Neuropeptide Y regulation of dental pulp neurogenic inflammation provoked by tooth bleaching agents

Neuropeptide Y regulation of dental pulp neurogenic inflammation provoked by tooth bleaching agents | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Neuropeptide Y regulation of dental pulp neurogenic inflammation provoked by tooth bleaching agents Javier Caviedes-Bucheli, Mario Pérez-Villota, Karolina Aucú-Miño, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5375900/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: To assess the neuropeptide Y (NPY) expression in healthy human dental pulp following tooth bleaching with three in-office hydrogen peroxide-based systems: Opalescence Boost © (Ultradent Products, South Jordan, UT), Pola Office © (SDI, Victoria, Australia), and Zoom © (Zoom! Bleaching System; Discuss Dental, Culver City, CA). Methods: This observational study was designed following the STROBE guidelines. Forty dental pulps were collected from healthy premolars scheduled for extraction for orthodontic reasons. Teeth were divided into four groups containing ten healthy premolars each: Control group (n= 10): the teeth were not exposed to dental bleaching agents (healthy pulps assessed for normal/basal NPY values). Pola Office system group (n= 10): application of Pola office (35% H2O2) for 8 minutes. Opalescent Boost system group (n= 10): application of Opalescent Boost (40% H2O2) for 20 minutes. Zoom system group (n= 10): application of Zoom! (25% H2O2 + cold blue light) for 15 minutes. We rigorously followed the manufacturer's instructions for all bleaching systems. Following the extractions, the pulpal tissue was collected, placed in a 4% formaldehyde solution in Eppendorf tubes, and processed. NPY levels were measured using enzyme-linked immunosorbent assay (ELISA). Results: The expression levels of NPY in the dental pulp of human premolars showed the lowest value in the control group (0.0263 pmol/mg of pulp tissue), followed by the Zoom group (0.0314 pmol/mg of pulp tissue). An increase in expression was observed in the Pola Office group (0.0399 pmol/mg of tissue), with the highest NPY expression found in the Opalescence Boost group (0.0441 pmol/mg of pulp tissue), which demonstrated a significant difference compared to the control group (P = 0.03). Conclusion: NPY - whose expression is closely correlated with the bleaching agent's concentration and the intensity of the inflammatory response - mediates the pulp's responses to tooth bleaching agent-induced neurogenic pulp inflammation. NPY is expressed in the dental pulp in both pathological and physiological conditions. The highest NPY expression values were found in OPALESCENCE BOOST ® followed by POLA OFFICE ® , and ZOOM ® . Trial registration: Clinical Trials.gov ID NCT06606236. Tooth bleaching Dental pulp Neurogenic inflammation Neuropeptide Y Figures Figure 1 Background Human dental pulp is a specialized, highly vascularized, and innervated ecto-mesenchymal tissue, containing an array of cell types such as immune cells, fibroblasts, and odontoblasts. The dental pulp can launch an assortment of biological mechanisms to preserve homeostasis and pulp vitality in the aftermath of noxious stimuli such as tooth-bleaching procedures [ 1 ]. Tooth-bleaching products contain hydrogen peroxide (H2O2) as the active ingredient (Minoux and Serfaty, 2008). When activated by chemicals or physical activators (i.e., enzymes, light, or heat), H2O2 dissociates into free radicals such as hydroxyl (OH), oxygen (O), and perhydroxyl (HO2) the most reactive free radical, responsible for the dentin's bleaching effect [ 2 , 3 ]. H2O2-based tooth bleaching systems such as Pola Office (35% H2O2, SDI, Victoria, Australia), Opalescence Boost (40% H2O2, Ultradent Products, South Jordan, UT) and Zoom (25% H2O2 + cold blue light, Zoom! Bleaching System; Discuss Dental, Culver City, CA) are currently, the most popular in-office bleaching products. The free radicals released by these bleaching agents can diffuse through the enamel and dentin to reach the dental pulp, thus triggering a transitory reversible inflammatory phenomenon that clinically manifests as dentinal hypersensitivity, with a prevalence of 15% − 78% of cases [ 4 , 5 ]. Dentinal hypersensitivity is the most common side effect of this type of procedure [ 6 ]. The extent of the inflammatory phenomenon that leads to this clinical scenario depends on the H2O2 concentration, the free radicals' interaction time with the substrates, and the activators' molecular weight in each particular bleaching system [ 7 ]. The inflammatory response that arises from the passage of free radicals into the pulp tissue is neurogenic in nature, as the nervous system governs the vascular and immune systems through the release of potent neuropeptides. Somatosensory nerve fibre stimulation causes the release of Calcitonin gene-related peptide (CGRP), Substance P (SP), and Neurokinin A (NKA), whereas sympathetic and parasympathetic fibres release Neuropeptide Y (NPY) and Vasoactive intestinal peptide (VIP) respectively [ 8 ]. The neurogenic inflammation involves both myelinated Aδ and unmyelinated C fibres. Aδ and C fibres anastomose at the pulp tissue's periphery, interacting with the vascular component to form the so-called Rashkow plexus, responsible for regulating blood flow through neuropeptide influence. The release of SP, CGRP, and NKA provokes vasodilation and drives the migration of immune cells via chemotaxis, thus setting up an inflammatory response [ 3 ]. As a result, and as a compensatory mechanism, NPY, a strong vasoconstrictor, and VIP, a regulatory vasodilator, are released to preserve tissue homeostasis and control the inflammatory process established by the somatosensory neuropeptides. Additionally, NPY and VIP promote pulp tissue repair by binding to specific cells, such as odontoblasts, fibroblasts, and immune and dendritic cells [ 9 ]. Neuropeptides are released in response to orthodromic and/or antidromic stimuli [ 10 ]. An orthodromic stimulus occurs under physiological conditions, such as temperature fluctuations or masticatory function. In such situations, A-delta fibres become activated, releasing NPY or VIP neuropeptides to sustain tissue homeostasis [ 11 ]. In contrast, H2O2 free radicals that attack dental pulp generate an antidromic signal by exciting type C nerve endings. This leads to the release of SP and CGRP and sets off a defensive response that increases blood flow and intra-pulpal pressure through significant vasodilation and immune and inflammatory cell chemotaxis [ 3 ]. Notably, the release of NPY due to sympathetic system activation occurs in response to the inflammatory phenomena triggered by somatosensory neuropeptides. NPY inhibits acetylcholine and SP-induced vasodilation and amplifies the post-synaptic effects of other vasoconstrictors such as noradrenaline [ 12 ]. NPY-induced vasoconstriction reduces blood flow and intra-pulpal pressure via a compensating defensive mechanism against neurogenic inflammation [ 13 ]. NPY vasoconstriction modulates the pro-inflammatory vasodilatory impact of SP and CGRP, preventing pulp volume expansion and driving fluid movement in an antidromic direction into the dentinal tubules, resulting in dentinal hypersensitivity [ 11 ]. Therefore, NPY operates as a defensive mechanism by regulating the inflammatory process on dental pulp, hence minimizing post-bleaching dentinal hypersensitivity. Similarly, it has been proposed that the presence of NPY-1 receptors in fibroblasts, undifferentiated cells, and odontoblasts may be linked to pulp mineralization processes as a defence mechanism [ 14 , 15 ]. In light of the preceding, this study aims to establish the NPY levels expressed in human dental pulps of recently extracted premolars, following the application of Pola Office, Opalescence Boost, and Zoom, bleaching systems to identify potential control mechanisms of neurogenic pulp inflammation and dentinal hypersensitivity that developed in response to tooth-bleaching procedures. Methods The present study was conducted using the CONSORT 2010 guidelines for clinical trials. It follows the recommendations of the Colombian Ministry of Health on ethical issues in research with human tissues. The bioethics committee approved the study under resolution BIO407—Acta No.12, May 25, 2023. Written informed consent was obtained from each patient who participated in the study. The study protocol was registered in Clinical Trials.gov ID NCT06606236. Forty (n = 40) dental pulps were collected from healthy non-smoking human donors aged 18 to 27 who underwent premolar extractions for orthodontic purposes. All premolars were caries and restoration-free, with complete