM), an activator of exchange protein directly activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 µM), an inhibitor of ryanodine receptors, and when cytosolic [Ca2+] was buffered by inclusion of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, Sigma (BAPTA) (50 µM) in the pipette solution, suggesting that the inhibition of INa was not due to Ca2+-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation V1/2, the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates INa in mouse ASM via Epac, but not PKA.NEW & NOTEWORTHY ß-adrenergic agonists are commonly used in inhalers to treat asthma and chronic obstructive pulmonary disease. These work by causing bronchodilation and reducing inflammation. The present study provides evidence that these drugs have an additional action, namely, to reduce sodium influx into airway smooth muscle cells via fast voltage-dependent channels. This may have the dual effect of promoting bronchodilation and reducing remodeling of the airways, which has a detrimental effect in these diseases.

Molecular biomarkers for objective assessment of symptomatic pulpitis: A systematic review and meta-analysis.

BACKGROUND: Inflammatory biomarkers are potentially useful targets for pulpal diagnostic tests that can identify pulp status and predict vital pulp treatment (VPT) outcome, however, their accuracy is unknown. OBJECTIVES: (1) Calculate sensitivity, specificity and diagnostic odds ratio (DOR) of previously investigated pulpitic biomarkers; (2) Determine if biomarker levels discriminate between clinical diagnoses of pulpitis based on the presence or absence of spontaneous pain (3) Evaluate if biomarker level can predict VPT outcome. METHODS: Searches: PubMed/MEDLINE, Ovid SP, Cochrane Central Register of Controlled Trials (CENTRAL), International Clinical Trials Registry Platform (ICTRP), ClinicalTrials.gov, Embase, Web of Science and Scopus in May 2023. INCLUSION: prospective and retrospective observational studies and randomized trials. Participants were humans with vital permanent teeth and a well-defined pulpal diagnosis. EXCLUSION: deciduous teeth, in vitro and animal studies. Risk of bias was assessed with modified-Downs and Black quality assessment checklist. Meta-analysis was performed using bivariate random effect model in Meta-DiSc 2.0 and RevMan and the quality of the evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation. RESULTS: Fifty-six studies were selected, reporting >70 individual biomolecules investigating pulpal health and disease at the gene and protein level. Most studies were of low and fair quality. Among the biomolecules investigated, IL-8 and IL-6 demonstrated a level of diagnostic accuracy with high sensitivity, specificity and DOR to discriminate between healthy pulps and those exhibiting spontaneous pain suggestive of IRP (low-certainty evidence). However, none was shown to have high DOR and the ability to discriminate between pulpitic states (very low certainty evidence). Limited data suggests high levels of matrix metalloproteinase 9 correlate with poorer outcomes of full pulpotomy. DISCUSSION: The inability of identified molecular inflammatory markers to discriminate between dental pulps with spontaneous and non-spontaneous pain should shift the focus to improved study quality or the pursuit of other molecules potentially associated with healing and repair. CONCLUSIONS: Low-quality evidence suggests IL-8 and IL-6 demonstrated level of diagnostic accuracy to discriminate between healthy pulps and those exhibiting spontaneous pain. There is a need for standardized biomarker diagnostic and prognostic studies focusing on solutions that can accurately determine the degree of pulp inflammation. REGISTRATION: PROSPERO CRD42021259305.

The Role of Extracellular Matrix (ECM) Adhesion Motifs in Functionalised Hydrogels.

To create functional tissue engineering scaffolds, biomaterials should mimic the native extracellular matrix of the tissue to be regenerated. Simultaneously, the survival and functionality of stem cells should also be enhanced to promote tissue organisation and repair. Hydrogels, but in particular, peptide hydrogels, are an emerging class of biocompatible scaffolds which act as promising self-assembling biomaterials for tissue engineering and regenerative therapies, ranging from articular cartilage regeneration at joint defects, to regenerative spinal cord injury following trauma. To enhance hydrogel biocompatibility, it has become imperative to consider the native microenvironment of the site for regeneration, where the use of functionalised hydrogels with extracellular matrix adhesion motifs has become a novel, emerging theme. In this review, we will introduce hydrogels in the context of tissue engineering, provide insight into the complexity of the extracellular matrix, investigate specific adhesion motifs that have been used to generate functionalised hydrogels and outline their potential applications in a regenerative medicine setting. It is anticipated that by conducting this review, we will provide greater insight into functionalised hydrogels, which may help translate their use towards therapeutic roles.

Oral health care in adult intensive care units: A national point prevalence study.

