Effectiveness of full Pulpotomy compared with Root canal treatment in managing teeth with signs and symptOms indicative of irreversible pulpitis: a protocol for prospectiVE meta-analysis of individual participant data of linked randomised clinical trials (PROVE).

BACKGROUND: Full pulpotomy has been proposed as an alternative to root canal treatment in teeth with signs and symptoms indicative of irreversible pulpitis (IRP), but the evidence is limited, relying on underpowered studies with a high risk of bias. The aim of this study is to conduct a prospective meta-analysis (PMA) of individual participant data of a series of individual randomised trials to provide robust evidence on the clinical and cost-effectiveness of pulpotomy compared with root canal treatment. METHODS: Individual participant data will be obtained from a series of randomised trials designed and conducted by a consortium of multi-national investigators with an interest in vital pulp treatment. These individualised trials will be conducted using a specified protocol, defined outcomes, and outcome measures. Ten parallel-group randomised trials currently being conducted in 10 countries will provide data from more than 500 participants. The primary outcome is a composite measure defined as (1) the absence of pain indicative of IRP, (2) the absence of signs and symptoms indicative of acute or chronic apical periodontitis, and (3) the absence of radiographic evidence of failure including radiolucency or resorption. Individual participant data will be obtained, assessed, and checked for quality by two independent reviewers prior to the PMA. Pooled estimates on treatment effects will be generated using a 2-stage meta-analysis approach. The first stage involves a standard regression analysis in each trial to produce aggregate data on treatment effect estimates followed by an inverse variance weighted meta-analysis to combine these aggregate data and produce summary statistics and forest plots. Cost-effectiveness analysis based on the composite outcome will be undertaken as a process evaluation to evaluate treatment fidelity and acceptability by patients and dentists. RESULTS: The research question and trial protocol were developed and approved by investigators in all 10 sites. All sites use shared resources including study protocols, data collection forms, participant information leaflets, and consent forms in order to improve flow, consistency, and reproducibility. Each site obtained its own Institutional Review Board approval, and trials were registered in appropriate open access platforms. Patient recruitment has started in most sites, as of July 2023. DISCUSSION: PMA offers a rigorous, flexible, and efficient methodology to answer this important research question and provide results with improved generalisability and external validity compared with traditional trials and retrospective meta-analyses. The results of this study will have implications for both the delivery of clinical practice and structured clinical guidelines' development. TRIAL REGISTRATION: PROSPERO CRD42023446809. Registered on 08 February 2023.

A protocol for the Development of Core Outcome Sets for Endodontic Treatment modalities (COSET): an international consensus process.

BACKGROUND: The outcome of endodontic treatment is generally assessed using a range of patient and clinician-centred, non-standardised clinical and radiographic outcome measures. This makes it difficult to synthesise evidence for systematic analysis of the literature and the development of clinical guidelines. Core outcome sets (COS) represent a standardised list of outcomes that should be measured and reported in all clinical studies in a particular field. Recently, clinical researchers and guideline developers have focussed on the need for the integration of a patient-reported COS with clinician-centred measures. This study aims to develop a COS that includes both patient-reported outcomes and clinician-centred measures for various endodontic treatment modalities to be used in clinical research and practice. METHODS: To identify reported outcomes (including when and how they are measured), systematic reviews and their included clinical studies, which focus on the outcome of endodontic treatment and were published between 1990 and 2020 will be screened. The COSs will be defined by a consensus process involving key stakeholders using semi-structured interviews and an online Delphi methodology followed by an interactive virtual consensus meeting. A heterogeneous group of key 'stakeholders' including patients, general dental practitioners, endodontists, endodontic teachers, clinical researchers, students and policy-makers will be invited to participate. Patients will establish, via interactive interviews, which outcomes they value and feel should be included in a COS. In the Delphi process, other stakeholders will be asked to prioritise outcomes identified from the literature and patient interviews and will have the opportunity at the end of the first round to add outcomes that are not included, but which they consider relevant. Feedback will be provided in the second round, when participants will be asked to prioritise the list again. If consensus is reached, the remaining outcomes will be discussed at an online meeting and agreement established via defined consensus rules of outcome inclusion. If consensus is not reached after the second round, a third round will be conducted with feedback, followed by the online meeting. Following the identification of a COS, we will proceed to identify how and when these outcomes are measured. DISCUSSION: Using a rigorous methodology, the proposed consensus process aims to develop a COS for endodontic treatment that will be relevant to stakeholders. The results of the study will be shared with participants and COS users. To increase COS uptake, it will also be actively shared with clinical guideline developers, research funders and the editors of general dental and endodontology journals. TRIAL REGISTRATION: COMET 1879. 21 May 2021.

