Photobiomodulation counteracts DAMP signaling to improve Odontoblast Survival for Dentin Repair

Photobiomodulation counteracts DAMP signaling to improve Odontoblast Survival for Dentin Repair | 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 Photobiomodulation counteracts DAMP signaling to improve Odontoblast Survival for Dentin Repair Fatemeh Tavakkoli, Zahra Yazdani, Praveen R. Arany This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8289723/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 Objectives Tooth vitality is driven by the odontoblast responses in the pulp-dentin complex. Low-dose light treatments, termed Photobiomodulation (PBM) therapy has been noted to induce odontoblast differentiation from dental pulp stem cells and promote dentin repair in a sterile, direct pulp capping approach. Its ability to direct repair in a routine clinical scenario post-infection or injury remains to be elucidated. Methods An odontoblast cell line, MDPC-23 were subjected to various stressors namely inflammation (TNF-α), bacterial infection (LPS), hypoxia (CoCl₂), nutrient deprivation (0.2% serum), and pH stress (pH 4 or 12). Cells were treated with four PBM wavelengths of 447 nm (blue), 532 nm (green), 658 nm (red), and 810 nm (near-infrared) delivered at doses of 0.03, 3, or 30 J/cm². To investigate potential mechanisms, small molecule inhibitors targeting ROS (N-Acetylcysteine, NAC), ATP (Sodium Azide, NaN₃), NFκB (BAY 11-7082), BCL-2 (anti-apoptotic), Caspase-3 (pro-apoptotic) were used. Cell viability was assessed with Alamar Blue and mitochondrial membrane potential was assessed with JC-1 fluorescence assay. Results We noted all four PBM wavelengths induced significant (n = 4, p < 0.05) odontoblast survival at 3 J/cm 2 at optimal cell density. All stressors, except LPS, reduced odontoblast viability significantly (n = 4, p < 0.05) that were rescued significantly (n = 4, p 0.05) affected by neutralization of ROS, BCL-2 or Caspase 3, but differentially affected by ATP deprivation and significantly (n = 4, p < 0.05) neutralized by NF-κB inhibition. The increased mitochondrial membrane potential to TNFα treatments were also differentially modulated by the two wavelengths significantly (n = 4, p < 0.05) suggesting there are divergences in individual signaling pathways mediating the overall PBM survival response. Conclusion These results demonstrate that discrete PBM wavelengths evoke context-dependent odontoblast proliferative responses. These findings highlight the therapeutic potential of PBM in modulating odontoblast responses to various damage stimuli that can be utilized to develop specific protocols for optimal clinical therapeutic clinical outcomes. Photobiomodulation Odontoblasts DAMP NFκB Inhibition Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Tissue regeneration in dentistry is premised on restoring the pulp-dentin vitality and tooth supporting periodontal tissues. Among them, pulp regeneration is focuses on restoring the pulp–dentin complex is primarily driven by odontoblasts responses that is supported by fibroblasts, endothelial and immune cells ( 1 ). The current clinical approach of disinfecting, obturating the root canal and restorations are prone to biomaterial failures besides loss of naturally protective proprio reception. Regenerative therapy seeks to re-establish the pulp’s natural architecture, vascularity, innervation, and cellular composition, thereby maintaining long term tooth vitality, sensory unction, and long-term structural integrity ( 2 ). These regenerative techniques rely on the coordinated use of stem cells, scaffolding materials, and suitable molecular signaling cues to rebuild a vital and functional pulp tissue ( 3 ). Within this regenerative framework, odontoblasts are central to both mineralized dentin formation and the maintenance of pulp vitality ( 4 ). They play a vital role in responding to external stimuli such as mechanical, thermal, and chemical insults by depositing reactionary dentin and orchestrating repair processes to protect the pulp tissue. The preservation and modulation of odontoblast function are therefore central to dental tissue repair and regeneration ( 5 ). Various factors such as trauma and caries can trigger a cascade of inflammatory responses within the tooth ( 6 , 7 ). Secreted factors from caries causing microbes can induce inflammatory responses and morphological changes in the pulp, even before the microbes themselves access the pulp cavity ( 7 ). These damage signals trigger disruption of blood flow and local hypoxia initiating potent inflammatory cascades ( 6 ). Given the rigid encasement of the pulp tissues, the otherwise routine inflammatory hyperemia can compromise repair and undermine integrity. Cellular injury (Damage-associated molecular patterns, DAMPs) or stressors such as microbial pathogens (pathogen-associated molecular patterns, PAMPs) are detected by pattern recognition receptors (PRRs) that promotes release of endogenous molecules ( 8 , 9 ). Recognition of damage triggers assembly of inflammasomes, which activate key signaling pathways, especially NFκB, that modulates progression or resolution of the inflammatory response ( 10 , 11 ). The activation of intracellular signaling cascades results in elaboration of cytokine mediators such as IL-1β, IL-6, and TNF-α that perpetuate the inflammation cascade ( 12 , 7 , 13 ). TNFα is a key pro-inflammatory cytokine that has been noted to be upregulated in in carious and inflamed pulps, where it contributes to nociceptor sensitization and heightened inflammatory activity ( 14 ). Thus, the interconnected roles of DAMPs/PAMPs, NFκB, and TNF-α signaling appears to mediate and sustain inflammation within dental tissues that needs to be resolved prior to initiating pulp-dentin repair ( 15 , 16 ). Photobiomodulation (PBM) in dentistry utilizes low dose light in the visible (400 nm) to near-infrared (1200 nm) spectrum to alleviate pain and inflammation while promoting tissue healing and regeneration ( 17 ). PBM has been noted to promote differentiation of dental pulp and mesenchymal bone marrow stem cells to odontoblasts via redox-mediated activation of latent TGF-β1 ( 18 , 19 ). These studies were performed in routine, physiological conditions, especially the in vivo pulp capping technique and PBM treatments were performed in sterile, controlled mechanical exposures. Other studies have noted activation of intracellular mitochondrial ATP, ROS and NFκB signaling that regulates inflammation, oxidative stress, cell proliferation, and upregulates mineralization markers such as BMP2, Runx2, and Osterix ( 20 – 22 ). A prior study noted PBM-generated redox activates NFκB leading to the expression of genes involved in cell survival and anti-apoptosis responses ( 23 ). Among them, the Bcl-2 family protects against cell death by inhibiting pro-apoptotic factors such as the cysteine protease, Caspases that blocks release of apoptogenic factors from the mitochondria ( 24 ). The Caspases are critical executioner of the orderly process of apoptosis, orchestrating programmed cell death ( 25 ). Thus, the ability of PBM treatments to modulate these coordinated signaling pathways could determine overall cell survival versus death. Despite these advances, the context-dependent effects of PBM treatments on odontoblasts under various stressors that would be clinically relevant have not been explored. This study examined the PBM responses in odontoblasts in simulated inflammation, infection, hypoxia, nutrient deprivation, and extreme pH. The major goal of this work is to gain mechanistic insight to inform optimal future clinical PBM treatment strategies for pulp-dentin complex preservation and regeneration. Materials and Methods Cell culture A murine odontoblast (MDPC-23) cell line provided by Tatiana Botero and Jacque Nor, University at Michigan, were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Optimal cell density To determine the optimal cell density for PBM assays, cells were seeded in 96-well flat-bottom plates (Corning, USA) at 1,000, 3,000, or 5,000 cells/well, allowed to adhere overnight prior to studies. Photobiomodulation treatment A diode laser at 447 nm (blue), 532 nm (green), 658 nm (red), and 810 nm (infrared) (Weber Medical, Germany) was used at power outputs of 1, 10, and 100 mW in continuous wave (CW) mode for a fixed duration of 300 sec that corresponds to energy 0.03, 3, and 30 J respectively. Distance and positioning of laser probe from cells were standardized and confirmed prior to each study with a sensor and power meter (S120CV and PM400, Thor labs). All control groups were handled identically but not exposed to laser illumination. Cell viability assessments : To assess cell viability, the Alamar Blue assay was performed at 24- or 72-hours post treatment. Briefly, culture medium was aspirated and replaced with fresh complete DMEM containing 10% (v/v) Alamar Blue reagent (Thermo Fisher Scientific). Plates were incubated at 37°C for one