root development, confirmed both visually and radiographically, with normal response to sensitivity testing, and no evidence of periodontal disease, traumatic occlusion, or previous orthodontic force application. Teeth with similar features were selected, to ensure the results' validity and reliability while limiting bias and any confounding factors. Samples were divided into four groups containing ten healthy premolars each (without discriminating between maxillary and mandibular premolars): (1) Control group (n = 10): the teeth were not exposed to dental bleaching agents (healthy pulps with normal/basal NPY values). (2) Pola Office system group (SDI, Victoria, Australia) (n = 10): application of Pola office (35% H2O2) for 8 minutes. (3) Opalescent Boost system group (Ultradent Products, South Jordan, UT) (n = 10): application of Opalescent Boost (40% H2O2) for 20 minutes. (4) Zoom system group (Zoom! Bleaching System; Discuss Dental, Culver City, CA) (n = 10): application of Zoom! (25% H2O2 + cold blue light) for 15 minutes. We rigorously followed the manufacturer's instructions for all bleaching systems (Fig. 1 ). A preliminary sensitivity test was conducted to assess the normal state of the pulp. This involved using a pulp tester to evaluate the pulp's response to an electrical stimulus. A cold test was performed using Endo Ice at -40 degrees Celsius to confirm the results further. These tests collectively verified that the sensitivity of the premolars was within normal limits. Clinical procedures and sample Collection Teeth were anaesthetized immediately following toot-bleaching, with 3% mepivacaine without vasoconstrictor infiltration injection for maxillary premolars and inferior alveolar nerve block injection for mandibular premolars. Ten minutes later, teeth were extracted by conventional methods using a supra-osseous luxation and extraction technique. The extraction process did not exceed five minutes. For the control group, extraction was also performed ten minutes after the anaesthetic application. After extraction, all teeth were rinsed with 5.25% sodium hypochlorite to remove any remaining periodontal ligament that could contaminate the pulp sample. The teeth were sectioned with a Zekrya bur (Dentsply, Tulsa, OK) in a high-speed handpiece irrigated with saline solution. Pulp tissue was collected using a sterile endodontic excavator, deposited on an Eppendorf tube with 1 ml 4% Paraformaldehyde, and stored at 70°C until use [ 13 ]. Enzyme-linked immunosorbent assay (ELISA) 50 µL of each standard, blank, and sample were added to the corresponding wells. All samples and standards were duplicated. The sample dilution was established by preliminary experiments. 50 µL/well of working solution with biotinylated antibody was added. The plate was covered with sealing film and incubated for 45 min at 37°C. After decanting the solution, each well was dispensed with 350 µL of Wash Buffer (1 minute/soak). Plates were washed three times and gently blotted before adding 100 µL horseradish peroxidase-conjugated working solution to each well. The plate was covered with a new sealing film and incubated at 37°C for 30 minutes. After washing plates five times as previously described, the substrate was added (50 µL/well). The plate was incubated for 15min at 37°C. The plate was protected from light by wrapping it in aluminium foil, which caused a colour change. 50 µL/well of stop solution was added in the same order as the substrate solution was previously added. Each well's optical density (OD) was determined with a microplate reader at 450 nm [ 16 ]. Statistical analysis For each of the bleaching techniques analyzed, mean and standard deviation, as well as minimum and maximum values for NPY, expressed in pmol/mg of pulp tissue, were calculated. A Kolmogorov-Smirnov test was applied to each series of values ​​corresponding to each bleaching technique to determine whether they were normally distributed. Finally, an ANOVA test was performed to determine if there were statistically significant differences between the different files evaluated, as well as post-hoc Tukey tests at a significant level of p < 0.05. Results Forty pulp tissue samples were analyzed, classified as follows: 10 from teeth bleached with Pola Office, 10 with Opalescence Boost, 10 with Zoom! and 10 from intact teeth that were used as the control group. Table 1 describes the NPY expression in pmol per mg of the pulp tissue, finding that the lowest expression was found in the control group, followed by the Zoom! Pola Office and Opalescence Boost systems respectively (Anova p = 0.03). Table 1 Expression of Neuropeptide Y in dental pulps after different bleaching techniques Bleaching Technique n Mean St. Dev. Minimum Maximum CONTROL GROUP 10 0.02626 0.0066 0.01241 0.04704 POLA OFFICE 10 0.03994 0.0165 0.01107 0.07509 OPALESCENCE BOOST 10 0.04407 0.0179 0.01924 0.08645 ¡ZOOM! 10 0.03142 0.0125 0.00592 0.6834 Anova p = 0.03 Table 2 shows the Tukey post-hoc comparisons, where it can be observed that the only statistically significant difference was between the control group and Opalescence Boost (p = 0.04). All other pairwise comparisons did not show statistically significant differences. Table 2 Post-hoc comparisons (Tukey Test) (I) Bleaching Agent (II) Bleaching Agent Sig. CONTROL GROUP POLA OFFICE 0.15 OPALESCENCE BOOST 0.04 ZOOM! 0.85 POLA OFFICE OPALESCENCE BOOST 0.91 ZOOM! 0.54 OPALESCENCE BOOST ZOOM! 0.20 Discussion Tooth bleaching, one of the most prevalent procedures in modern dentistry, is proven to elicit a reversible pulpal inflammatory response, as free radicals dissociated from H2O2 diffuse through enamel and dentin into the pulp tissue. Such inflammatory response, clinically recognised as post-bleaching dentinal hypersensitivity, is strongly related to the bleaching system's H2O2 concentration, exposure time, and activation mode [ 1 , 2 , 4 , 5 , 8 ]. Tooth bleaching results from the dissociation of free radicals from H2O2. Free radicals such as O, OH, and HO2 permeate the enamel and dentin, oxidizing the chromophores, and breaking the double bonds of organic chemical dyes that stain teeth [ 17 ]. H2O2 free radicals activate somatosensory nerve terminals in the Rashkow plexus [ 9 ], leading to the up-regulated expression of SP, CGRP, and NKA from Aδ y C fibres, resulting in transient pulp inflammation. If the inflammatory phenomenon is not properly regulated, it can lead to odontoblast and fibroblast apoptosis, resulting in premature pulp ageing, which jeopardizes the pulp tissue's protective mechanisms and homeostasis [ 4 , 8 , 18 ]. The inflammatory pulp phenomenon brought on by tooth bleaching procedures elicits an antidromic stimulus, characterized by rising blood flow and intrapulpal pressure, thereby increasing the dental pulp volume and promoting fluid flow towards the dentinal tubules. Clinically, this condition is described as post-bleaching dentinal hypersensitivity [ 19 ]. NPY is released by exocytosis from sympathetic Aδ and type C fibres as a compensatory anti-inflammatory mechanism and to modulate dentinal hypersensitivity. NPY binds to target cells via the NPY-1 receptor, expressed in fibroblasts, endothelial cells, macrophages, odontoblasts, and undifferentiated dental pulp cells, competing and antagonizing with pro-inflammatory somatosensory neuropeptides such as SP, CGRP, and NKA [ 9 , 11 , 12 , 20 ]. NPY exerts a vasoconstrictor effect, aided by norepinephrine at the post-synaptic level, thus counteracting vasodilation generated by SP and CGRP [ 13 ]. Furthermore, NPY promotes pulp tissue repair by attaching to its receptor NPY-1 in target cells such as macrophages responsible for degrading H2O2 free radicals [ 21 ]. Likewise, NPY drives endothelial cells to promote angiogenesis, fibroblasts to renew and repair the extracellular matrix, and dental pulp stem cells to undergo odontoblastic differentiation, with the ultimate aim of producing tertiary dentin even up to 21 days after exposure to H2O2 based-agents [ 18 , 20 ]. All of these regulatory and repairing mechanisms lead the inflammatory reaction to decline a few days post-tooth-bleaching, resulting in the control of dentinal hypersensitivity by restricting fluid flow inside the dentinal tubules [ 19 ]. Finally, when dental pulp is exposed to H2O2 free radicals, the up-regulated expression of dismutase and catalase, which enzymatically break down the peridroxyl ion, provides a protective impact on the pulp cells and preserves pulp vitality over time [ 9 ]. Three different in-office tooth bleaching systems — POLA OFFICE® (SDI, Victoria, Australia), OPALESCENCE BOOST® (Ultradent Products, South Jordan, UT), and Zoom® (Zoom! Bleaching System; Discuss