Outcome measures to assess the effectiveness of endodontic treatment for pulpitis and apical periodontitis for use in the development of European Society of Endodontology (ESE) S3 level clinical practice guidelines: a protocol.

The European Society of Endodontology (ESE) is in the process of developing S3Level Clinical Practice Guidelines for the treatment of pulpal and apical disease for the benefit of clinicians and patients. In order to ensure a homogenous review process in the development of the clinical practice guidelines, it is essential that the core outcomes for all endodontic treatments are standardized and recommendations are made regarding minimum follow-up time specific to each outcome measure. In the absence of a recognized core outcome set in Endodontics, the current project aimed to follow an established consensus process to define the most appropriate clinician and patient-reported outcomes. As part of the project, recommendations will also be agreed regarding an acceptable minimum follow-up period for studies by literature review and group discussion. The selected outcome measures and follow-up periods will be used in subsequent systematic analyses of the literature to investigate the effectiveness of endodontic treatment to alleviate pulpitis and apical periodontitis. In this paper, previous reviews, ESE Guidelines and Position Statements were searched in order to compile a list of potentially important outcome measures for the treatment of pulpitis (working group 1), the nonsurgical treatment of apical periodontitis (working group 2), the surgical treatment of apical periodontitis (working group 3) and the regenerative treatment of apical periodontitis (working group 4). Initially, the two S3 guideline leads selected two independent senior clinical academics with experience of evidence-based dentistry to lead each of the four working groups forming a 10-member steering group. The working group leads in turn selected 32 academics with experience of evidence-based dentistry to lead the individual systematic reviews contained within the respective working groups. These 42 individuals make up the Guideline Development Group (GDG). Prior to the selected systematic reviewers commencing writing and submitting the review protocol, the complete list of outcome variables identified in this document will be ranked by the 42 members of the GDG in their importance to the individual patient using a 9-point Likert scale. A summary of the survey scores will thereafter be shared with the members of the group and the final list of clinician and patient-reported outcome measures rated as critical for decision making (7-9 on Likert scale by majority of survey participants) to guide systematic reviews will be consented and confirmed during an online meeting of the steering group. In this online meeting, another aspect with regard to meaningfulness of clinical trial results to be addressed in systematic reviews will be consented: length of follow-up. In order to develop high quality guidelines, it is suggested that the follow-up period after treatment should be related to the specific outcome measure being addressed; however, a minimum of one year for assessing the effectiveness of treatments for pulpitis and apical periodontitis should be considered. It is accepted, that selected research questions that focus on pain, swelling, medication taken or investigating diagnostic accuracy are likely to have shorter follow-up periods. As a result of the GDG consensus process, the outcome measures and length of follow-up will, alongside the use of standard instruments to assess the methodological quality of clinical trials and other comparative studies, be applied to all the commissioned systematic reviews that will inform the subsequent process when developing the ESE S3 Level Clinical Practice Guidelines.

Odontoblast cell death induces NLRP3 inflammasome-dependent sterile inflammation and regulates dental pulp cell migration, proliferation and differentiation.