hour, until a visible pink color change was observed. Fluorescence was assessed at Ex/Em: 520/590 nm using a microplate reader (iMax3, Molecular Devices). Cell stress (DAMP and PAMP) induction : Various simulated damage and pathogen associated molecular patterns (DAMPs and PAMPs) were generated prior to PBM treatments namely bacterial infection ( E coli O26:B6 LPS 1 µg/ml, Sigma), inflammation (TNF-α, 1 µg/mL, Biosciences), serum deprivation (0.2% FBS, Atlas), hypoxia (CoCl₂ Ward Science), and extreme pH - acidosis (pH 4) or alkalosis (pH 9 or 12) in HEPES-buffered DMEM. For each condition, cells were exposed to the respective treatment solution for 2 hours at 37°C prior to PBM therapy. Only for the pH-treated cells, the pH adjusted HEPES media was replaced with complete media before PBM treatment. All other media conditions were maintained for 24 hours. Pathway specific-inhibitors Small molecule inhibitors against TNF-α (BAY 11-7082 1mM Cayman Chemical, USA), BCL-2 (10 mM, Abcam, USA), Caspase-3 (1 mM Thermo Fisher Scientific), N-Acetylcysteine (1 mM NAC, Sigma), and sodium azide (NaN₃ 1 mM, Sigma) were preincubated 30 min prior to PBM treatments in appropriate media conditions in each group. JC-1 Assay To evaluate mitochondrial membrane potential, JC-1 dye (2µM, Fisher Scientific) was added immediately after PBM treatments. As a positive control, carbonyl cyanide m-chlorophenyl hydrazone (CCCP 50 µM, Fiser Scientific) was used to induce complete mitochondrial depolarization. Fluorescence intensity for red fluorescence (J-aggregates) at 535/595 nm (Ex/Em), and green fluorescence (monomers) at 485/535 nm were assessed using a microplate reader (iMax3, Molecular Devices) or inverted fluorescence microscope (Zoe, Biorad). All imaging parameters were kept constant across groups and digital image analysis was performed with NIH ImageJ software (r91, NIH, USA). Statistical Analysis All experiments were performed in quadriplicates and repeated repeated independently at least three times. Data were presented as mean ± standard deviation (SD) and statistical comparisons were performed using one-way ANOVA GraphPad Prism (v10, Dotmatics, USA), where p-value of < 0.05 was considered statistically significant. Results Role of cell density in determining optimal PBM responses We first examined the effects of odontoblast density on the PBM responses by seeding cells in 1000, 3000, and 500 cells in a 96-well dish. Among the PBM treatments at 3 J/cm 2 , the 3,000 cells/well group exhibited significantly (n = 4, p < 0.05) higher viability compared to other two seeding densities ( Figure 1A-C ). The 1,000 cells/well group had sparsely seeded cells and minimal response while the 5,000 cells/well group showed signs of early contact inhibition and reduced proliferation. These results indicated 3,000 cells per well were an optimal cell density to assess consistent and robust PBM responses. Effect of various PBM wavelengths on Odontoblasts Next, we performed a PBM dose-escalation study at 3000 cells/well with 0.3, 3, and 30 J/cm². All wavelengths induced a significant (n = 4, p < 0.05) increase in cell proliferation at 3 J/cm 2 compared to 0.03 and 30 J/cm² ( Figure 1D-G ). The most prominent responses were observed with red (660 nm) and blue (447 nm) wavelengths. Similar trends were noted at 48 hours ( data not shown ) although cell viability showed modest temporal variation at the longer timepoint. Odontoblasts cell stress responses to PBM treatments In the simulated DAMPs and PAMPs conditions, PBM responses were evaluated with all wavelengths to assess optimal efficacy. We first simulated inflammation-mimicking DAMP with TNFα treatments and observed odontoblasts demonstrated significantly (n = 4, p < 0.5) decreased cell viability compared to untreated controls ( Figure 2A ). PBM treatments led to a statistically significant recovery with red (658 nm, n = 4, p < 0.5) and green (532 nm, n = 4, p 0.5). We next simulated infection-mimicking PAMP with LPS treatments and observed LPS significantly (n = 4, p 0.5) ( Figure 2B ). We examined low serum-induced cell stress that caused a substantial (n = 4, p < 0.5) decline in odontoblast viability, confirming the induction of nutrient stress. PBM treatments at 3 J/cm² with red, green, and infrared groups showed significantly (n = 4, p 0.5). Next, we examined more potent, but transient, stressors. As cells have varying sensitivity to hypoxia, we first performed a dose escalation of chemically-induced hypoxia with varying concentrations of CoCl₂ from 15 mM to 1500 mM. We noted 15 mM of CoCl 2 potently (n = 4, p < 0.5) that was significantly (n = 4, p 0.5) response. Both tissue damage and restorative materials induce extreme pH that has been shown to modulate the dentin reparative responses (26). To simulate these, we used HEPES-buffered DMEM media that was freshly adjusted to acidic or alkaline pH and verified prior to cell treatments for 2 hrs. At pH 4, odontoblast viability was significantly (n = 4, p < 0.5) reduced, reflecting acid-induced stress. PBM treatment with the green laser (532 nm) resulted in a statistically significant (n = 4, p 0.5), odontoblast rescue. Under physiological pH 7, PBM again produced the highest viability increase in the green laser group, reaching statistical significance (p < 0.05), while other wavelengths yielded modest, non-significant improvements. Under mild (pH 9) and strong alkaline (pH 12) conditions, odontoblast viability was also reduced significantly (n = 4, p 0.5) improvements. PBM treatments with the green laser showed significant (n = 4, p < 0.5) recovery. At pH 12, all four laser wavelengths elicited a significant (n = 4, p < 0.5) increase in odontoblast viability relative to non-treated control ( Figure 2F ). These results indicate that odontoblast stressor responses vary with individual DAMPs or PAMPs ( Figure 2G ) implying specific PBM clinical parameters are necessary to evoke optimal therapeutic benefits. Role of specific PBM-evoked signaling pathways in odontoblast survival The variance in the PBM treatment efficacy to individual stressors suggest there are discrete operative mechanisms in individual pathophysiological scenarios. For these set of studies, we chose to pursue the red and green PBM treatments in odontoblasts that showed maximal efficacy following the inflammation simulating TNFa treatments. We resorted to carefully investigating the known PBM evoked mitochondrial pathways, namely the photoabsorption by Cytochrome C Oxidase (CCO) generated ROS and ATP generation. Co-treatment with NAC , a well-characterized ROS scavenger, significantly (n = 4, p < 0.5) reduced TNF-α–induced odontoblast cytotoxicity ( Figure 3A ). The significantly (n = 4, p 0.5), neutralized by NAC pre-treatments. Neutralization with So dium Azide (NaN 3 ) , a mitochondrial complex IV inhibitor, also markedly (n = 4, p < 0.5) improved cell viability following TNF-α treatments ( Figure 3B ). PBM treatments demonstrated improved odontoblast viability that was neutralized significantly (n = 4, p 0.5) with the green laser. These results suggest that both PBM generated ATP and ROS are partly contributing to the improved odontoblast survival response during inflammation. Next, we examined the role of NFkB, a central inflammation signaling mediator integrating odontoblast survival and death responses. We noted that the NFkB inhibitor, BAY 11-7082 treatments significantly (n = 4, p < 0.5) reduced odontoblast viability and potently neutralized (n = 4, p < 0.5) both red and green PBM treatment induced odontoblast survival ( Figure 3C ). We then utilized two apoptosis pathway modulators, namely BCL-2 (anti-apoptotic) and Caspase 3 (pro-apoptotic). Odontoblast treated with a BCL-2 inhibitor, that promotes apoptosis, showed no significant (n = 4, p > 0.5) effects following TNFa treatments ( Figure 3D ). However, they subtly improved PBM mediated survival by both wavelengths, although these were not statistically significant (n = x, p > 0.5). Finally, Caspase 3 inhibitor, an apoptosis blocker, significantly (n = 4, p > 0.5) improved TNFa cytotoxicity, but did not have any appreciable (n = 4, p > 0.5) efficacy in PBM improved odontoblast survival ( Figure 3E ). These results indicate that PBM-induced NFkB signaling is a key mediator in odontoblast survival in inflammation that primarily involves induction of cell survival signaling rather than inhibiting cell death (apoptosis) pathway. Role of mitochondrial signaling in PBM-evoked odontoblast survival To examine the mechanism of PBM-improved odontoblast survival, we assessed the effects of PBM treatments and pathway-specific inhibitors on the mitochondrial membrane potential using a fluorescent dye, JC-1. Odontoblasts probed with JC1 demonstrated high mitochondrial membrane potential with robust accumulation of the dye forming red fluorescence (J-aggregates) and minimal green fluorescence (J-monomers) indicating healthy mitochondrial functions. TNFa treatments reduced red and increased