BACKGROUND: The importance of good oral hygiene for patients in Intensive Care Units (ICUs) is well recognized, however, the most effective way to achieve good oral care in the ICU is unclear. AIM: This study aimed to provide a national picture of oral care practices in adult ICUs in the United Kingdom (UK) to identify areas for improvement. STUDY DESIGN: A national one-day point prevalence study was undertaken in adult ICUs in the UK in the period from 30th September to 14th October 2021. Data were collected on all patients in the ICU on the date of data collection. Using a validated electronic data collection form, anonymised data were collected on methods and frequency of oral care provided, and the use of oral care protocols within the ICU. Data were analysed using descriptive analysis. RESULTS: Data from 195 patients in 15 ICUs in England, Wales and Northern Ireland were collected. Written oral care protocols were available for use in the care of 65% (n = 127) of patients. 73% (n = 142) of patients received oral care within the 24-h period. Oral care methods included toothbrushing 41% (n = 79), foam sticks 3% (n = 5), moisturizing the oral cavity 10% (n = 19) and mouth rinse with chlorhexidine 3% (n = 5) and other oral care methods not specified 12% (n = 23). 44% (n = 85) of patients had an oral assessment within the 24-h period and variable assessment methods were used. CONCLUSION: There is large variability in oral care provision and methods for intubated ICU patients and a lack of consensus was revealed in the study. Oral assessment is conducted less frequently using multiple tools. Optimal oral care standards and further research into oral care provision is pivotal to address this important patient-relevant practice. RELEVANCE TO CLINICAL PRACTICE: Oral care is a fundamental part of care for ICU patients, however, there is a large degree of variability, and oral care is often not based upon oral assessment. The use of an oral care protocol and oral assessments would help to improve patient care, ease of use for staff and provide a tailored oral care plan for patients, improving efficiency and preventing wasted resources.

The association of denture wearing with reduced lung function and increased airflow limitation in 58-72 year old men.

OBJECTIVE: To investigate the association between denture wearing and airflow limitation in men in Northern Ireland enrolled in the Prospective Epidemiological Study of Myocardial Infarction (PRIME) study. METHODS: A case-control design was used to study partially dentate men. Cases were men aged 58-72 years who were confirmed as denture wearers. Controls were never denture wearers who were matched by age (± 1 month) and smoking habit to the cases. The men had a periodontal assessment and completed a questionnaire detailing their medical history, dental history and behaviours, social circumstances, demographic background and tobacco use. Physical examination and spirometry measurements of forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were also undertaken. Spirometry data for edentulous men who wore complete dentures were compared with that recorded for the partially dentate men studied. RESULTS: There were 353 cases who were partially dentate and were confirmed denture wearers. They were matched for age and smoking habit to never denture wearer controls. The cases had an FEV1 that was on average 140 ml lower than the controls, p = 0.0013 and a 4% reduction in percent predicted FEV1, p = 0.0022. Application of the GOLD criteria indicated that 61 (17.3%) of the cases had moderate to severe airflow limitation compared with 33 (9.3%) of controls, p = 0.0051. Fully adjusted multivariable analysis showed that partially dentate men who were denture wearers were significantly more likely (p = 0.01) to have moderate to severe airflow reduction with an adjusted odds ratio (OR) of 2.37 (95% confidence intervals 1.23-4.55). In the 153 edentulous men studied moderate to severe airflow limitation was recorded in 44 (28.4%), which was significantly higher than in the partially dentate denture wearers (p = 0.017), and the men who had never worn a denture (p<0.0001). CONCLUSION: Denture wearing was associated with an increased risk of moderate to severe airflow limitation in the cohort of middle-aged Western European men studied.

Subgingival microbial diversity and respiratory decline: A cross-sectional study.

AIM: To investigate whether there is an association between subgingival microbial diversity and reduced respiratory function. MATERIALS AND METHODS: A group of dentate 58-72-year-old men in Northern Ireland had a comprehensive periodontal examination including subgingival plaque sampling. DNA was extracted from plaque samples and the V1-V3 regions of the 16S rRNA gene were analysed by high-throughput sequencing and a microbial diversity index (MDI) was derived. Spirometry measurements were made using a wedge bellows spirometer. The primary outcome variable of interest was the percentage of predicted forced expiratory volume in 1 s (% predicted FEV1 ). Analysis included multiple linear regression with adjustment for various confounders. RESULTS: Five-hundred and seven men were included in the analysis. The mean age was 63.6 years (SD = 3.1). Of these, 304 (60.0%) men had no or mild periodontitis, 105 (20.7%) had moderate periodontitis and 98 (19.3%) had severe periodontitis. Multiple linear regression analysis showed that a one unit increase in MDI was associated with a 0.71% loss (95% confidence interval: 0.06%-1.35%; p = .03) in % predicted FEV1 after adjustment for all confounders. CONCLUSIONS: In this group of dentate men from Northern Ireland, subgingival microbial diversity was associated with reduced respiratory function.