AIM: To investigate the ability of dead odontoblasts to initiate NLRP3 inflammasome-dependent sterile inflammation and to explore the effect on dental pulp cell (DPCs) migration, proliferation and odontogenic differentiation. METHODS: Odontoblast-like cells were subjected to freezing-thawing cycles to produce odontoblast necrotic cell lysate (ONCL). DPCs were treated with ONCL to assess proliferation and migration. THP-1 differentiated macrophages stimulated with ONCL and live cell imaging and western blotting were used to assess NLRP3 inflammasome activation. Cytokines were measured with multiplex arrays and ELISA. qPCR, alkaline phosphatase and Alizarin red assays were used to assess odontogenic differentiation of DPCs. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at P ≤ 0.05. RESULTS: ONCL induced migration and proliferation of DPCs. Treatment of THP-1 macrophages with ONCL resulted in the release of the inflammatory cytokines IL-1ß, IL-6, IL-8, TNFα, IFN-γ, CCL2 and angiogenic growth factors, angiogenin and angiopoietin. This inflammatory response was associated with activation of NFκB, p38MAPK and NLRP3 inflammasome. To confirm that ONCL induced inflammatory response is NLRP3 inflammasome-dependent, treatment with a caspase-1 inhibitor and a specific NLRP3 inhibitor significantly reduced IL-1ß release in THP-1 macrophages (P = 0.01 and 0.001). Inflammasome activation product, IL-1ß, induced odontogenic differentiation of DPCS as evident by the increase in odontogenic genes expression DMP-1, RUNX-2, DSPP and SPP, alkaline phosphatase activity and mineralization. CONCLUSION: Dead odontoblasts induced NLRP3 inflammasome-dependent sterile inflammation and activated the migration, proliferation and differentiation of DPCs.

Efficacy of direct pulp capping for management of cariously exposed pulps in permanent teeth: a systematic review and meta-analysis.

BACKGROUND: The outcome of vital pulp treatment after carious pulp exposure is multifactorial and related to the procedure, biomaterial and pre-operative pulpal diagnosis. OBJECTIVES: To conduct a systematic review and meta-analysis determining the outcome of direct pulp capping (DPC) in mature permanent teeth with a cariously exposed pulp and a clinical diagnosis of reversible pulpitis, and ascertain whether the capping material influences the outcome. METHODS: Sources: MEDLINE Ovid-SP, Cochrane Central Register of Controlled Trials (CENTRAL), International Clinical Trials Registry Platform (ICTRP), ClinicalTrials.gov, Embase and Web of Science until April 2020. Inclusion: Prospective, retrospective cohort studies and randomized trials investigating DPC outcome or comparing different capping materials after carious pulp exposure. Exclusion: Primary teeth, mechanical, traumatic or not specified pulp exposure, teeth with irreversible pulpitis or no pulpal diagnosis. Risk of bias assessed using Cochrane and modified Downs and Black quality assessment checklist. Meta-analysis on combined clinical/radiographic outcome was performed using a random effect model. Success was defined as absence of signs and symptoms of irreversible pulpitis, apical periodontitis or loss of pulp vitality. RESULTS: Quality assessment highlighted four non-randomized studies to be of fair and five of poor quality. Four randomized trials had a high risk of bias. The pooled success rate differed based on material and follow-up. Calcium hydroxide success rate was 74% at 6-months, 65% at 1-year, 59% at 2-3 years and 56% at 4-5 years. Mineral trioxide aggregate (MTA) success was 91%, 86%, 84% and 81% at the same time points. Biodentine success was 96% at 6-months, 86% at 1 year and 86% at 2-3 years. The meta-analysis revealed MTA had better success than calcium hydroxide at 1-year (OR 2.66, 95% CI; 1.46- 4.84, P = 0.001) and 2- to 3-year follow-up (OR 2.21, 95% CI; 1.42-3.44, P = 0.0004). There was no difference between MTA and Biodentine. DISCUSSION: These results were based on poor methodological quality studies. The effect size for of MTA vs Ca(OH)2, although modest, was consistent with narrow CI. CONCLUSIONS: Low-quality evidence suggests a high success rate for direct pulp capping in teeth with cariously exposed pulps with better long-term outcomes for MTA and Biodentine compared with calcium hydroxide.

A connectivity mapping approach predicted acetylsalicylic acid (aspirin) to induce osteo/odontogenic differentiation of dental pulp cells.