Dental, Culver City, CA) — were used in this study to quantify NPY expression in human dental pulp following tooth bleaching procedures. We rigorously followed the manufacturer's instructions, as the detrimental impacts of H2O2 on pulp tissue are proportional to the concentration, application time, and activation method [ 22 ]. Depending on NPY expression, it may be feasible to infer how this neuropeptide controls the neurogenic inflammatory phenomenon following tooth bleaching procedures, as well as provide a scientific explanation for the nature of post-bleaching dentinal hypersensitivity and its regulatory mechanisms [ 19 ]. The ELISA test was performed to quantify NPY expression. It has been established that the ELISA test is sensitive enough to quantify neuropeptides in pmol/mg of pulp weight, the most generally used unit of measurement for neuropeptide expression in freshly extracted human dental pulp [ 13 , 23 ]. Considering the lack of previous research on NPY expression following tooth bleaching, a direct comparison was conducted between basal levels (pre-bleaching) and pulp inflammation (post-bleaching) in healthy premolars requiring extraction for orthodontic reasons [ 13 ]. All groups received 3% mepivacaine without a vasoconstrictor as a local anaesthetic to prevent alpha-adrenergic agonists from minimising neuropeptide expression. To allow NPY expression, a 10-minute interval was set aside following removing the bleaching agent [ 23 ]. Teeth were extracted using a supra-osseous luxation and extraction technique, which should not take longer than five minutes to prevent endogenous endopeptidases from degrading the neuropeptide and to quantify the NPY expression linked to the bleaching procedure rather than the dental extraction [ 3 , 13 ]. The control group exhibited an overall NPY expression of 0.026 pmol/mg of pulp tissue demonstrating that NPY is present in human dental pulp under physiological conditions, performing homeostatic and regulatory activities in inflammatory reactions. These values are consistent with previous studies employing radioimmunoassay for assessing NPY expression [ 13 ]. The ZOOM system group (25% H2O2) achieved an average NPY expression of 0.031 pmol/mg of pulp tissue, after 15 minutes of application and activation with a cold blue light. However, there was no significant difference ( P ˃ 0.05) compared to the control group's reference values. These results can be explained by the fact that the ZOOM bleaching system uses the lowest concentration of the bleaching agents evaluated and its activation medium is a cold light lamp. Notably, findings from this study differ from previous research in which SP expression in human dental pulp was assessed in response to 25% ZOOM bleaching with hot light activation. The ZOOM system was found to elicit the highest SP expression, most likely due to the increased release of H2O2 free radicals by the activation source [ 3 , 24 ]. Confirming that the activation method affects the expression of neuropeptides in the dental pulp and therefore the severity of neurogenic inflammation [ 3 , 25 ]. NPY expression is directly proportional to the amounts of H2O2 free radicals that enter the pulp tissue. The Pola Office system group (35% H2O2) with 8 minutes of application and the Opalescence Boost system group (40% H2O2) with 20 minutes of application displayed an average expression of 0.039 pmol/mg of pulp tissue and 0.044 pmol/mg of pulp tissue respectively. There was no significant difference ( P ˃ 0.05) between these two groups. Notably, an increased NPY expression compared to the control group was evident. Opalescence Boost was the only group that showed statistically significant differences ( P ˂ 0.05) compared to the control group, implying that the NPY expression is directly proportional to the concentration of the bleaching agent rather than the exposure time. Further, it can be inferred that the Pola Office and Opalescence Boost system groups trigger a more intense inflammatory phenomenon, as a higher NPY expression occurs, attempting to preserve pulp tissue homeostasis [ 3 , 8 ]. The release of NPY counteracts the harmful effects of free radicals, hence reducing the inflammatory response. NPY promotes protective vasoconstriction, thus decreasing blood flow, intrapulpal pressure, and inflammatory cell chemotaxis, while also inhibiting neural activity by blocking SP and CGRP. Since the dental pulp does not expand in response to the neurogenic inflammatory phenomena, the antidromic pressure of dentinal fluid is lowered, therefore, minimizing post-bleaching dentinal hypersensitivity [ 11 , 19 , 26 ]. High bleaching agent concentrations trigger neurogenic inflammation with antidromic effects, leading to fluid movement from the pulp to the dentinal tubules. This stimulates type C and Aδ somatosensory fibres which release SP and CGRP. As a control and compensation mechanism for the inflammatory phenomenon, NPY is released to oppose somatosensory neuropeptides, thus making pulp inflammation reversible. However, as a long-term adverse effect, it may result in premature ageing of dental pulp by eliciting pro-angiogenic defensive reactions [ 1 , 23 ]. Similarly, it has been proposed that NPY binding to its specific receptor NPY-1 in fibroblasts, undifferentiated mesenchymal cells, and odontoblasts may be linked to pulp mineralization processes as a defence mechanism. Apoptosis of these cells has also been observed when the effect of free radicals generated by dental bleaching surpasses the pulp tissue's tolerance [ 8 , 9 , 27 ]. If the aggression of free radicals from tooth bleaching systems exceeds the tolerance of the pulp tissue's defence mechanisms, as a result of the use of high concentrations of H2O2 and improper application times, which perpetuate the inflammatory phenomenon and thus the antidromic stimuli, irreversible pulpitis associated with the overexpression of NKA, SP, and CGRP may develop. In this scenario, neither parasympathetic nor sympathetic neuropeptides, like VIP and NPY can control the inflammatory response. This can lead to the destruction of immature odontoblasts and undifferentiated cells' DNA [ 4 , 8 , 18 ], the degeneration of pulp fibroblasts, and even irreversible damage of immune cells such as macrophages, which can prevent pulp tissue from being repaired and prompt asymptomatic pulp necrosis [ 27 ]. Results from this study in terms of NPY expression and its potential relationship to post-bleaching dentinal hypersensitivity are supported by previous clinical studies that compared the level of dentinal hypersensitivity experienced by patients following tooth bleaching therapies with Zoom, Pola Office, and Opalecense boost. Following the Opalescence boost application, a lower percentage (15%) of dentinal hypersensitivity was observed, which is consistent with the findings of this study, in which this bleaching agent showed the highest levels of NPY expression, confirming that NPY expression is directly related to inflammation and dentinal hypersensitivity control. The percentage of dentinal hypersensitivity was higher with Pola Office (39%), which agrees with the NPY expression value of this study, which was lower than that of Opalecense boost. Finally, the zoom group had the highest percentage of dentinal hypersensitivity (91%), which agrees with the lower expression of NPY in this study [ 4 , 28 ]. The foregoing results suggest NPY regulates neurogenic inflammation triggered by tooth bleaching, which manifests as dentinal hypersensitivity. Future research on this topic should focus on quantifying and comparing the expression of NPY to that of its receptor, NPY-Y1, and linking these expressions to clinical studies assessing post-bleaching dentinal hypersensitivity aiming at inferring its control mechanisms. Conclusion Within the limitations of this in-vivo study, it can be established that NPY - whose expression is closely correlated with the bleaching agent's concentration and the intensity of the inflammatory response - mediates the pulp's responses to tooth bleaching agent-induced neurogenic pulp inflammation. Results of this study show that NPY is expressed in the dental pulp in pathological and physiological conditions. The highest NPY expression values ​​were found in OPALESCENCE BOOST® followed by POLA OFFICE®, and ZOOM®. Abbreviations Hydrogen peroxide (H2O2), Hydroxyl (OH), Oxygen (O), Perhydroxyl (HO2), Calcitonin gene-related peptide (CGRP), Substance P (SP), Neurokinin A (NKA), Neuropeptide Y (NPY), Vasoactive intestinal peptide (VIP), Optical density (OD), Enzyme-linked immunosorbent assay (ELISA). Declarations Ethics approval and consent to participate The study was approved by the bioethics committee, under resolution BIO407- Acta No.12, May 25, 2023. All participants provided written informed consent before the study procedures. The study was conducted according to principles outlined in the Helsinki Declaration. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding The authors received no specific funding for this work. Authors Contribution J. C: Conceptualization, Methodology, Project Administration, Supervision, Writing. M.A: Investigation, Resources, Software. K. A: Investigation, Resources, Software. D. M: Investigation, Resources, Software. H. D. M: Data curation, Resources, Supervision. N. R: Formal Analysis, Validation, Writing-original draft preparation. J. F. G: Formal Analysis, Validation, Writing-original draft preparation. L. E. D: Investigation, Methodology. E. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5375900","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":377216267,"identity":"9cb66d9e-9e16-4566-8fd9-7cc0906ca06c","order_by":0,"name":"Javier Caviedes-Bucheli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBUlEQVRIiWNgGAWjYBACxgYGhgNgFjMDgwQDgw1IrPEAKVrSwGJ4taAAoJbDYAZeLcztZwwPfNxjJ29wnPngjQ9/ztutbT8MtKXGJhqnw3pyDA7OeJZsuOEwW7LlzLbbydvOJAK1HEvLbcDpl7SEwzwHDjDObOYxk+ZtuJ1sdgCohbHhMG4t/c8SDv85cMB+ZjP/N+k/f84lm51/SEDLjOQDhxkOHEjsZ+Zhk2ZgO2BndoOQLTMeHzjYcyA5uZ+Zzdiyty05wewG0JYEPH4x7E9s/vDjgJ1tG//hhzd+/LGzNzuf/vDBhxob3FrQJRLBAgk4lIOAPLqAPR7Fo2AUjIJRMEIBAKNBaey4BXaDAAAAAElFTkSuQmCC","orcid":"","institution":"Pontificia Universidad Javeriana","correspondingAuthor":true,"prefix":"","firstName":"Javier","middleName":"","lastName":"Caviedes-Bucheli","suffix":""},{"id":377216268,"identity":"5ecbdcca-ec60-4c18-b48b-72ddb6cfee3b","order_by":1,"name":"Mario Pérez-Villota","email":"","orcid":"","institution":"Cooperative University of Colombia","correspondingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Pérez-Villota","suffix":""},{"id":377216269,"identity":"112e95fc-bcbb-4bb8-919c-9ed539aa01c0","order_by":2,"name":"Karolina Aucú-Miño","email":"","orcid":"","institution":"Cooperative University of Colombia","correspondingAuthor":false,"prefix":"","firstName":"Karolina","middleName":"","lastName":"Aucú-Miño","suffix":""},{"id":377216270,"identity":"e455b573-ee0f-442b-a87f-5c53f2285ae1","order_by":3,"name":"Diana Escobar-Mafla","email":"","orcid":"","institution":"Cooperative University of Colombia","correspondingAuthor":false,"prefix":"","firstName":"Diana","middleName":"","lastName":"Escobar-Mafla","suffix":""},{"id":377216271,"identity":"93305ab3-5158-4ec5-94c2-f155e9d7edee","order_by":4,"name":"Hernan Dario Muñoz-Alvear","email":"","orcid":"","institution":"Cooperative University of Colombia","correspondingAuthor":false,"prefix":"","firstName":"Hernan","middleName":"Dario","lastName":"Muñoz-Alvear","suffix":""},{"id":377216272,"identity":"714a35d6-9549-4338-aad0-1fb56069c7c2","order_by":5,"name":"Nestor Rios-Osorio","email":"","orcid":"","institution":"El Bosque University","correspondingAuthor":false,"prefix":"","firstName":"Nestor","middleName":"","lastName":"Rios-Osorio","suffix":""},{"id":377216273,"identity":"e951be01-5150-4407-bd43-37c4b4f2cc2c","order_by":6,"name":"Jose Francisco Gomez-Sosa","email":"","orcid":"","institution":"Instituto Venezolano de Investigaciones Científicas","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"Francisco","lastName":"Gomez-Sosa","suffix":""},{"id":377216274,"identity":"0dbb154b-dcb3-4269-973b-3626e7e2dca1","order_by":7,"name":"Luis Eduardo Diaz-Barrera","email":"","orcid":"","institution":"University of La Sabana","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"Eduardo","lastName":"Diaz-Barrera","suffix":""},{"id":377216275,"identity":"148f5ab3-f4c6-4502-b3a1-d58c103ed6c8","order_by":8,"name":"Edgar Güiza – Cristancho","email":"","orcid":"","institution":"Cooperative University of Colombia","correspondingAuthor":false,"prefix":"","firstName":"Edgar","middleName":"Güiza –","lastName":"Cristancho","suffix":""},{"id":377216276,"identity":"e40d7106-a12b-4c06-90b2-53ea83dd44e0","order_by":9,"name":"Hugo Roberto Munoz","email":"","orcid":"","institution":"University of San Carlos of Guatemala","correspondingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"Roberto","lastName":"Munoz","suffix":""}],"badges":[],"createdAt":"2024-11-02 01:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5375900/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5375900/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69923168,"identity":"46867970-e94c-4099-aefe-8e02400d6e49","added_by":"auto","created_at":"2024-11-26 15:50:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart. \u003c/strong\u003eThe sample size was calculated using parameters from earlier studies \u003cstrong\u003e[3].\u003c/strong\u003e Forty samples of human dental pulp were collected from healthy premolars scheduled for extraction for orthodontic reasons.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5375900/v1/4e1d85cdb67ef51c95b57cae.png"},{"id":74032770,"identity":"a9fb2111-837a-4be6-b364-192c22852e19","added_by":"auto","created_at":"2025-01-17 07:09:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":710884,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5375900/v1/1b868983-a5c1-4d2a-a3a7-33938fad1f89.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neuropeptide Y regulation of dental pulp neurogenic inflammation provoked by tooth bleaching agents","fulltext":[{"header":"Background","content":"\u003cp\u003eHuman dental pulp is a specialized, highly vascularized, and innervated ecto-mesenchymal tissue, containing an array of cell types such as immune cells, fibroblasts, and odontoblasts. The dental pulp can launch an assortment of biological mechanisms to preserve homeostasis and pulp vitality in the aftermath of noxious stimuli such as tooth-bleaching procedures [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTooth-bleaching products contain hydrogen peroxide (H2O2) as the active ingredient (Minoux and Serfaty, 2008). When activated by chemicals or physical activators (i.e., enzymes, light, or heat), H2O2 dissociates into free radicals such as hydroxyl (OH), oxygen (O), and perhydroxyl (HO2) the most reactive free radical, responsible for the dentin's bleaching effect [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eH2O2-based tooth bleaching systems such as Pola Office (35% H2O2, SDI, Victoria, Australia), Opalescence Boost (40% H2O2, Ultradent Products, South Jordan, UT) and Zoom (25% H2O2\u0026thinsp;+\u0026thinsp;cold blue light, Zoom! Bleaching System; Discuss Dental, Culver City, CA) are currently, the most popular in-office bleaching products. The free radicals released by these bleaching agents can diffuse through the enamel and dentin to reach the dental pulp, thus triggering a transitory reversible inflammatory phenomenon that clinically manifests as dentinal hypersensitivity, with a prevalence of 15% \u0026minus;\u0026thinsp;78% of cases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Dentinal hypersensitivity is the most common side effect of this type of procedure [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The extent of the inflammatory phenomenon that leads to this clinical scenario depends on the H2O2 concentration, the free radicals' interaction time with the substrates, and the activators' molecular weight in each particular bleaching system [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe inflammatory response that arises from the passage of free radicals into the pulp tissue is neurogenic in nature, as the nervous system governs the vascular and immune systems through the release of potent neuropeptides. Somatosensory nerve fibre stimulation causes the release of Calcitonin gene-related peptide (CGRP), Substance P (SP), and Neurokinin A (NKA), whereas sympathetic and parasympathetic fibres release Neuropeptide Y (NPY) and Vasoactive intestinal peptide (VIP) respectively [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The neurogenic inflammation involves both myelinated Aδ and unmyelinated C fibres. Aδ and C fibres anastomose at the pulp tissue's periphery, interacting with the vascular component to form the so-called Rashkow plexus, responsible for regulating blood flow through neuropeptide influence. The release of SP, CGRP, and NKA provokes vasodilation and drives the migration of immune cells \u003cem\u003evia\u003c/em\u003e chemotaxis, thus setting up an inflammatory response [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As a result, and as a compensatory mechanism, NPY, a strong vasoconstrictor, and VIP, a regulatory vasodilator, are released to preserve tissue homeostasis and control the inflammatory process established by the somatosensory