An exploratory study of maternal diabetes and offspring use of dental services-Northern Ireland national cohort study.

INTRODUCTION: A small number of literature has posited a link between prenatal exposure to gestational diabetes mellitus and an increased risk of developmental defects in the enamel of offspring. However, the evidence remains inconclusive. AIMS: This study examined the relationship between the diabetes status of mothers and the use of dental services by offspring to that pregnancy. MATERIAL AND METHODS: Anonymised data from a cohort of mothers who carried a child to term in Northern Ireland between 2012 and 2017 and service use by the child were taken from administrative databases from March 2015 to September 2021. Descriptive statistics, differences in means and regression analyses were used to examine the relationship between service use and maternal diabetes status, controlling for covariates. RESULTS: In multivariate analyses that controlled inter alia for age and deprivation, diabetes status was negatively related to restoration, extraction, prevention, and total service use. In the analysis of the COVID period, pre-COVID prevention was negatively related to extractions, restorations, prevention and services in general. CONCLUSION: The relationship between maternal diabetic status and aspects of offspring use of dental services was contrary to that suggested in previous studies and warrants more detailed investigation using this valuable data resource.

Quantitative oral health assessments in mechanically ventilated patients: A scoping review.

BACKGROUND: Oral health is a key contributor to a person's overall health. Previous studies indicate that oral health deteriorates throughout ventilation and may contribute to the development of ventilator-associated pneumonia (VAP). Oral health at the time of initial ventilation may impact on this deterioration. AIMS: To determine the quantitative clinical assessment methods used to measure oral health and what is currently known regarding the oral health of patients at the time of initial ventilation. STUDY DESIGN: A systematic literature search using electronic bibliographic databases MEDLINE/PubMed, Embase, CINAHL, and the Cochrane Library was undertaken for this scoping review. Studies were included if patients were >18 years old and mechanically ventilated for <48 h at the time of the first oral assessment. RESULTS: In total, 12 studies were included. The review demonstrates a limited understanding of clinical oral health at the time of initial ventilation. Significant variation in both assessment methods and reporting of oral health makes comparison of results difficult resulting in a poor overall understanding of oral health at the time of intubation. CONCLUSION: Standardized assessment and reporting methods may improve clinical application of findings and help direct future research. We suggest developing a core outcome set to ensure consistent use of assessment tools as well as standardized reporting of results. RELEVANCE TO CLINICAL PRACTICE: It is essential that a good understanding of oral health at the time of initial ventilation is gained so that patients receive more targeted oral hygiene intervention in ICU, potentially leading to improved patient outcomes.

Regulation of caries-induced pulp inflammation by NLRP3 inflammasome: A laboratory-based investigation.

AIM: To evaluate the expression and function of the nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome in caries induced pulpitis. METHODOLOGY: NLRP3 expression was determined with immunohistochemistry in the dental pulp and qPCR in dental pulp cells (DPCs). THP-1 macrophages expressing the apoptosis-related speck-like protein (ASC) and green fluorescent protein (GFP) fusion protein were used to assess NLRP3 inflammasome activation by live cell imaging, following treatment with lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Caspase I inhibitor was used to confirm inflammasome activation. An ex-vivo pulpitis model in which the DPCs were co-cultured with THP-1 macrophages was used to study the effect of the NLRP3 inflammasome inhibitor (MCC950), and cytokines were measured using ELISA and multiplex array. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at p ≤ .05. RESULTS: NLRP3 inflammasome was differentially expressed in dental pulp of sound and carious teeth. Treatment of DPCs with LTA significantly upregulates NLRP3 and IL-1 ß-expression (p < .05) and in induces more ASC specks formation compared to LPS. IL-ß release in response to LTA treatment is significantly reduced with Caspase I inhibitor suggesting inflammasome dependent mechanism (p < .01). NLRP3-specific inhibitor, MCC950, significantly reduced IL-1ß and IL-6 in an ex-vivo pulpitis model (p < .01) but had no effect on IL-8 or matrix metalloproteinase-9 (MMP-9). CONCLUSIONS: Expression and upregulation of NLRP3 inflammasome with caries and LTA treatment suggest a role in caries-induced pulpitis. NLRP3 inhibitor attenuated the release of selective inflammatory cytokines and could be a potential treatment target that merit further investigation.