green fluorescence, indicating mitochondrial depolarization and reduced membrane potential ( Figure 4A ). Odontoblasts treated with CCCP induced robust depolarization that was also evident with TNF-a treatments. The increased membrane potential was not affected by treatments with the NFkB inhibitor, BAY 11-7082. The red laser PBM appeared to increase the membrane potential that was significantly (n = 4, p < 0.5) reduced by NFkB inhibition while the green laser potently (n = 4, p < 0.5) reduced membrane potential that appears to be independent of NFkB inhibition. These trends were further verified with fluorescence imaging for the red to green fluorescence based on their ratios that were digitally quantified ( Figure 4B ). Overall, it appears the two laser wavelengths have distinctly different effects on the mitochondrial potential perturbed by inflammation. Discussion The pulp-dentin complex contains odontoblasts and stem cells that maintain its health and vitality. These cells are subjected to various forms of damage or stressors, such as mechanical injury, infections, radiation, or chemical exposures that trigger represent damage-associated molecular patterns (DAMPs) (27). DAMPs are endogenous molecules released from stressed or dying cells, either from the intracellular or extracellular space, and are key mediators implicated in inflammation and inflammatory diseases. (28) The present study noted the ability of discrete wavelengths for photobiomodulation (PBM) are capable of protecting odontoblasts from a broad range of DAMPs including inflammation, hypoxia, nutrient deprivation, and extreme pH. The ability of red and green wavelengths to significantly improve cell viability under conditions by promoting cell survival signaling via NFkB and TGF-b signaling appears to be distinct from the cell death (BCL2 and Caspase) signaling indicating specificity of the therapeutic pathway s evoked. These observations support the use of PBM for vital pulp therapy and pulp-dentin regenerative strategies. PBM has been historically explored for accelerating healing and reducing inflammation in soft tissues. (29-33) While most clinical studies have focused on specific disease contexts, in vitro studies rarely simulate these pathophysiological conditions, specifically the unique nature and sequalae of disease patterns (DAMPs or PAMPs). Attempts at optimizing PBM dosing and delivery techniques could significantly improve with suitable contextualized in vitro models, also termed microphysiological systems (MPS) utilizing organoids or organ-on-a-chip (OoC) systems (34-38). Several experts have suggested PBM could add significant utility in regenerative pulp-dentin tissue engineering (39, 40) These reviews emphasize the ability of PBM to stimulate cell proliferation in different cell lineages of the pulp-dentin complex including odontoblasts, fibroblasts, and endothelial cells (41-43). However, the precise signaling mediating this response remains to be elucidated. (44) The clinical scenarios simulated in this study focused on relevant stressors from caries progression, pulpal inflammation, or physicochemical tissue injuries (45). These included TNF-α (inflammation), E. coli LPS (bacterial infection), CoCl₂ (hypoxia), low serum (nutrient deprivation), and extreme pH contextualizing the injury stimuli. The rationale here is that the basal cell response signaling network would be perturbed in these pathophysiological contexts and hence, require specific modulation to therapeutically benefit from PBM treatments (46). While each injurious agent would generate varying degrees of cell stress response, the ultimate output would be survival or death. Hence, the ability of a therapeutic intervention to target a conserved stress response would modulate the magnitude and specificity of the evoked pathways and ultimately affect cell survival. Hence, we chose this as our primary endpoint. In this study, we noted inflammation, hypoxia, nutrition and alkaline pH stress were effectively counteracted by PBM treatments while infection and acidic pH were not modulated. We first investigated the effect of cell density on odontoblast response to PBM treatments as cell-cell contact mediated paracrine signaling plays a vital role in integrating extrinsic perturbations including stressors like hypoxia, serum starvation, and oxidative stress, among others (47-50). We noted that providing a semi-confluent (40-60%) culture condition evokes optimal cell proliferative response to a 3J/cm 2 PBM dose that is in agreement with prior studies (41). Lower or higher confluency were ineffectual. While this could be attributed to biological cellular microenvironment, it may also be due to non-optimal photon distribution based on the Arndt-Schultz curve implying lower confluent cells getting too much PBM dose and higher getting too little (51, 52). Nonetheless, optimizing physiological cell response appears to be a key step prior to exploring pathological stimuli contexts, especially in normal versus transformed cell responses to PBM treatments (53). To further investigate the improved survival with PBM treatments, we sought to examine any perturbations to cell death pathway. We looked at BCL-2 (anti-apoptotic) and Caspase-3 (pro-apoptotic) that play crucial and opposing roles in the regulation of apoptosis, a key cell death response to stress (54). BCL-2 preserves cell survival by maintaining mitochondrial integrity and inhibiting the release of cytochrome c, preventing activation of the caspase cascade. This function is essential for reducing oxidative stress and controlling calcium flux within cells. In our studies, the BCL-2 inhibitor did not rescue the reduced odontoblast survival in response to inflammation and did not significantly affect the PBM rescue, although there was a trend towards improvement. A study by Zhang et al. observed genetic manipulation of anti-apoptotic proteins like BCL-2 can enhance odontoblast survival and promote dentin repair. (55) This could be attributed to the physiological context and endogenous regulation but could be investigated further. Caspase-3 serves as a central executioner of apoptosis; once activated, it cleaves various cellular substrates and promotes the morphological and biochemical changes characteristic of programmed cell death. Notably, caspase-3 can cleave BCL-2 itself, disabling its protective effects and reinforcing the apoptotic process. This interplay between BCL-2 and caspase-3 represents a critical balance between cell survival and death. (56-58) In the current study, the Caspase-3 inhibitor significantly rescued the inflammation induced odontoblast cell death, but it also did not significantly modulate the PBM enhanced odontoblast survival. As neither inhibitor modulated the PBM-induced rescue, it appears the PBM-induced survival signaling is likely upstream of the apoptotic (BCL-2 and Caspase-3) signaling responses. We noted the most prominent odontoblast response was with the NF-κB inhibitor, a central pathway in mediating inflammation (59-61). The complete abrogation of the PBM rescue as well as further depression of odontoblast survival to TNFa suggested NF-kB is a key PBM cytoprotective signaling pathway. Direct activation of NF-kB signal transduction via redox generation by PBM treatments has been demonstrated (23). While TNF-a increased mitochondrial membrane potential (ΔΨm) as anticipated, the two wavelengths had discretely different responses, with the red laser showing an increase, while green laser showed a decrease. Further, inhibiting NF-kB signaling had no effect on basal inflammatory or reduced mitostress by green laser. Most intriguingly, the red wavelength not only increased mitostress, but was also modulated significantly by NF-kB inhibition. As both wavelengths effectively improve odontoblast survival during simulated inflammation that are neutralized by NF-kB inhibition, these observations together provide significant insights into integrated survival signaling induced by PBM treatments on odontoblasts. A major finding in this study was the selectivity of the evoked therapeutic PBM wavelengths. Prior studies have observed the clinical efficacy of discrete wavelengths for PBM treatments (18, 62-66). We noted both red and green most consistently improved odontoblast survival across various DAMPs. Blue showed no efficacy in serum, inflammation or acidic pH while NIR only showed efficacy in alkaline DAMPs condition suggesting there are wavelength-specific evoked responses. The clinical use of visible or infrared wavelengths or their combination has been employed based on anatomical target location. This has been premised on the former treating more superficial targets while infrared penetrates to deeper tissues while their combination aimed at treating the volume more effectively. The rationalized use of individual wavelength evoked responses, likely based on selective absorption, have not been investigated in PBM treatments thus far. Moreover, the use of clinically effective infrared wavelengths, especially 800 to 1200 nm has raised important questions on photoabsorption and inelastic scattering as the thermodynamic basis for biophotonics energy transfer (67, 68). Despite some valuable mechanistic insights, this study has several limitations. The pulp dentin complex has cells of various lineages. Although odontoblast has a key role in dentin regeneration, fibroblasts, endothelial and macrophages play important roles. Future in vitro and in vivo studies could validate their responses to PBM in various DAMP conditions. This study outlined distinct role for two individual wavelengths red (658 nm) and green (532 nm), but their combinations can be further explored. Future studies employing higher resolution analytical techniques such as proteomics, metabolomics, and single-cell analysis, and spatial transcriptomics could unravel mechanistic foundation of PBM-mediated protection. Alternative mechanisms of PBM cytoprotection could explore activation of transient receptor potential (TRP) channels, including TRPV1, TRPV2, and TRPV4, resulting in increased calcium influx and downstream signaling activation.