neuropeptides. Additionally, NPY and VIP promote pulp tissue repair by binding to specific cells, such as odontoblasts, fibroblasts, and immune and dendritic cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNeuropeptides are released in response to orthodromic and/or antidromic stimuli [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. An orthodromic stimulus occurs under physiological conditions, such as temperature fluctuations or masticatory function. In such situations, A-delta fibres become activated, releasing NPY or VIP neuropeptides to sustain tissue homeostasis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, H2O2 free radicals that attack dental pulp generate an antidromic signal by exciting type C nerve endings. This leads to the release of SP and CGRP and sets off a defensive response that increases blood flow and intra-pulpal pressure through significant vasodilation and immune and inflammatory cell chemotaxis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, the release of NPY due to sympathetic system activation occurs in response to the inflammatory phenomena triggered by somatosensory neuropeptides. NPY inhibits acetylcholine and SP-induced vasodilation and amplifies the post-synaptic effects of other vasoconstrictors such as noradrenaline [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. NPY-induced vasoconstriction reduces blood flow and intra-pulpal pressure \u003cem\u003evia\u003c/em\u003e a compensating defensive mechanism against neurogenic inflammation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNPY vasoconstriction modulates the pro-inflammatory vasodilatory impact of SP and CGRP, preventing pulp volume expansion and driving fluid movement in an antidromic direction into the dentinal tubules, resulting in dentinal hypersensitivity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, NPY operates as a defensive mechanism by regulating the inflammatory process on dental pulp, hence minimizing post-bleaching dentinal hypersensitivity. Similarly, it has been proposed that the presence of NPY-1 receptors in fibroblasts, undifferentiated cells, and odontoblasts may be linked to pulp mineralization processes as a defence mechanism [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn light of the preceding, this study aims to establish the NPY levels expressed in human dental pulps of recently extracted premolars, following the application of Pola Office, Opalescence Boost, and Zoom, bleaching systems to identify potential control mechanisms of neurogenic pulp inflammation and dentinal hypersensitivity that developed in response to tooth-bleaching procedures.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e The present study was conducted using the CONSORT 2010 guidelines for clinical trials. It follows the recommendations of the Colombian Ministry of Health on ethical issues in research with human tissues. The bioethics committee approved the study under resolution \u003cb\u003eBIO407\u0026mdash;Acta No.12, May 25, 2023.\u003c/b\u003e Written informed consent was obtained from each patient who participated in the study. The study protocol was registered in Clinical Trials.gov \u003cb\u003eID NCT06606236.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eForty (n\u0026thinsp;=\u0026thinsp;40) dental pulps were collected from healthy non-smoking human donors aged 18 to 27 who underwent premolar extractions for orthodontic purposes. All premolars were caries and restoration-free, with complete root development, confirmed both visually and radiographically, with normal response to sensitivity testing, and no evidence of periodontal disease, traumatic occlusion, or previous orthodontic force application. Teeth with similar features were selected, to ensure the results' validity and reliability while limiting bias and any confounding factors.\u003c/p\u003e \u003cp\u003eSamples were divided into four groups containing ten healthy premolars each (without discriminating between maxillary and mandibular premolars): (1) Control group (n\u0026thinsp;=\u0026thinsp;10): the teeth were not exposed to dental bleaching agents (healthy pulps with normal/basal NPY values). (2) Pola Office system group (SDI, Victoria, Australia) (n\u0026thinsp;=\u0026thinsp;10): application of Pola office (35% H2O2) for 8 minutes. (3) Opalescent Boost system group (Ultradent Products, South Jordan, UT) (n\u0026thinsp;=\u0026thinsp;10): application of Opalescent Boost (40% H2O2) for 20 minutes. (4) Zoom system group (Zoom! Bleaching System; Discuss Dental, Culver City, CA) (n\u0026thinsp;=\u0026thinsp;10): application of Zoom! (25% H2O2\u0026thinsp;+\u0026thinsp;cold blue light) for 15 minutes. We rigorously followed the manufacturer's instructions for all bleaching systems (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA preliminary sensitivity test was conducted to assess the normal state of the pulp. This involved using a pulp tester to evaluate the pulp's response to an electrical stimulus. A cold test was performed using Endo Ice at -40 degrees Celsius to confirm the results further. These tests collectively verified that the sensitivity of the premolars was within normal limits.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical procedures and sample Collection\u003c/h2\u003e \u003cp\u003eTeeth were anaesthetized immediately following toot-bleaching, with 3% mepivacaine without vasoconstrictor infiltration injection for maxillary premolars and inferior alveolar nerve block injection for mandibular premolars. Ten minutes later, teeth were extracted by conventional methods using a supra-osseous luxation and extraction technique. The extraction process did not exceed five minutes. For the control group, extraction was also performed ten minutes after the anaesthetic application.\u003c/p\u003e \u003cp\u003eAfter extraction, all teeth were rinsed with 5.25% sodium hypochlorite to remove any remaining periodontal ligament that could contaminate the pulp sample. The teeth were sectioned with a Zekrya bur (Dentsply, Tulsa, OK) in a high-speed handpiece irrigated with saline solution. Pulp tissue was collected using a sterile endodontic excavator, deposited on an Eppendorf tube with 1 ml 4% Paraformaldehyde, and stored at 70\u0026deg;C until use [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h3\u003e\n\u003cp\u003e50 \u0026micro;L of each standard, blank, and sample were added to the corresponding wells. All samples and standards were duplicated. The sample dilution was established by preliminary experiments. 50 \u0026micro;L/well of working solution with biotinylated antibody was added. The plate was covered with sealing film and incubated for 45 min at 37\u0026deg;C. After decanting the solution, each well was dispensed with 350 \u0026micro;L of Wash Buffer (1 minute/soak). Plates were washed three times and gently blotted before adding 100 \u0026micro;L horseradish peroxidase-conjugated working solution to each well. The plate was covered with a new sealing film and incubated at 37\u0026deg;C for 30 minutes. After washing plates five times as previously described, the substrate was added (50 \u0026micro;L/well). The plate was incubated for 15min at 37\u0026deg;C. The plate was protected from light by wrapping it in aluminium foil, which caused a colour change. 50 \u0026micro;L/well of stop solution was added in the same order as the substrate solution was previously added. Each well's optical density (OD) was determined with a microplate reader at 450 nm [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor each of the bleaching techniques analyzed, mean and standard deviation, as well as minimum and maximum values for NPY, expressed in pmol/mg of pulp tissue, were calculated. A Kolmogorov-Smirnov test was applied to each series of values ​​corresponding to each bleaching technique to determine whether they were normally distributed. Finally, an ANOVA test was performed to determine if there were statistically significant differences between the different files evaluated, as well as post-hoc Tukey tests at a significant level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eForty pulp tissue samples were analyzed, classified as follows: 10 from teeth bleached with Pola Office, 10 with Opalescence Boost, 10 with Zoom! and 10 from intact teeth that were used as the control group. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e describes the NPY expression in pmol per mg of the pulp tissue, finding that the lowest expression was found in the control group, followed by the Zoom! Pola Office and Opalescence Boost systems respectively (Anova p\u0026thinsp;=\u0026thinsp;0.03).