AIM: To use connectivity mapping, a bioinformatics approach, to identify compounds that could induce odontogenic differentiation of dental pulp cells (DPCs) and to experimentally validate this effect. A subsidiary aim was to investigate the anti-inflammatory effect of any identified compound. METHODOLOGY: The Gene Expression Omnibus (GEO) database was searched for microarray data sets assessing odontogenic differentiation of human DPCs. An odontogenic gene expression signature was generated by differential expression analysis. The statistical significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. DPCs were treated with the compound identified, and osteo/odontogenic differentiation was assessed by Alizarin red staining, alkaline phosphatase activity and expression of osteo/odontogenic genes ALPL, RUNX2, COL1A1, DSPP, DMP1 and SPP1 by RT-PCR. The anti-inflammatory effect of the compound was assessed using an ex vivo pulpitis model, and cytokine levels were measured with multiplex assay. Means were compared using the t-test or ANOVA followed by a Bonferroni post hoc test with the level of significance set at P ≤ 0.05. RESULTS: The GEO database search identified a specific gene expression signature for osteo/odontogenic differentiation. Analysis using ssCMap found that acetylsalicylic acid [(ASA)/aspirin] was the drug with the strongest correlation with that gene signature. The treatment of DPCs with 0.05 mmol L-1 ASA showed increased alkaline phosphatase activity (P < 0.001), mineralization (P < 0.05), and increased the expression of the osteo/odontogenic genes, DMP1 and DSPP (P < 0.05). Low concentration (0.05 mmol L-1 ) ASA reduced inflammatory cytokines IL-6 (P < 0.001), CCL21 (P < 0.05) and MMP-9 (P < 0.05) in an ex vivo pulpitis model. CONCLUSIONS: Connectivity mapping, a web-based informatics method, was successfully used to identify aspirin as a candidate drug that could modulate the differentiation of DPCs. Aspirin was shown to induce odontogenic differentiation in DPCs in vitro and this, together with its anti-inflammatory effects, makes it a potential candidate for vital pulp therapies.

European Society of Endodontology position statement: Management of deep caries and the exposed pulp.

This position statement on the management of deep caries and the exposed pulp represents the consensus of an expert committee, convened by the European Society of Endodontology (ESE). Preserving the pulp in a healthy state with sustained vitality, preventing apical periodontitis and developing minimally invasive biologically based therapies are key themes within contemporary clinical endodontics. The aim of this statement was to summarize current best evidence on the diagnosis and classification of deep caries and caries-induced pulpal disease, as well as indicating appropriate clinical management strategies for avoiding and treating pulp exposure in permanent teeth with deep or extremely deep caries. In presenting these findings, areas of controversy, low-quality evidence and uncertainties are highlighted, prior to recommendations for each area of interest. A recently published review article provides more detailed information and was the basis for this position statement (Bjørndal et al. 2019, International Endodontic Journal, doi:10.1111/iej.13128). The intention of this position statement is to provide the practitioner with relevant clinical guidance in this rapidly developing area. An update will be provided within 5 years as further evidence emerges.

An audit on technical quality of root fillings performed by undergraduate students.

AIM: To evaluate radiographically the technical quality of root fillings performed by undergraduate dental students and to assess whether students were exposed to an appropriate endodontic case mix during their clinical training. METHODOLOGY: A retrospective audit was undertaken evaluating the clinical records of patients who underwent endodontic procedures during the period from September 2015 to June 2016 in the Dental School at Queen's University Belfast, UK. Two final-year dental students were trained and calibrated to evaluate postoperative intra-oral periapical radiographs of completed root canal treatments using specific assessment criteria. Data were presented as frequencies, percentage and mean ± standard deviation (SD). Comparisons of treatment outcomes between groups (posterior and anterior teeth) were calculated using Fisher's exact test, and the level of significance was set at P < 0.05. Intra- and interexaminer reproducibility was assessed by Kappa statistics. RESULTS: A total of 222 teeth and 381 canals were assessed, and of those, 253 (66%) of the root fillings were found to be acceptable in all the assessment parameters, namely taper, length and lateral adaptation of the root filling. Subanalysis of individual root filling parameters revealed that 372 canals (97%) exhibited good taper, and 275 canals (72%) were considered to be of an appropriate length, with 89 canals (23%) found to be underfilled and 17 canals (5%) overfilled. Overall 346 (91%) of canals had good lateral condensation. Students treated both single and multirooted teeth, and there was no significant association between tooth type and the quality of root filling provided (P > 0.05). CONCLUSIONS: In the majority of the teeth treated by undergraduate students at Queen's University Belfast, the technical quality of the root filling was acceptable and students were exposed to an appropriate case mix for endodontic training.