(69, 70) PBM has also been shown to promote photodissociation of nitric oxide (NO), increasing its bioavailability and improving microcirculation to stressed cells.(71, 72) Finally, PBM activation of intracellular cell survival signaling such as JAK/STAT, PI3K/Akt and MAPK pathways that are central to promoting cell proliferation, enhancing survival, and facilitating the resolution of inflammation could be explored.(73-76) It remains to be seen if these pathways operate independently or in concert with the classical PBM induced ROS:ATP:NF-κB axis providing additional avenues exploited by PBM-mediated cellular protection. Conclusion This study noted the ability of PBM treatments to significantly enhance odontoblasts under various stress conditions. This can contribute to their role in pulp-dentin repair and regeneration. Elucidation of specific intracellular signaling pathways following PBM treatments that modulate mitochondrial functions, oxidative stress responses, and inflammatory signaling could enable precision programmed approach for PBM in clinical regenerative endodontics. Declarations Acknowledgement The authors acknowledge the University at Buffalo faculty funds for providing support for these studies. Conflict of interest The authors declare they have no conflicts of interest with the current work. Author Contributions FT: designed and performed the studies, analyzed the data, prepared the draft manuscript, ZY: assisted with data analysis, writing and reviewing the manuscript PRA: conceptualized and designed study, revised and finalized manuscript Data Availability All requests for primary data will be made available upon reasonable request References Feigin, K. and B. Shope (2017) Regenerative Endodontics. 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09:25:17","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":174846,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8289723/v1/8efcd4e6abdf0a6b8a291e9c.html"},{"id":97865277,"identity":"975123f8-9372-49cf-96f8-284e65f23c2c","added_by":"auto","created_at":"2025-12-10 09:25:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":581099,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of cell density and wavelengths on odontoblast PBM responses. MDPC-23s were seeded at \u003cstrong\u003eA.\u003c/strong\u003e 1000 cells/well, \u003cstrong\u003eB\u003c/strong\u003e. 3000 cells/well and C. 5000 cells/well in a 96-well plate and first treated with four laser wavelengths namely 810 nm near-infrared, 658 nm red, 532 nm green, and 447 nm blue at 3J/cm\u003csup\u003e2\u003c/sup\u003e and cell viability was assessed. PBM treatments were performed at 3 doses namely 0.3, 3, and 30 J/cm\u003csup\u003e2 \u003c/sup\u003ewith each of the wavelength namely \u003cstrong\u003eD\u003c/strong\u003e. 810 nm, \u003cstrong\u003eE\u003c/strong\u003e. 660 nm, \u003cstrong\u003eF\u003c/strong\u003e. 5 nm green, and \u003cstrong\u003eG\u003c/strong\u003e. 447 nm blue. Data is presented as mean ± SD (n = 4) of atleast 3 independent repetitions, statistical significance is denoted as * p \u0026lt; 0.05, **p \u0026lt; 0.01, n.s. - not significant. \u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289723/v1/4582d41ce7db964b14db6a41.png"},{"id":97865252,"identity":"8d15f471-0d73-4a9d-b3b9-8f94507173ed","added_by":"auto","created_at":"2025-12-10 09:25:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":787033,"visible":true,"origin":"","legend":"\u003cp\u003eThe role of PBM in mitigating DAMP signaling. Odontoblasts were treated with various agents for 2 hours namely \u003cstrong\u003eA.\u003c/strong\u003e Inflammatory signaling was simulated by TNFa treatments, \u003cstrong\u003eB.\u003c/strong\u003e Infection was simulated with \u003cem\u003eE Coli\u003c/em\u003e lipopolysaccharide, \u003cstrong\u003eC\u003c/strong\u003e. Nutritional stress was simulated by reducing serum to 0.2%, \u003cstrong\u003eD. \u003c/strong\u003ehypoxia was simulated with cobalt chloride treatments, \u003cstrong\u003eE.\u003c/strong\u003e alkaline pH 12 was simulated by adjusting HEPES buffer with sodium hydroxide and \u003cstrong\u003eF.\u003c/strong\u003e acidic pH 3 was simulated by adjusting HEPES buffer with hydrochloric acid. This was followed by PBM treatments at 3 J/cm\u003csup\u003e2\u003c/sup\u003e with all four wavelengths and cell viability was assessed using AlamarBlue.\u0026nbsp; \u003cstrong\u003eG.\u003c/strong\u003e Summary of PBM responses in various DAMP signaling. Data is presented as mean ± SD (n = 4) of atleast 3 independent repetitions, statistical significance is denoted as * p \u0026lt; 0.05, **p \u0026lt; 0.01, n.s. - not significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8289723/v1/fecdd9acec5332c1b91bee41.png"},{"id":97865261,"identity":"c1073456-1d9d-4fee-8d30-c873ecc2730c","added_by":"auto","created_at":"2025-12-10 09:25:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":809147,"visible":true,"origin":"","legend":"\u003cp\u003ePBM induced cell survival signaling pathways. Odontoblasts were preincubated with A. N-Acetylcysteine to inhibit redox signaling, B. Sodium azide (NaN₃) to block ATP generation C. BAY 11-7082 to inhibit NF-κB signaling D. BCL-2 inihbitor to promote apoptosis and E. Caspase 3 inhibtior to prevent apoptosis followed by PBM treatments with red (658 nm) or green (532 nm) for cell viability assessment at 24 hours. Data is presented as mean ± SD (n = 4) of atleast 3 independent repetitions, statistical significance is denoted as * p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, n.s. - not significant.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8289723/v1/4eb54e92461ffd1dffeb0a2f.png"},{"id":97900501,"identity":"5ea1e8cd-517b-4034-a66a-5470e5fcf35c","added_by":"auto","created_at":"2025-12-10 15:45:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4884283,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial function following inflammation and PBM treatments. \u003cstrong\u003eA.\u003c/strong\u003eOdontoblasts were treated with TNFa, BAY 11-7082 + TNF-α, and PBM with red or green laser and JC-1 assay was performed to assess mitochondrial membrane potential. The red (normal) versus green (stress) fluorescence ratio was quantitated with microplate reader. Data is presented as mean ± SD (n = 4), statistical significance is denoted as * p \u0026lt; 0.05, **p \u0026lt; 0.01, n.s. - not significant, \u003cstrong\u003eB\u003c/strong\u003e. JC-1 staining of MDPC-23 cells with fluorescence microscopy for each condition is shown. Bar = 100 mM\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8289723/v1/f34442e0f87ea5449d1e487b.png"},{"id":99796772,"identity":"eb4fe27d-8a27-4877-a9ac-bf5b5b3ab395","added_by":"auto","created_at":"2026-01-08 13:43:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6812109,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8289723/v1/a044a648-8c18-432c-9f9a-34f409f8fb75.