M2 Muscarinic Receptor-Dependent Contractions of Airway Smooth Muscle are Inhibited by Activation of ß-Adrenoceptors.

Beta-adrenoceptor (ß-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting ß-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of ß-ARs. M2R-dependent contractions were apparent at low frequency (2Hz) electric field stimulation (EFS) and short (10s) stimulus intervals. The ß1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 s and was more effective at inhibiting contractions evoked by EFS at 2 than 20 Hz. Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the ß3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of ß1-ARs and that genetic ablation of M2Rs decreased the efficacy of ß-AR agonists on cholinergic contractions.

Functional expression of NaV1.7 channels in freshly dispersed mouse bronchial smooth muscle cells.

Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na+ was substituted with N-Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The V1/2s of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC50 = 1.5 nM) and the NaV1.7 subtype-selective blocker, PF-05089771 (IC50 = 8.6 nM), consistent with NaV1.7 as the underlying pore-forming α subunit. Two NaV1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-NaV antibody. RT-PCR, performed on groups of ∼15 isolated SMCs, revealed transcripts for NaV1.7 in 7/8 samples. Veratridine (30 µM), a nonselective NaV channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE2 (prostaglandin E2), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express NaV1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.

Outcomes reporting in systematic reviews on vital pulp treatment: A scoping review for the development of a core outcome set.

BACKGROUND: A large number of research reports on vital pulp treatment (VPT) has been published over the last two decades. However, heterogeneity in reporting outcomes of VPT is a significant challenge for evidence synthesis and clinical decision-making. OBJECTIVES: To identify outcomes assessed in VPT studies and to evaluate how and when outcomes are measured. A subsidiary aim was to assess evidence for selective reporting bias in the included studies. The results of this review will be used to inform the development of a core outcome set (COS) for endodontic treatments. METHODS: Multiple healthcare bibliographic databases, including PubMed/MEDLINE, Ovid EMBASE, Scopus, Cochrane Database of Systematic Reviews and Web of Science were searched for systematic reviews published between 1990 and 2020, reporting on VPT. Screening, data extraction and risk of bias assessment were completed independently by two reviewers. Outcomes' information was extracted and aligned with a healthcare taxonomy into five core areas: survival, clinical/physiological changes, life impact, resource use and adverse events. RESULTS: Thirty-six systematic reviews were included, 10 reporting on indirect pulp capping or selective caries removal, nine on direct pulp capping, eight on pulpotomy and nine on combined VPTs. There was considerable variation in the outcomes reported in these reviews and their included studies. Clinician-reported outcomes were used considerably more often than patient-reported outcomes. A range of instruments and time points were used for measuring outcomes. Several of the reviews were assessed as having low risk of selective reporting bias, but many did not specifically report this domain, whilst others did not provide risk of bias assessment at all. DISCUSSION: Considerable variation in selection of outcomes and how and when they are measured and reported was evident, and this heterogeneity has implications for evidence synthesis and clinical decision-making. CONCLUSIONS: Whilst there is a lack of consistency, several potentially important outcomes for VPT, including pulp survival, incidence of post-operative pain and need for further intervention, have been identified which could inform the development of a COS for endodontic treatment. REGISTRATION: Core Outcome Measures in Effectiveness Trials (COMET) (No. 1879).

Novel Antibacterial Properties of the Human Dental Pulp Multipotent Mesenchymal Stromal Cell Secretome.

It is well recognized that clearance of bacterial infection within the dental pulp precedes pulpal regeneration. However, although the regenerative potential of the human dental pulp has been investigated extensively, its antimicrobial potential remains to be examined in detail. In the current study bactericidal assays were used to demonstrate that the secretome of dental pulp multipotent mesenchymal stromal cells (MSCs) has direct antibacterial activity against the archetypal Gram-positive and Gram-negative bacteria, Staphylococcus aureus and Escherichia coli, respectively, as well as the oral pathogens Streptococcus mutans, Lactobacillus acidophilus, and Fusobacterium nucleatum. Furthermore, a cytokine/growth factor array, enzyme-linked immunosorbent assays, and antibody blocking were used to show that cytokines and growth factors present in the dental pulp MSC secretome, including hepatocyte growth factor, angiopoietin-1, IL-6, and IL-8, contribute to this novel antibacterial activity. This study elucidated a novel and diverse antimicrobial secretome from human dental pulp MSCs, suggesting that these cells contribute to the antibacterial properties of the dental pulp. With this improved understanding of the secretome of dental pulp MSCs and its novel antibacterial activity, new evidence for the ability of the dental pulp to fight infection and restore functional competence is emerging, providing further support for the biological basis of pulpal repair and regeneration.