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExpression of Neuropeptide Y in dental pulps after different bleaching techniques\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBleaching Technique\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSt. Dev.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCONTROL GROUP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.02626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0066\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.01241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.04704\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePOLA OFFICE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03994\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.01107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.07509\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOPALESCENCE BOOST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.04407\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.01924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.08645\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026iexcl;ZOOM!\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.6834\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eAnova p\u0026thinsp;=\u0026thinsp;0.03\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the Tukey post-hoc comparisons, where it can be observed that the only statistically significant difference was between the control group and Opalescence Boost (p\u0026thinsp;=\u0026thinsp;0.04). All other pairwise comparisons did not show statistically significant differences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePost-hoc comparisons (Tukey Test)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e(I) Bleaching Agent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(II) Bleaching Agent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSig.\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCONTROL GROUP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePOLA OFFICE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOPALESCENCE BOOST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZOOM!\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePOLA OFFICE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOPALESCENCE BOOST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZOOM!\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOPALESCENCE BOOST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZOOM!\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTooth bleaching, one of the most prevalent procedures in modern dentistry, is proven to elicit a reversible pulpal inflammatory response, as free radicals dissociated from H2O2 diffuse through enamel and dentin into the pulp tissue. Such inflammatory response, clinically recognised as post-bleaching dentinal hypersensitivity, is strongly related to the bleaching system's H2O2 concentration, exposure time, and activation mode [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTooth bleaching results from the dissociation of free radicals from H2O2. Free radicals such as O, OH, and HO2 permeate the enamel and dentin, oxidizing the chromophores, and breaking the double bonds of organic chemical dyes that stain teeth [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eH2O2 free radicals activate somatosensory nerve terminals in the Rashkow plexus [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], leading to the up-regulated expression of SP, CGRP, and NKA from Aδ y C fibres, resulting in transient pulp inflammation. If the inflammatory phenomenon is not properly regulated, it can lead to odontoblast and fibroblast apoptosis, resulting in premature pulp ageing, which jeopardizes the pulp tissue's protective mechanisms and homeostasis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe inflammatory pulp phenomenon brought on by tooth bleaching procedures elicits an antidromic stimulus, characterized by rising blood flow and intrapulpal pressure, thereby increasing the dental pulp volume and promoting fluid flow towards the dentinal tubules. Clinically, this condition is described as post-bleaching dentinal hypersensitivity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNPY is released by exocytosis from sympathetic Aδ and type C fibres as a compensatory anti-inflammatory mechanism and to modulate dentinal hypersensitivity. NPY binds to target cells via the NPY-1 receptor, expressed in fibroblasts, endothelial cells, macrophages, odontoblasts, and undifferentiated dental pulp cells, competing and antagonizing with pro-inflammatory somatosensory neuropeptides such as SP, CGRP, and NKA [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. NPY exerts a vasoconstrictor effect, aided by norepinephrine at the post-synaptic level, thus counteracting vasodilation generated by SP and CGRP [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, NPY promotes pulp tissue repair by attaching to its receptor NPY-1 in target cells such as macrophages responsible for degrading H2O2 free radicals [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Likewise, NPY drives endothelial cells to promote angiogenesis, fibroblasts to renew and repair the extracellular matrix, and dental pulp stem cells to undergo odontoblastic differentiation, with the ultimate aim of producing tertiary dentin even up to 21 days after exposure to H2O2 based-agents [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All of these regulatory and repairing mechanisms lead the inflammatory reaction to decline a few days post-tooth-bleaching, resulting in the control of dentinal hypersensitivity by restricting fluid flow inside the dentinal tubules [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Finally, when dental pulp is exposed to H2O2 free radicals, the up-regulated expression of dismutase and catalase, which enzymatically break down the peridroxyl ion, provides a protective impact on the pulp cells and preserves pulp vitality over time [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThree different in-office tooth bleaching systems \u0026mdash; POLA OFFICE\u0026reg; (SDI, Victoria, Australia), OPALESCENCE BOOST\u0026reg; (Ultradent Products, South Jordan, UT), and Zoom\u0026reg; (Zoom! Bleaching System; Discuss Dental, Culver City, CA) \u0026mdash; were used in this study to quantify NPY expression in human dental pulp following tooth bleaching procedures. We rigorously followed the manufacturer's instructions, as the detrimental impacts of H2O2 on pulp tissue are proportional to the concentration, application time, and activation method [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Depending on NPY expression, it may be feasible to infer how this neuropeptide controls the neurogenic inflammatory phenomenon following tooth bleaching procedures, as well as provide a scientific explanation for the nature of post-bleaching dentinal hypersensitivity and its regulatory mechanisms [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ELISA test was performed to quantify NPY expression. It has been established that the ELISA test is sensitive enough to quantify neuropeptides in pmol/mg of pulp weight, the most generally used unit of measurement for neuropeptide expression in freshly extracted human dental pulp [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Considering the lack of previous research on NPY expression following tooth bleaching, a direct comparison was conducted between basal levels (pre-bleaching) and pulp inflammation (post-bleaching) in healthy premolars requiring extraction for orthodontic reasons [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll groups received 3% mepivacaine without a vasoconstrictor as a local anaesthetic to prevent alpha-adrenergic agonists from minimising neuropeptide expression. To allow NPY expression, a 10-minute interval was set aside following removing the bleaching agent [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Teeth were extracted using a supra-osseous luxation and extraction technique, which should not take longer than five minutes to prevent endogenous endopeptidases from degrading the neuropeptide and to quantify the NPY expression linked to the bleaching procedure rather than the dental extraction [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe control group exhibited an overall NPY expression of 0.026 pmol/mg of pulp tissue demonstrating that NPY is present in human dental pulp under physiological conditions, performing homeostatic and regulatory activities in inflammatory reactions. These values are consistent with previous studies employing radioimmunoassay for assessing NPY expression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe ZOOM system group (25% H2O2) achieved an average NPY expression of 0.031 