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Photobiomodulation counteracts DAMP signaling to improve Odontoblast Survival for Dentin Repair","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTissue regeneration in dentistry is premised on restoring the pulp-dentin vitality and tooth supporting periodontal tissues. Among them, pulp regeneration is focuses on restoring the pulp\u0026ndash;dentin complex is primarily driven by odontoblasts responses that is supported by fibroblasts, endothelial and immune cells (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The current clinical approach of disinfecting, obturating the root canal and restorations are prone to biomaterial failures besides loss of naturally protective proprio reception. Regenerative therapy seeks to re-establish the pulp\u0026rsquo;s natural architecture, vascularity, innervation, and cellular composition, thereby maintaining long term tooth vitality, sensory unction, and long-term structural integrity (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). These regenerative techniques rely on the coordinated use of stem cells, scaffolding materials, and suitable molecular signaling cues to rebuild a vital and functional pulp tissue (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Within this regenerative framework, odontoblasts are central to both mineralized dentin formation and the maintenance of pulp vitality (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). They play a vital role in responding to external stimuli such as mechanical, thermal, and chemical insults by depositing reactionary dentin and orchestrating repair processes to protect the pulp tissue. The preservation and modulation of odontoblast function are therefore central to dental tissue repair and regeneration (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eVarious factors such as trauma and caries can trigger a cascade of inflammatory responses within the tooth (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Secreted factors from caries causing microbes can induce inflammatory responses and morphological changes in the pulp, even before the microbes themselves access the pulp cavity (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). These damage signals trigger disruption of blood flow and local hypoxia initiating potent inflammatory cascades (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Given the rigid encasement of the pulp tissues, the otherwise routine inflammatory hyperemia can compromise repair and undermine integrity. Cellular injury (Damage-associated molecular patterns, DAMPs) or stressors such as microbial pathogens (pathogen-associated molecular patterns, PAMPs) are detected by pattern recognition receptors (PRRs) that promotes release of endogenous molecules (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Recognition of damage triggers assembly of inflammasomes, which activate key signaling pathways, especially NFκB, that modulates progression or resolution of the inflammatory response (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The activation of intracellular signaling cascades results in elaboration of cytokine mediators such as IL-1β, IL-6, and TNF-α that perpetuate the inflammation cascade (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). TNFα is a key pro-inflammatory cytokine that has been noted to be upregulated in in carious and inflamed pulps, where it contributes to nociceptor sensitization and heightened inflammatory activity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Thus, the interconnected roles of DAMPs/PAMPs, NFκB, and TNF-α signaling appears to mediate and sustain inflammation within dental tissues that needs to be resolved prior to initiating pulp-dentin repair (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhotobiomodulation (PBM) in dentistry utilizes low dose light in the visible (400 nm) to near-infrared (1200 nm) spectrum to alleviate pain and inflammation while promoting tissue healing and regeneration (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). PBM has been noted to promote differentiation of dental pulp and mesenchymal bone marrow stem cells to odontoblasts via redox-mediated activation of latent TGF-β1 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). These studies were performed in routine, physiological conditions, especially the \u003cem\u003ein vivo\u003c/em\u003e pulp capping technique and PBM treatments were performed in sterile, controlled mechanical exposures. Other studies have noted activation of intracellular mitochondrial ATP, ROS and NFκB signaling that regulates inflammation, oxidative stress, cell proliferation, and upregulates mineralization markers such as BMP2, Runx2, and Osterix (\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). A prior study noted PBM-generated redox activates NFκB leading to the expression of genes involved in cell survival and anti-apoptosis responses (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Among them, the Bcl-2 family protects against cell death by inhibiting pro-apoptotic factors such as the cysteine protease, Caspases that blocks release of apoptogenic factors from the mitochondria (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The Caspases are critical executioner of the orderly process of apoptosis, orchestrating programmed cell death (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Thus, the ability of PBM treatments to modulate these coordinated signaling pathways could determine overall cell survival versus death.\u003c/p\u003e\u003cp\u003eDespite these advances, the context-dependent effects of PBM treatments on odontoblasts under various stressors that would be clinically relevant have not been explored. This study examined the PBM responses in odontoblasts in simulated inflammation, infection, hypoxia, nutrient deprivation, and extreme pH. The major goal of this work is to gain mechanistic insight to inform optimal future clinical PBM treatment strategies for pulp-dentin complex preservation and regeneration.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003cp\u003eA murine odontoblast (MDPC-23) cell line provided by Tatiana Botero and Jacque Nor, University at Michigan, were cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. Cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eOptimal cell density\u003c/strong\u003e\u003cp\u003eTo determine the optimal cell density for PBM assays, cells were seeded in 96-well flat-bottom plates (Corning, USA) at 1,000, 3,000, or 5,000 cells/well, allowed to adhere overnight prior to studies.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePhotobiomodulation treatment\u003c/strong\u003e\u003cp\u003eA diode laser at 447 nm (blue), 532 nm (green), 658 nm (red), and 810 nm (infrared) (Weber Medical, Germany) was used at power outputs of 1, 10, and 100 mW in continuous wave (CW) mode for a fixed duration of 300 sec that corresponds to energy 0.03, 3, and 30 J respectively. Distance and positioning of laser probe from cells were standardized and confirmed prior to each study with a sensor and power meter (S120CV and PM400, Thor labs). All control groups were handled identically but not exposed to laser illumination.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell viability assessments\u003c/b\u003e: To assess cell viability, the Alamar Blue assay was performed at 24- or 72-hours post treatment. Briefly, culture medium was aspirated and replaced with fresh complete DMEM containing 10% (v/v) Alamar Blue reagent (Thermo Fisher Scientific). Plates were incubated at 37\u0026deg;C for one hour, until a visible pink color change was observed. Fluorescence was assessed at Ex/Em: 520/590 nm using a microplate reader (iMax3, Molecular Devices).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCell stress (DAMP and PAMP) induction\u003c/b\u003e: Various simulated damage and pathogen associated molecular patterns (DAMPs and PAMPs) were generated prior to PBM treatments namely bacterial infection (\u003cem\u003eE coli\u003c/em\u003e O26:B6 LPS 1 \u0026micro;g/ml, Sigma), inflammation (TNF-α, 1 \u0026micro;g/mL, Biosciences), serum deprivation (0.2% FBS, Atlas), hypoxia (CoCl₂ Ward Science), and extreme pH - acidosis (pH 4) or alkalosis (pH 9 or 12) in HEPES-buffered DMEM. For each condition, cells were exposed to the respective treatment solution for 2 hours at 37\u0026deg;C prior to PBM therapy. Only for the pH-treated cells, the pH adjusted HEPES media was replaced with complete media before PBM treatment. All other media conditions were maintained for 24 hours.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePathway specific-inhibitors\u003c/strong\u003e\u003cp\u003eSmall molecule inhibitors against TNF-α (BAY 11-7082 1mM Cayman Chemical, USA), BCL-2 (10 mM, Abcam, USA), Caspase-3 (1 mM Thermo Fisher Scientific), N-Acetylcysteine (1 mM NAC, Sigma), and sodium azide (NaN₃ 1 mM, Sigma) were preincubated 30 min prior to PBM treatments in appropriate media conditions in each group.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eJC-1 Assay\u003c/strong\u003e\u003cp\u003eTo evaluate mitochondrial membrane potential, JC-1 dye (2\u0026micro;M, Fisher Scientific) was added immediately after PBM treatments. As a positive control, carbonyl cyanide m-chlorophenyl hydrazone (CCCP 50 \u0026micro;M, Fiser Scientific) was used to induce complete mitochondrial depolarization. Fluorescence intensity for red fluorescence (J-aggregates) at 535/595 nm (Ex/Em), and green fluorescence (monomers) at 485/535 nm were assessed using a microplate reader (iMax3, Molecular Devices) or inverted fluorescence microscope (Zoe, Biorad). All imaging parameters were kept constant across groups and digital image analysis was performed with NIH ImageJ software (r91, NIH, USA).