Putative periodontal pathogens in the subgingival plaque of Sudanese subjects with aggressive periodontitis.

BACKGROUND AND OBJECTIVES: There has been limited study of the bacterial species associated with aggressive periodontitis (AgP) in high-risk populations in Africa. The aim of this study was to investigate and quantify the presence of four putative periodontal pathogens in the subgingival plaque of Sudanese subjects with AgP. A secondary aim was to investigate the effect of varying the detection threshold on the reported prevalence of the bacterial species investigated. MATERIALS AND METHODS: Subgingival plaque samples were collected from AgP cases (n=73) and healthy controls (n=71). Bacterial DNA was extracted and analyzed by quantitative polymerase chain reaction for the detection and quantification of four putative periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola and Tannerella forsythia. RESULTS: At the lowest detection threshold (>101 cells), P. gingivalis (p<0.0001) was more prevalent in AgP cases than controls. T. forsythia and T. denticola had a high prevalence (>70%) in AgP cases at all detection levels. While T. forsythia was significantly more frequently identified in AgP than in controls at all detection thresholds, this was only the case for T. denticola at the intermediate threshold (>102 cells). A. actinomycetemcomitans was identified less frequently than the other bacterial species with no difference in its prevalence between AgP cases and controls. CONCLUSION: The prevalence of the putative periodontal pathogens investigated varied considerably in Sudanese subjects with AgP and in periodontally healthy controls depending on the detection thresholds applied. T. forsythia was identified as having the strongest association with AgP.

Matrix metalloproteinase-8 activity in gingival crevicular fluid: development of a novel assay.

BACKGROUND AND OBJECTIVE: The matrix metalloproteinases (MMPs) play a role in regulating turnover and metabolism of connective tissues in health but they have also been implicated in a wide variety of pathological conditions, including periodontal disease. MMP-8 has been extensively studied in periodontal health and disease using ELISA, although this technique is limited by its inability to determine enzyme activity. The aim was to develop an assay specifically to measure MMP-8 activity and to demonstrate its use in the analysis of gingival crevicular fluid samples. MATERIAL AND METHODS: A specific antibody was used to coat black 96-well microtitre plates to capture MMP-8 selectively. The activity of bound MMP-8 was measured using a fluorogenic substrate. Gingival crevicular fluid samples, from healthy and periodontally diseased sites, were collected using PerioPaper strips and tested for MMP-8 activity. RESULTS: Significantly higher MMP-8 activity was demonstrated in gingival crevicular fluid from periodontally diseased sites compared with healthy sites that exhibited basal or no MMP-8 activity. No cross-reactivity with other MMPs was noted. CONCLUSION: We show, for the first time, that MMP-8 activity can be specifically detected and quantified in gingival crevicular fluid samples. Measurement of MMP-8 activity could prove to be useful in monitoring periodontal disease progression.

PAR-2 regulates dental pulp inflammation associated with caries.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated enzymatically by proteolysis of an N-terminal domain. The cleavage and activation of PARs by serine proteases represent a novel mechanism by which such enzymes could influence the host inflammatory response. The aim of this study was to determine whether PAR-2 expression and activation were increased in dental caries. Using immunohistochemistry, we showed PAR-2 to be localized to pulp cells subjacent to caries lesions, but minimally expressed by healthy pulp tissue. Trypsin and the PAR-2 agonist (PAR2-AP) activated PAR-2 in an in vitro functional assay. Endogenous molecules present in pulp cell lysates from carious teeth specifically activated PAR-2, but those from healthy teeth failed to do so. The activation of PAR-2 in vitro was shown to increase the expression of the pro-inflammatory mediator cyclo-oxygenase-2 (COX-2), providing a mechanism whereby PAR-2 could modulate pulpal inflammation.

Quantum mechanical calculations of the cephalosporin nucleus.