Ficus septica exudate, a traditional medicine used in Papua New Guinea for treating infected cutaneous ulcers: in vitro evaluation and clinical efficacy assessment by cluster randomised trial.

BACKGROUND AND OBJECTIVES: Infected cutaneous ulcers are major health problems for children living in rural areas of Papua New Guinea. The inaccessibility of affected populations and lack of access to basic healthcare, make a local plant-based therapy an attractive treatment option. We assessed Ficus septica exudate in biological assays relevant to wound healing. We then carried out a clinical trial to determine the exudate's efficacy in healing small cutaneous ulcers compared with Savlon antiseptic cream, and soap and water washing. METHODS: Pre-clinical in vitro assessment of the exudate was carried out using assays to monitor the pro-inflammatory responses of M1 macrophages and neutrophils, antibacterial assays using known ulcer pathogens, an Ames test for mutagenicity and LC-MS chemical analysis of the exudate. An open label cluster-randomised clinical trial was performed, enrolling participants from three different clusters with skin lesions less than 1 cm in diameter. Each cluster comprising 50 participants was randomly assigned to one of three treatment arms namely topical exudate, topical Savlon antiseptic cream, and standard care (soap and water treatment), all administered daily for 2 days. The primary outcome was clinical healing/improvement measured at days 7 and 14, assessed by three dermatologists using blinded photographs. The primary analysis was assessed as non-inferiority of F. septica treatment based on the risk difference for healing/improvement. RESULTS: In vitro, the exudate which is rich in the alkaloid ficuseptine, was found to be non-mutagenic whilst also inhibiting pro-inflammatory responses and exhibiting antibacterial activity. When administered to participants enrolled in the clinical trial, no significant differences were observed between the healing efficacy of F. septica exudate and the two comparator treatments (Savlon antiseptic cream and soap/water treatment). At day 14, but not at day 7, the efficacy of F. septica exudate for healing/improving the ulcers was non-inferior to Savlon antiseptic cream or water/soap treatment. CONCLUSIONS: F. septica exudate is non-mutagenic and has both bactericidal and anti-inflammatory properties. When applied topically to small cutaneous ulcers, the exudate has a healing effect that is non-inferior to Savlon antiseptic cream and standard treatment with soap and water at day 14. Our findings, which should be confirmed in larger clinical trials, have important public health implications.

Differential regulation of NPY and SP receptor expression in STRO-1+ve PDLSCs by inflammatory cytokines.

OBJECTIVES: The aims of this study were to investigate neuropeptide receptor expression regulation on STRO-1 +ve periodontal ligament stem cells (PDLSCs) in response to inflammatory cytokines and to investigate a potential osteogenic effect of neuropeptides. BACKGROUND: Nerve fibres innervating the periodontal tissues in humans contain several neuropeptides including neuropeptide Y and substance P. The role of neuropeptide receptors on PDLSCs, including their response to the local inflammatory environment of periodontitis, is currently unknown. METHODS: A homogenous population of STRO-1 +ve PDLSCs was prepared by immunomagnetic separation of cells obtained by the tissue out-growth method from healthy premolar teeth from a single donor. Regulation of gene expression of the neuropeptide Y Y1 receptor and substance P receptor tachykinin receptor 1 was investigated. A potential osteogenic effect of neuropeptide Y and substance P was also investigated by measuring alkaline phosphatase (ALP) activity, Alizarin red staining and quantifying osteogenic gene expression. RESULTS: Treatment of STRO-1 +ve PDLSCs with tumour necrosis factor-alpha or interleukin 1-beta up-regulated the expression of the neuropeptide Y's Y1 receptor, but down-regulated substance P's receptor. Significantly increased ALP activity was observed in STRO-1 +ve PDLSCs treated with neuropeptide Y but not substance P. Further studies showed that neuropeptide Y had a modest osteogenic effect on cells at both a functional level and a gene level. CONCLUSIONS: Expression of the neuropeptide Y Y1 receptor gene on STRO-1 +ve PDLSCs was sensitive to local inflammatory cytokines. Treatment of cells with neuropeptide Y was found to produce a modest enhanced osteogenic effect.