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003cp\u003eAll experiments were performed in quadriplicates and repeated repeated independently at least three times. Data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and statistical comparisons were performed using one-way ANOVA GraphPad Prism (v10, Dotmatics, USA), where p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRole of cell density in determining optimal PBM responses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first examined the effects of\u0026nbsp;odontoblast density on the PBM responses by seeding cells in 1000, 3000, and 500 cells in a 96-well dish. Among the PBM treatments at 3 J/cm\u003csup\u003e2\u003c/sup\u003e, the\u0026nbsp;3,000 cells/well\u0026nbsp;group exhibited significantly (n = 4, p \u0026lt; 0.05) higher viability compared to other two seeding densities (\u003cstrong\u003eFigure 1A-C\u003c/strong\u003e). The 1,000 cells/well group had sparsely seeded cells and minimal response while the 5,000 cells/well group showed signs of early contact inhibition and reduced proliferation. These results indicated 3,000 cells per well were an optimal cell density to assess consistent and robust PBM responses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of various PBM wavelengths on Odontoblasts\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we performed a PBM dose-escalation study at 3000 cells/well with 0.3, 3, and 30 J/cm\u0026sup2;. All wavelengths induced a significant (n = 4, p \u0026lt; 0.05) increase in cell proliferation at 3 J/cm\u003csup\u003e2\u003c/sup\u003e compared to 0.03 and 30 J/cm\u0026sup2; (\u003cstrong\u003eFigure 1D-G\u003c/strong\u003e). The most prominent responses were observed with red (660 nm) and blue (447 nm) wavelengths. Similar trends were noted at 48 hours \u0026nbsp;(\u003cem\u003edata not shown\u003c/em\u003e) although cell viability showed modest temporal variation at the longer timepoint. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOdontoblasts cell stress responses to PBM treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the simulated DAMPs and PAMPs conditions, PBM responses were evaluated with all wavelengths to assess optimal efficacy. We first simulated inflammation-mimicking DAMP with\u003cem\u003e\u0026nbsp;\u003c/em\u003eTNF\u0026alpha; treatments and observed odontoblasts demonstrated significantly (n = 4, p \u0026lt; 0.5) decreased cell viability compared to untreated controls (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). PBM treatments led to a statistically significant recovery with red (658 nm, n = 4, p \u0026lt; 0.5) and green (532 nm, n = 4, p \u0026lt; 0.5) groups. The blue and near-infrared groups demonstrated moderate increases in viability but were not statistically significant (n = 4, p \u0026gt; 0.5). We next simulated infection-mimicking PAMP with LPS\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003etreatments\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eand observed\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eLPS significantly (n = 4, p \u0026lt; 0.5) reduced odontoblasts viability. None of the PBM treatment wavelengths showed any recovery compared to the LPS-only group (n = 4, p \u0026gt; 0.5) (\u003cstrong\u003eFigure 2B\u003c/strong\u003e). We examined low serum-induced cell stress that caused a substantial (n = 4, p \u0026lt; 0.5) decline in odontoblast viability, confirming the induction of nutrient stress. PBM treatments at 3 J/cm\u0026sup2; with red, green, and infrared groups showed significantly (n = 4, p \u0026lt; 0.5) enhanced odontoblast viability (\u003cstrong\u003eFigure 2C\u003c/strong\u003e). The blue wavelength group showed a trend toward increased viability, but this was not statistically significant (n = 4, p \u0026gt; 0.5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we examined more potent, but transient, stressors. As cells have varying sensitivity to hypoxia, we first performed a dose escalation of chemically-induced hypoxia with varying concentrations of CoCl₂ from 15 mM to 1500 mM. \u0026nbsp; We noted 15 mM of CoCl\u003csub\u003e2\u003c/sub\u003e potently (n = 4, p \u0026lt; 0.5) that was significantly (n = 4, p \u0026lt; 0.5) rescued by PBM treatments with red, green, and blue groups (\u003cstrong\u003eFigure 2D\u003c/strong\u003e). Infrared PBM treatments did not induce any significant (n = 4, p \u0026gt; 0.5) response. Both tissue damage and restorative materials induce extreme pH that has been shown to modulate the dentin reparative responses (26). To simulate these, we used HEPES-buffered DMEM media that was freshly adjusted to acidic or alkaline pH and verified prior to cell treatments for 2 hrs. At\u0026nbsp;pH 4, odontoblast viability was significantly (n = 4, p \u0026lt; 0.5) reduced, reflecting acid-induced stress. PBM treatment with the\u0026nbsp;green laser (532 nm)\u0026nbsp;resulted in a statistically significant (n = 4, p \u0026lt; 0.5) increase in viability compared to the pH-matched non-treated controls (\u003cstrong\u003eFigure 2E\u003c/strong\u003e). Other wavelengths showed moderate, but non-significant (n = 4, p \u0026gt; 0.5), odontoblast rescue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnder\u0026nbsp;physiological pH 7, PBM again produced the highest viability increase in the\u0026nbsp;green laser group, reaching statistical significance (p \u0026lt; 0.05), while other wavelengths yielded modest, non-significant improvements. Under mild (pH 9)\u0026nbsp;and\u0026nbsp;strong alkaline (pH 12)\u0026nbsp;conditions, odontoblast viability was also reduced significantly (n = 4, p \u0026lt; 0.5), though to a lesser extent than in pH 4. PBM treatment across all, but green, wavelengths resulted in a trend, but non-significant (n = 4, p \u0026gt; 0.5) improvements. PBM treatments with the\u0026nbsp;green laser\u0026nbsp;showed significant (n = 4, p \u0026lt; 0.5) recovery. At\u0026nbsp;pH 12, all four laser wavelengths elicited a significant (n = 4, p \u0026lt; 0.5) increase in odontoblast viability relative to non-treated control (\u003cstrong\u003eFigure 2F\u003c/strong\u003e). These results indicate that odontoblast stressor responses vary with individual DAMPs or PAMPs (\u003cstrong\u003eFigure 2G\u003c/strong\u003e) implying specific PBM clinical parameters are necessary to evoke optimal therapeutic benefits. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of specific PBM-evoked signaling pathways in odontoblast survival\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe variance in the PBM treatment efficacy to individual stressors suggest there are discrete operative mechanisms in individual pathophysiological scenarios. For these set of studies, we chose to pursue the red and green PBM treatments in odontoblasts that showed maximal efficacy following the inflammation simulating TNFa treatments. We resorted to carefully investigating the known PBM evoked mitochondrial pathways, namely the photoabsorption by Cytochrome C Oxidase (CCO) generated ROS and ATP generation. Co-treatment with \u003cstrong\u003eNAC\u003c/strong\u003e, a well-characterized ROS scavenger, significantly\u0026nbsp;(n = 4, p \u0026lt; 0.5)\u0026nbsp;reduced TNF-\u0026alpha;\u0026ndash;induced\u0026nbsp;odontoblast\u0026nbsp;cytotoxicity (\u003cstrong\u003eFigure 3A\u003c/strong\u003e). The significantly\u0026nbsp;(n = 4, p \u0026lt; 0.5) improved viability noted with PBM treatments was partly, but statistically significantly\u0026nbsp;(n = 4, p \u0026gt; 0.5),\u0026nbsp;neutralized by NAC pre-treatments. Neutralization with So\u003cstrong\u003edium Azide (NaN\u003csub\u003e3\u003c/sub\u003e)\u003c/strong\u003e, a mitochondrial complex IV inhibitor, also markedly\u0026nbsp;(n = 4, p \u0026lt; 0.5) improved cell viability following TNF-\u0026alpha; treatments (\u003cstrong\u003eFigure 3B\u003c/strong\u003e). PBM treatments demonstrated improved odontoblast viability that was neutralized significantly (n = 4, p \u0026lt; 0.5) with red lasers, and only partially, not statistically significantly (n = 4, p \u0026gt; 0.5) with the green laser. These results suggest that both PBM generated ATP and ROS are partly contributing to the improved odontoblast survival response during inflammation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we examined the role of NFkB, a central inflammation signaling mediator integrating odontoblast survival and death responses. We noted that the NFkB inhibitor, BAY 11-7082 treatments significantly (n = 4, p \u0026lt; 0.5) reduced odontoblast viability and potently neutralized (n = 4, p \u0026lt; 0.5) both red and green PBM treatment induced odontoblast survival (\u003cstrong\u003eFigure 3C\u003c/strong\u003e). We then utilized two apoptosis pathway modulators, namely BCL-2 (anti-apoptotic) and Caspase 3 (pro-apoptotic). Odontoblast treated with a\u0026nbsp;BCL-2 inhibitor, that promotes apoptosis, showed no significant (n = 4, p \u0026gt; 0.5) effects following TNFa\u0026nbsp;treatments\u0026nbsp;(\u003cstrong\u003eFigure 3D\u003c/strong\u003e). However, they subtly improved PBM mediated survival by both wavelengths, although these were not statistically significant (n = x, p \u0026gt; 0.5). Finally, Caspase 3 inhibitor, an apoptosis blocker, significantly (n = 4, p \u0026gt; 0.5) improved TNFa\u0026nbsp;cytotoxicity, but did not have any appreciable (n = 4, p \u0026gt; 0.5) efficacy in PBM improved odontoblast survival\u0026nbsp;(\u003cstrong\u003eFigure 3E\u003c/strong\u003e). These results indicate that PBM-induced NFkB signaling is a key mediator in odontoblast survival in inflammation that primarily involves induction of cell survival signaling rather than inhibiting cell death (apoptosis) pathway. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of mitochondrial signaling in PBM-evoked odontoblast survival\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the mechanism of PBM-improved odontoblast survival, we assessed the effects of PBM treatments and pathway-specific inhibitors on the mitochondrial membrane potential using a fluorescent dye, JC-1. Odontoblasts probed with JC1 demonstrated high mitochondrial membrane potential with robust accumulation of the dye forming red fluorescence (J-aggregates) and minimal green fluorescence (J-monomers) indicating healthy mitochondrial functions. TNFa\u0026nbsp;treatments reduced red and increased green fluorescence, indicating mitochondrial depolarization and reduced membrane potential (\u003cstrong\u003eFigure 4A\u003c/strong\u003e). Odontoblasts treated with CCCP induced robust depolarization that was also evident with TNF-a treatments. The increased membrane potential was not affected by treatments with the NFkB inhibitor, BAY 11-7082. The red laser PBM appeared to increase the membrane potential that was significantly (n = 4, p \u0026lt; 0.5) reduced by NFkB inhibition while the green laser potently (n = 4, p \u0026lt; 0.5) reduced membrane potential that appears to be independent of NFkB inhibition. These trends were further verified with fluorescence imaging for the red to green fluorescence based on their ratios that were digitally quantified (\u003cstrong\u003eFigure 4B\u003c/strong\u003e). Overall, it appears the two laser wavelengths have distinctly different effects on the mitochondrial potential perturbed by inflammation. \u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe pulp-dentin complex contains odontoblasts and stem cells that maintain its health and vitality. These cells are subjected to various forms of damage or stressors, such as mechanical injury, infections, radiation, or chemical exposures that trigger represent damage-associated molecular patterns (DAMPs) (27). DAMPs are endogenous molecules released from stressed or dying cells, either from the intracellular or extracellular space, and are key mediators implicated in inflammation and inflammatory diseases. (28) The present study noted the ability of discrete wavelengths for photobiomodulation (PBM) are capable of protecting odontoblasts from a broad range of DAMPs including inflammation, hypoxia, nutrient deprivation, and extreme pH. The ability of red and green wavelengths to significantly improve cell viability under conditions by promoting cell survival signaling via NFkB and TGF-b\u0026nbsp;signaling appears to be distinct from the cell death (BCL2 and Caspase) signaling indicating specificity of the therapeutic pathway s evoked. These observations support the use of PBM for vital pulp therapy and pulp-dentin regenerative strategies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBM has been historically explored for accelerating healing and reducing inflammation in soft tissues. (29-33) While most clinical studies have focused on specific disease contexts, \u003cem\u003ein vitro\u003c/em\u003e studies rarely simulate these pathophysiological conditions, specifically the unique nature and sequalae of disease patterns (DAMPs or PAMPs). Attempts at optimizing PBM dosing and delivery techniques could significantly improve with suitable contextualized in vitro models, also termed microphysiological systems (MPS) utilizing organoids or organ-on-a-chip (OoC) systems (34-38). Several experts have suggested PBM could add significant utility in regenerative pulp-dentin tissue engineering (39, 40) These reviews emphasize the\u0026nbsp;ability of PBM to stimulate cell proliferation in different cell lineages of the pulp-dentin complex including odontoblasts, fibroblasts, and endothelial cells\u0026nbsp;(41-43). However, the precise signaling mediating this response remains to be elucidated.\u0026nbsp;(44)\u0026nbsp;The clinical scenarios simulated in this study focused on relevant stressors from caries progression, pulpal inflammation, or physicochemical tissue injuries\u0026nbsp;(45). These included TNF-\u0026alpha; (inflammation), \u003cem\u003eE. coli\u003c/em\u003e LPS (bacterial infection), CoCl₂ (hypoxia), low serum (nutrient deprivation), and extreme pH contextualizing the injury stimuli.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe rationale here is that the basal cell response signaling network would be perturbed in these pathophysiological contexts and hence, require specific modulation to therapeutically benefit from PBM treatments (46). While each injurious agent would generate varying degrees of cell stress response, the ultimate output would be survival or death. Hence, the ability of a therapeutic intervention to target a conserved stress response would modulate the magnitude and specificity of the evoked pathways and ultimately affect cell survival. Hence, we chose this as our primary endpoint. In this study, we noted inflammation, hypoxia, nutrition and alkaline pH stress were effectively counteracted by PBM treatments while infection and acidic pH were not modulated. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe first investigated the effect of cell density on odontoblast response to PBM treatments as cell-cell contact mediated paracrine signaling plays a vital role in integrating extrinsic perturbations including stressors like hypoxia, serum starvation, and oxidative stress, among others (47-50). We noted that providing a semi-confluent (40-60%) culture condition evokes optimal cell proliferative response to a 3J/cm\u003csup\u003e2\u003c/sup\u003e PBM dose that is in agreement with prior studies (41). Lower or higher confluency were ineffectual. While this could be attributed to biological cellular microenvironment, it may also be due to non-optimal photon distribution based on the Arndt-Schultz curve implying lower confluent cells getting too much PBM dose and higher getting too little\u0026nbsp;(51, 52). Nonetheless,\u0026nbsp;optimizing physiological cell response appears to be a key step prior to exploring pathological stimuli contexts, especially in normal versus transformed cell responses to PBM treatments\u0026nbsp;(53).\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To further investigate the improved survival with PBM treatments, we sought to examine any perturbations to cell death pathway. We looked at BCL-2 (anti-apoptotic) and Caspase-3 (pro-apoptotic) that play crucial and opposing roles in the regulation of apoptosis, a key cell death response to stress (54). BCL-2 preserves cell survival by maintaining mitochondrial integrity and inhibiting the release of cytochrome c, preventing activation of the caspase cascade. This function is essential for reducing oxidative stress and controlling calcium flux within cells. In our studies, the BCL-2 inhibitor did not rescue the reduced odontoblast survival in response to inflammation and did not significantly affect the PBM rescue, although there was a trend towards improvement. A study by Zhang et \u003cem\u003eal.\u003c/em\u003e observed genetic manipulation of anti-apoptotic proteins like BCL-2 can enhance odontoblast survival and promote dentin repair. (55) This could be attributed to the physiological context and endogenous regulation but could be investigated further. Caspase-3 serves as a central executioner of apoptosis; once activated, it cleaves various cellular substrates and promotes the morphological and biochemical changes characteristic of programmed cell death. Notably, caspase-3 can cleave BCL-2 itself, disabling its protective effects and reinforcing the apoptotic process. This interplay between BCL-2 and caspase-3 represents a critical balance between cell survival and death. (56-58) In the current study, the Caspase-3 inhibitor significantly rescued the inflammation induced odontoblast cell death, but it also did not significantly modulate the PBM enhanced odontoblast survival. As neither inhibitor modulated the PBM-induced rescue, it appears the PBM-induced survival signaling is likely upstream of the apoptotic (BCL-2 and Caspase-3) signaling responses. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe noted the most prominent odontoblast response was with the NF-\u0026kappa;B inhibitor, a central pathway in mediating inflammation (59-61). The complete abrogation of the PBM rescue as well as further depression of odontoblast survival to TNFa\u0026nbsp;suggested NF-kB is a key PBM cytoprotective signaling pathway. Direct activation of NF-kB signal transduction via redox generation by PBM treatments has been demonstrated\u0026nbsp;(23). While TNF-a\u0026nbsp;increased mitochondrial membrane potential (\u0026Delta;\u0026Psi;m) as anticipated, the two wavelengths had discretely different responses, with the red laser showing an increase, while green laser showed a decrease. Further, inhibiting NF-kB signaling had no effect on basal inflammatory or reduced mitostress by green laser. Most intriguingly, the red wavelength not only increased mitostress, but was also modulated significantly by NF-kB inhibition. As both wavelengths effectively improve odontoblast survival during simulated inflammation that are neutralized by NF-kB inhibition, these observations together provide significant insights into integrated survival signaling induced by PBM treatments on odontoblasts. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA major finding in this study was the selectivity of the evoked therapeutic PBM wavelengths. Prior studies have observed the clinical efficacy of discrete wavelengths for PBM treatments (18, 62-66). We noted both red and green most consistently improved odontoblast survival across various DAMPs. Blue showed no efficacy in serum, inflammation or acidic pH while NIR only showed efficacy in alkaline DAMPs condition suggesting there are wavelength-specific evoked responses. The clinical use of visible or infrared wavelengths or their combination has been employed based on anatomical target location. This has been premised on the former treating more superficial targets while infrared penetrates to deeper tissues while their combination aimed at treating the volume more effectively. The rationalized use of individual wavelength evoked responses, likely based on selective absorption, have not been investigated in PBM treatments thus far. Moreover, the use of clinically effective infrared wavelengths, especially 800 to 1200 nm has raised important questions on photoabsorption and inelastic scattering as the thermodynamic basis for biophotonics energy transfer (67, 68). \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite some valuable mechanistic insights, this study has several limitations. The pulp dentin complex has cells of various lineages. Although odontoblast has a key role in dentin regeneration, fibroblasts, endothelial and macrophages play important roles. Future \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e studies could validate their responses to PBM in various DAMP conditions. This study outlined distinct role for two individual wavelengths red (658 nm) and green (532 nm), but their combinations can be further explored. Future studies employing higher resolution analytical techniques such as proteomics, metabolomics, and single-cell analysis, and spatial transcriptomics could unravel mechanistic foundation of PBM-mediated protection. Alternative mechanisms of PBM cytoprotection could explore activation of transient receptor potential (TRP) channels, including TRPV1, TRPV2, and TRPV4, resulting in increased calcium influx and downstream signaling activation.(69, 70) PBM has also been shown to promote photodissociation of nitric oxide (NO), increasing its bioavailability and improving microcirculation to stressed cells.(71, 72) Finally, PBM activation of intracellular cell survival signaling such as JAK/STAT, PI3K/Akt and MAPK pathways that are central to promoting cell proliferation, enhancing survival, and facilitating the resolution of inflammation could be explored.(73-76) It remains to be seen if these pathways operate independently or in concert with the classical PBM induced ROS:ATP:NF-\u0026kappa;B axis providing additional avenues exploited by PBM-mediated cellular protection.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study noted the ability of PBM treatments to significantly enhance odontoblasts under various stress conditions. This can contribute to their role in pulp-dentin repair and regeneration. Elucidation of specific intracellular signaling pathways following PBM treatments that modulate mitochondrial functions, oxidative stress responses, and inflammatory signaling could enable precision programmed approach for PBM in clinical regenerative endodontics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the University at Buffalo faculty funds for providing support for these studies. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no conflicts of interest with the current work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFT: designed and performed the studies, analyzed the data, prepared the draft manuscript, \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZY: assisted with data analysis, writing and reviewing the manuscript\u003c/p\u003e\n\u003cp\u003ePRA: conceptualized and designed study, revised and finalized manuscript \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll requests for primary data will be made available upon reasonable request\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFeigin, K. and B. 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Arany (2020) Photobiomodulation therapy in diabetic wound healing. \u003cem\u003eWound Healing, Tissue Repair, and Regeneration in Diabetes\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e 305-321.\u003c/li\u003e\n\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":"Photobiomodulation, Odontoblasts, DAMP, NFκB Inhibition","lastPublishedDoi":"10.21203/rs.3.rs-8289723/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8289723/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eTooth vitality is driven by the odontoblast responses in the pulp-dentin complex. Low-dose light treatments, termed Photobiomodulation (PBM) therapy has been noted to induce odontoblast differentiation from dental pulp stem cells and promote dentin repair in a sterile, direct pulp capping approach. Its ability to direct repair in a routine clinical scenario post-infection or injury remains to be elucidated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eAn odontoblast cell line, MDPC-23 were subjected to various stressors namely inflammation (TNF-α), bacterial infection (LPS), hypoxia (CoCl₂), nutrient deprivation (0.2% serum), and pH stress (pH 4 or 12). Cells were treated with four PBM wavelengths of 447 nm (blue), 532 nm (green), 658 nm (red), and 810 nm (near-infrared) delivered at doses of 0.03, 3, or 30 J/cm\u0026sup2;. To investigate potential mechanisms, small molecule inhibitors targeting ROS (N-Acetylcysteine, NAC), ATP (Sodium Azide, NaN₃), NFκB (BAY 11-7082), BCL-2 (anti-apoptotic), Caspase-3 (pro-apoptotic) were used. Cell viability was assessed with Alamar Blue and mitochondrial membrane potential was assessed with JC-1 fluorescence assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe noted all four PBM wavelengths induced significant (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) odontoblast survival at 3 J/cm\u003csup\u003e2\u003c/sup\u003e at optimal cell density. All stressors, except LPS, reduced odontoblast viability significantly (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) that were rescued significantly (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with the red and green wavelengths most consistently. Both wavelengths were not significantly (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) affected by neutralization of ROS, BCL-2 or Caspase 3, but differentially affected by ATP deprivation and significantly (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) neutralized by NF-κB inhibition. The increased mitochondrial membrane potential to TNFα treatments were also differentially modulated by the two wavelengths significantly (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) suggesting there are divergences in individual signaling pathways mediating the overall PBM survival response.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese results demonstrate that discrete PBM wavelengths evoke context-dependent odontoblast proliferative responses. These findings highlight the therapeutic potential of PBM in modulating odontoblast responses to various damage stimuli that can be utilized to develop specific protocols for optimal clinical therapeutic clinical outcomes.\u003c/p\u003e","manuscriptTitle":"Photobiomodulation counteracts DAMP signaling to improve Odontoblast Survival for Dentin Repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 09:24:41","doi":"10.21203/rs.3.rs-8289723/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":"3a97c644-e722-4d9b-9a63-f1df3b7b38c1","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-07T13:39:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-10 09:24:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8289723","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8289723","identity":"rs-8289723","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}