pmol/mg of pulp tissue, after 15 minutes of application and activation with a cold blue light. However, there was no significant difference (\u003cem\u003eP\u003c/em\u003e ˃ 0.05) compared to the control group's reference values. These results can be explained by the fact that the ZOOM bleaching system uses the lowest concentration of the bleaching agents evaluated and its activation medium is a cold light lamp. Notably, findings from this study differ from previous research in which SP expression in human dental pulp was assessed in response to 25% ZOOM bleaching with hot light activation. The ZOOM system was found to elicit the highest SP expression, most likely due to the increased release of H2O2 free radicals by the activation source [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Confirming that the activation method affects the expression of neuropeptides in the dental pulp and therefore the severity of neurogenic inflammation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNPY expression is directly proportional to the amounts of H2O2 free radicals that enter the pulp tissue. The Pola Office system group (35% H2O2) with 8 minutes of application and the Opalescence Boost system group (40% H2O2) with 20 minutes of application displayed an average expression of 0.039 pmol/mg of pulp tissue and 0.044 pmol/mg of pulp tissue respectively. There was no significant difference (\u003cem\u003eP\u003c/em\u003e ˃ 0.05) between these two groups. Notably, an increased NPY expression compared to the control group was evident. Opalescence Boost was the only group that showed statistically significant differences (\u003cem\u003eP\u003c/em\u003e ˂ 0.05) compared to the control group, implying that the NPY expression is directly proportional to the concentration of the bleaching agent rather than the exposure time. Further, it can be inferred that the Pola Office and Opalescence Boost system groups trigger a more intense inflammatory phenomenon, as a higher NPY expression occurs, attempting to preserve pulp tissue homeostasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe release of NPY counteracts the harmful effects of free radicals, hence reducing the inflammatory response. NPY promotes protective vasoconstriction, thus decreasing blood flow, intrapulpal pressure, and inflammatory cell chemotaxis, while also inhibiting neural activity by blocking SP and CGRP. Since the dental pulp does not expand in response to the neurogenic inflammatory phenomena, the antidromic pressure of dentinal fluid is lowered, therefore, minimizing post-bleaching dentinal hypersensitivity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. High bleaching agent concentrations trigger neurogenic inflammation with antidromic effects, leading to fluid movement from the pulp to the dentinal tubules. This stimulates type C and Aδ somatosensory fibres which release SP and CGRP. As a control and compensation mechanism for the inflammatory phenomenon, NPY is released to oppose somatosensory neuropeptides, thus making pulp inflammation reversible. However, as a long-term adverse effect, it may result in premature ageing of dental pulp by eliciting pro-angiogenic defensive reactions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Similarly, it has been proposed that NPY binding to its specific receptor NPY-1 in fibroblasts, undifferentiated mesenchymal cells, and odontoblasts may be linked to pulp mineralization processes as a defence mechanism. Apoptosis of these cells has also been observed when the effect of free radicals generated by dental bleaching surpasses the pulp tissue's tolerance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. If the aggression of free radicals from tooth bleaching systems exceeds the tolerance of the pulp tissue's defence mechanisms, as a result of the use of high concentrations of H2O2 and improper application times, which perpetuate the inflammatory phenomenon and thus the antidromic stimuli, irreversible pulpitis associated with the overexpression of NKA, SP, and CGRP may develop. In this scenario, neither parasympathetic nor sympathetic neuropeptides, like VIP and NPY can control the inflammatory response. This can lead to the destruction of immature odontoblasts and undifferentiated cells' DNA [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], the degeneration of pulp fibroblasts, and even irreversible damage of immune cells such as macrophages, which can prevent pulp tissue from being repaired and prompt asymptomatic pulp necrosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResults from this study in terms of NPY expression and its potential relationship to post-bleaching dentinal hypersensitivity are supported by previous clinical studies that compared the level of dentinal hypersensitivity experienced by patients following tooth bleaching therapies with Zoom, Pola Office, and Opalecense boost. Following the Opalescence boost application, a lower percentage (15%) of dentinal hypersensitivity was observed, which is consistent with the findings of this study, in which this bleaching agent showed the highest levels of NPY expression, confirming that NPY expression is directly related to inflammation and dentinal hypersensitivity control. The percentage of dentinal hypersensitivity was higher with Pola Office (39%), which agrees with the NPY expression value of this study, which was lower than that of Opalecense boost. Finally, the zoom group had the highest percentage of dentinal hypersensitivity (91%), which agrees with the lower expression of NPY in this study [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The foregoing results suggest NPY regulates neurogenic inflammation triggered by tooth bleaching, which manifests as dentinal hypersensitivity.\u003c/p\u003e \u003cp\u003eFuture research on this topic should focus on quantifying and comparing the expression of NPY to that of its receptor, NPY-Y1, and linking these expressions to clinical studies assessing post-bleaching dentinal hypersensitivity aiming at inferring its control mechanisms.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWithin the limitations of this \u003cem\u003ein-vivo\u003c/em\u003e study, it can be established that NPY - whose expression is closely correlated with the bleaching agent's concentration and the intensity of the inflammatory response - mediates the pulp's responses to tooth bleaching agent-induced neurogenic pulp inflammation. Results of this study show that NPY is expressed in the dental pulp in pathological and physiological conditions. The highest NPY expression values ​​were found in OPALESCENCE BOOST\u0026reg; followed by POLA OFFICE\u0026reg;, and ZOOM\u0026reg;.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHydrogen peroxide (H2O2), Hydroxyl (OH), Oxygen (O), Perhydroxyl (HO2), Calcitonin gene-related peptide (CGRP), Substance P (SP), Neurokinin A (NKA), Neuropeptide Y (NPY), Vasoactive intestinal peptide (VIP), Optical density (OD), Enzyme-linked immunosorbent assay (ELISA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the bioethics committee, under resolution BIO407- Acta No.12, May 25, 2023. All participants provided written informed consent before the study procedures. The study was conducted according to principles outlined in the Helsinki Declaration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors received no specific funding for this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ. C: Conceptualization, Methodology, Project Administration, Supervision, Writing. M.A: Investigation, Resources, Software. K. A: Investigation, Resources, Software. D. M: Investigation, Resources, Software. H. D. M: Data curation, Resources, Supervision. N. R: Formal Analysis, Validation, Writing-original draft preparation. J. F. G: Formal Analysis, Validation, Writing-original draft preparation. L. E. D: Investigation, Methodology. E. G: \u0026nbsp; Investigation, Methodology. H. R. M: Conceptualization, Formal Analysis, Validation, Writing-review and editing\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCaviedes-Bucheli J, Gomez-Sosa JF, Azuero-Holguin MM, Orme\u0026ntilde;o-Gomez M, Pinto-Pascual V, Munoz HR. Angiogenic mechanisms of human dental pulp and their relationship with substance P expression in response to occlusal trauma. Int Endod J. 2017;50:339\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun L, Liang S, Sa Y, Wang Z, Ma X, Jiang T, et al. Surface alteration of human tooth enamel subjected to acidic and neutral 30% hydrogen peroxide. J Dent. 2011;39:686\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaviedes-Bucheli J, Ariza-Garc\u0026iacute;a G, Restrepo-M\u0026eacute;ndez S, R\u0026iacute;os-Osorio N, Lombana N, Mu\u0026ntilde;oz HR. The effect of tooth bleaching on substance P expression in human dental pulp. J Endod. 2008;34:1462\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasting RT, Amaral FLB, Fran\u0026ccedil;a FMG, Fl\u0026oacute;rio FM. Clinical comparative study of the effectiveness of and tooth sensitivity to 10% and 20% carbamide peroxide home-use and 35% and 38% hydrogen peroxide in-office bleaching materials containing desensitizing agents. Oper Dent. 2012;37:464\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinoux M, Serfaty R. Vital tooth bleaching: biologic adverse effects-a review. Quintessence Int. 2008;39:645\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeireles SS, Goettems ML, Dantas RVF, Bona \u0026Aacute;, Della, Santos IS, Demarco FF. Changes in oral health related quality of life after dental bleaching in a double-blind randomized clinical trial. J Dent. 2014;42:114\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBharti R, Wadhwani KK. Spectrophotometric evaluation of peroxide penetration into the pulp chamber from whitening strips and gel: An in vitro study. J Conserv Dent. 2013;16:131.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaviedes-Bucheli J, Mu\u0026ntilde;oz HR, Azuero-Holgu\u0026iacute;n MM, Ulate E. Neuropeptides in Dental Pulp: The Silent Protagonists. J Endod. 2008;34:773\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKina JF, Huck C, Riehl H, Martinez TC, Sacono NT, Ribeiro APD, et al. Response of human pulps after professionally applied vital tooth bleaching. Int Endod J. 2010;43:572\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlgart L, Edwall L, Fried K. Cat dental pulp after denervation and subsequent re-innervation: changes in blood-flow regulation and distribution of neuropeptide-, GAP-43- and low-affinity neurotrophin receptor-like immunoreactivity. Brain Res. 1993;625:109\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim S. Neurovascular interactions in the dental pulp in health and inflammation. J Endod. 1990;16:48\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLundy FT, Linden GJ. Neuropeptides and neurogenic mechanisms in oral and periodontal inflammation. Crit Rev Oral Biol Med. 2004;15:82\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaviedes-Bucheli J, Lombana N, Azuero-Holgu\u0026iacute;n MM, Munoz HR. Quantification of neuropeptides (calcitonin gene-related peptide, substance P, neurokinin A, neuropeptide Y and vasoactive intestinal polypeptide) expressed in healthy and inflamed human dental pulp. Int Endod J. 2006;39:394\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKillough SA, Lundy FT, Irwin CR. Dental pulp fibroblasts express neuropeptide Y Y1 receptor but not neuropeptide Y. Int Endod J. 2010;43:835\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRethnam S, Raju B, Fristad I, Berggreen E, Heyeraas KJ. Differential expression of neuropeptide Y Y1 receptors during pulpal inflammation. Int Endod J. 2010;43:492\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNurhapsari A, Cilmiaty R, Prayitno A, Purwanto B, Soetrisno S. The Role of Asiatic Acid in Preventing Dental Pulp Inflammation: An in-vivo Study. Clin Cosmet Investig Dent. 2023;15:109\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma H, Sharma DS. Detection of Hydroxyl and Perhydroxyl Radical Generation from Bleaching Agents with Nuclear Magnetic Resonance Spectroscopy. J Clin Pediatr Dent. 2017;41:126\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReis-Prado AH dos, Grossi IR, Chaves HG dos, Andr\u0026eacute; S, Morgan CB, Briso LF dos et al. ALF,. Influence of Hydrogen Peroxide on Mineralization in Dental Pulp Cells: A Systematic Review. Frontiers in Dental Medicine. 2021;2:689537.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePashley DH. How can sensitive dentine become hypersensitive and can it be reversed? J Dent. 2013;41 Suppl 404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Karim I, Lundy FT, Linden GJ, Lamey PJ. Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue. Arch Oral Biol. 2003;48:249\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaller KM, Weber M, Korkmaz Y, Widbiller M, Feuerer M. Inflammatory Response Mechanisms of the Dentine-Pulp Complex and the Periapical Tissues. Int J Mol Sci. 2021;22:1\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonato MV, dos Reis-Prado AH, Abreu LG, de Arantes LC, Goto J, Chaves HG dos. Influence of dental bleaching on the pulp tissue: A systematic review of in vivo studies. 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J Dent. 2012;40:644\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandes AMM, Vilela PGF, Valera MC, Bolay C, Hiller KA, Schweikl H, et al. Effect of bleaching agent extracts on murine macrophages. Clin Oral Investig. 2018;22:1771\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKugel G, Ferreira S, Sharma S, Barker ML, Gerlach RW. Clinical trial assessing light enhancement of in-office tooth whitening. J Esthet Restor Dent. 2009;21:336\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tooth bleaching, Dental pulp, Neurogenic inflammation, Neuropeptide Y","lastPublishedDoi":"10.21203/rs.3.rs-5375900/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5375900/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eTo assess the neuropeptide Y (NPY) expression\u0026nbsp;in healthy human dental pulp following tooth bleaching with three in-office hydrogen peroxide-based systems: Opalescence Boost\u003csup\u003e©\u003c/sup\u003e (Ultradent Products, South Jordan, UT), Pola Office\u003csup\u003e©\u003c/sup\u003e (SDI, Victoria, Australia), and Zoom\u003csup\u003e©\u003c/sup\u003e (Zoom! Bleaching System; Discuss Dental, Culver City, CA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u0026nbsp;\u003c/strong\u003eThis observational study was designed following the STROBE guidelines.\u003cstrong\u003e \u003c/strong\u003eForty\u0026nbsp;dental\u0026nbsp;pulps\u0026nbsp;were collected from healthy premolars scheduled for extraction for orthodontic reasons. Teeth were divided into four groups containing ten healthy premolars each: Control group (n= 10): the teeth were not exposed to dental bleaching agents (healthy pulps assessed for normal/basal NPY values). Pola Office system group (n= 10): application of Pola office (35% H2O2) for 8 minutes. Opalescent Boost system group (n= 10): application of Opalescent Boost (40% H2O2) for 20 minutes. Zoom system group (n= 10): application of Zoom! (25% H2O2 + cold blue light) for 15 minutes. We rigorously followed the manufacturer's instructions for all bleaching systems. Following the extractions, the pulpal tissue was collected, placed in a 4% formaldehyde solution in Eppendorf tubes, and processed. NPY levels were measured using enzyme-linked immunosorbent assay (ELISA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003eThe expression levels of NPY in the dental pulp of human premolars showed the lowest value in the control group (0.0263\u0026nbsp;pmol/mg of pulp tissue), followed by the Zoom group (0.0314\u0026nbsp;pmol/mg of pulp tissue). An increase in expression was observed in the Pola Office group (0.0399\u0026nbsp;pmol/mg of tissue), with the highest NPY expression found in the Opalescence Boost group (0.0441\u0026nbsp;pmol/mg of pulp tissue), which demonstrated a significant difference compared to the control group (P = 0.03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u0026nbsp;\u003c/strong\u003eNPY - whose expression is closely correlated with the bleaching agent's concentration and the intensity of the inflammatory response - mediates the pulp's responses to tooth bleaching agent-induced neurogenic pulp inflammation. NPY is expressed in the dental pulp in both pathological and physiological conditions. \u0026nbsp;The highest NPY expression values were found in OPALESCENCE BOOST\u003csup\u003e®\u003c/sup\u003e followed by POLA OFFICE\u003csup\u003e®\u003c/sup\u003e, and ZOOM\u003csup\u003e®\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration: \u003c/strong\u003eClinical Trials.gov ID NCT06606236.\u003c/p\u003e","manuscriptTitle":"Neuropeptide Y regulation of dental pulp neurogenic inflammation provoked by tooth bleaching agents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 15:50:50","doi":"10.21203/rs.3.rs-5375900/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9cc7e1c0-afd4-444e-9899-3c0d920f79cc","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-17T07:09:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-26 15:50:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5375900","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5375900","identity":"rs-5375900","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}