A computational study using several ab initio methods, HF, DFT and MP2 at different basis set levels (6-31G*, 6-31G**, 6-311G** and 6-311++G**) has been performed to determine the possible stable conformations attained by the cephalosporin nucleus. The calculations were carried out in three stages by studying the conformational analysis of the 3-cephem nucleus, the 3-cephem-4-carboxylic acid nucleus and the acetylamino group of the 7-acetylamino-3cephem-4-carboxylic acid. In the first two stages, the potential energy surfaces indicated two minima that correspond to the S1-up and C2-up conformations, with the S1-up being more stable. The energy required for the interconversion of the S1-up to the C2-up is around 7 kcal/mol, indicating the feasibility of interconversion between the two conformers. In the third stage, the acetylamino group attained two conformations with respect to the 3-cephem nucleus. All the geometric parameters obtained in this study were found to be in reasonably good agreement with available X-ray diffraction data, even upon using a simple basis set.

Neuropeptide Y Y1 receptor in human dental pulp cells of noncarious and carious teeth.

AIM: To determine the distribution of the NPY Y1 receptor in carious and noncarious human dental pulp tissue using immunohistochemistry. A subsidiary aim was to confirm the presence of the NPY Y1 protein product in membrane fractions of dental pulp tissue from carious and noncarious teeth using western blotting. METHODOLOGY: Twenty two dental pulp samples were collected from carious and noncarious extracted teeth. Ten samples were processed for immunohistochemistry using a specific antibody to the NPY Y1 receptor. Twelve samples were used to obtain membrane extracts which were electrophoresed, blotted onto nitrocellulose and probed with NPY Y1 receptor antibody. Kruskal-Wallis one-way analysis of variance was employed to test for overall statistical differences between NPY Y1 levels in noncarious, moderately carious and grossly carious teeth. RESULTS: Neuropeptide Y Y1 receptor immunoreactivity was detected on the walls of blood vessels in pulp tissue from noncarious teeth. In carious teeth NPY Y1 immunoreactivity was observed on nerve fibres, blood vessels and inflammatory cells. Western blotting indicated the presence and confirmed the variability of NPY Y1 receptor protein expression in solubilised membrane preparations of human dental pulp tissue from carious and noncarious teeth. CONCLUSIONS: Neuropeptide Y Y1 is expressed in human dental pulp tissue with evidence of increased expression in carious compared with noncarious teeth, suggesting a role for NPY Y1 in modulation of caries induced pulpal inflammation.

Dental erosion among 12-14 year old school children in Khartoum: a pilot study.

OBJECTIVES: To investigate dental erosion among 12-14 year old Sudanese school children and evaluate the associated risk factors. BASIC RESEARCH DESIGN: Cross sectional survey in secondary schools in Khartoum city, Sudan. METHOD AND PARTICIPANTS: A sample of 157 school children was obtained from both private and public schools. Erosion on the labial and palatal surfaces of maxillary incisors was measured by criterion based on the Smith and Knight Tooth Wear Index. Dietary intake and other related factors were assessed using a questionnaire. RESULTS: The overall erosion prevalence in this group was 66.9%, of which 45.2% was mild and 21.7% was moderate erosion. A strong association was found between erosion and private schooling (higher socioeconomic groups), carbonated drinks, herbal hibiscus drink and traditional acidic food consumption. CONCLUSION: There was a high prevalence of dental erosion among Sudanese school children which was mild to moderate in severity and was strongly associated with acidic dietary intake

Personality analysis of patients complaining of sialorrhoea.

BACKGROUND: Sialorrhoea, the symptom of apparent excessive secretion of saliva is a relatively uncommon complaint. Some authors consider that in the absence of clinical findings, then these patients have a psychiatric disorder masquerading as a physical illness. However, there is little evidence in the literature to support this conclusion and a detailed psychological assessment of this population has not previously been reported. METHODS: In total, 18 patients and 18 age- and sex-matched controls were studied. All had a history of a complaint of excess salivation in the absence of any oral mucosal or systemic abnormality. All patients completed an Eysenck Personality Questionnaire. RESULTS: There were no differences in the extroversion of psychoticism scores between the study and control group. However, the result showed significant increases in the neuroticism and Lie Scale score in the patient group. CONCLUSIONS: The overall results of this study indicate that the complaint of sialorrhoea in otherwise healthy individuals does not have an organic basis and suggest that sialorrhoea is associated with high levels of neuroticism and a tendency to dissimulate.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue.

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted.