Inflammatory, Oxidative, and Neurotrophic Profiles in Monozygotic Twins Discordant for Pain-related TMD

Inflammatory, Oxidative, and Neurotrophic Profiles in Monozygotic Twins Discordant for Pain-related TMD | 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 Inflammatory, Oxidative, and Neurotrophic Profiles in Monozygotic Twins Discordant for Pain-related TMD Lais Valencise Magri, Melissa Oliveira Melchior, Cecília Nogueira Tavares Peixeiro, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9336568/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract This study investigated the biological mechanisms underlying temporomandibular disorder (TMD) pain in monozygotic twins discordant for the condition, isolating environmental factors from genetic influence. Twenty women (ten pairs of discordant twins) underwent standardized examinations and venous plasma analysis. Inflammatory biomarkers (IL-6, IL-10), oxidative markers (MDA, SOD, catalase), matrix remodeling proteins (MMP-2, MMP-9, TIMP-1, TIMP-2), and neurotrophic factors (BDNF, β-NGF, α2M) were measured. Twins with painful TMD demonstrated greater pain severity, functional interference, and mechanical sensitivity compared to discordant controls. Significant within-pair differences were identified in IL-6, IL-6/IL-10 ratio, MDA/SOD ratio, MMP-9, TIMP-2, and BDNF levels. Pro-inflammatory and oxidative indices positively correlated with pain intensity and palpation sensitivity, while reduced BDNF associated with greater symptom burden. Principal component analysis revealed a dominant inflammatory-oxidative profile that discriminated painful twins from controls. In genetically identical individuals, painful TMD associates with selective peripheral biological alterations involving inflammation, oxidative imbalance, and extracellular matrix remodeling. These findings demonstrate that environmental and experiential factors biologically determine vulnerability to chronic orofacial pain, highlighting the importance of epigenetic mechanisms in TMD pathogenesis beyond genetic predisposition. This article identifies specific peripheral biomarker profiles distinguishing monozygotic twins with and without painful TMD, revealing that inflammation, oxidative imbalance, matrix remodeling, and reduced neurotrophic support characterize the painful phenotype. These mechanistic insights could enable clinicians to develop targeted, biologically informed therapeutic strategies and potentially predict pain vulnerability in genetically susceptible individuals. Temporomandibular Disorders Orofacial Pain Monozygotic Twins Inflammation Oxidative Stress Neurotrophic Factors Biomarkers Chronic Pain Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Temporomandibular disorders (TMD) are among the most frequent chronic orofacial pain conditions and arise from the interaction of biological, emotional, and environmental influences. 1 – 4 Large cohort studies have shown that individuals with painful TMD often present heightened mechanical sensitivity and altered nociceptive modulation, indicating contributions from both peripheral and central pain mechanisms. 1 – 3 , 5 Despite the advances in characterizing clinical phenotypes, the mechanisms underlying why some individuals develop persistent pain while others with comparable life experiences remain pain-free are not fully understood. Psychological and behavioral factors appear to shape pain experience and disability in TMD. 1 , 2 , 5 , 6 Patients frequently exhibit greater hyperalgesia, sleep disturbances, emotional distress, and attentional patterns directed toward pain, and these features are linked to more intense symptoms and functional compromise. 1 , 3 , 5 , 6 Clinically, this includes increased muscle tenderness, familiar pain evoked during palpation, and pain with mandibular movement, supporting an interaction between somatic sensitivity and behavioral responses. 1 – 3 , 6 These findings reinforce the importance of considering pain perception, behavior, and clinical presentation as interrelated domains in TMD. Twin and family studies have consistently demonstrated that a proportion of TMD pain risk is attributable to inherited factors, supporting a heritable component to the disorder while also highlighting the importance of environmental and experiential influences. 4 , 7 – 9 Monozygotic twins, in particular, provide a unique research model given their identical genetic profile, and discordance in pain presentation within twin pairs suggests that biological processes related to life experiences, stress exposure, and behavioral adaptation may contribute meaningfully to disease expression. 1 , 5 , 7 , 8 Current research has increasingly focused on biological systems associated with pain persistence, including immune mediators, oxidative processes, and neurobiological adaptations. 1 , 3 , 6 , 10 Epigenetic mechanisms such as DNA methylation and chromatin modifications have been shown to influence gene expression relevant to nociception and inflammatory signaling. 2 , 3 , 10 , 11 Biomarker-based investigations have identified inflammatory cytokines, oxidative stress indicators, and matrix-regulating molecules associated with TMD 12 – 15 , although evidence remains heterogeneous and few studies have adopted genetically controlled approaches. 12 , 14 – 16 Despite important advances, the specific biological characteristics associated with painful TMD in individuals who share identical genetic backgrounds are still unclear. Our previous research with monozygotic twins discordant for painful TMD demonstrated differences in clinical pain responses and behavioral features even in the absence of genetic variability. 2 , 5 – 7 Building on these findings, the present study investigates whether peripheral inflammatory markers, oxidative stress indicators, and neurotrophic factors differ between genetically identical women discordant for painful TMD. We hypothesize that twins with painful TMD will show higher inflammatory activity and altered biomarker patterns relative to their pain-free co-twins. Therefore, the objective of this study was to characterize biological signatures associated with painful TMD in monozygotic twins and relate these molecular findings to clinical pain expression. METHODS Study design and ethical approval This investigation employed a cross-sectional, discordant monozygotic twin design to examine biological and clinical correlates of painful temporomandibular disorder (TMD). By comparing genetically identical individuals who differ in pain status, this design minimizes confounding from inherited susceptibility, population stratification, and shared early-life environment, thereby strengthening inferences regarding environmentally shaped biological variation. Although cross-sectional in nature and therefore not suited for causal inference, the discordant twin approach enhances internal validity by isolating phenotypic divergence that arises after the period of shared genetic and early developmental influences. The present study expands upon a previously published analysis in the same cohort that characterized cognitive-emotional and somatosensory features of the painful phenotype 5 , providing an integrated biological extension focused on peripheral biomarker profiles. The study protocol was approved by the Research Ethics Committee of XXXXXXXXXX, which includes two representatives of patients who are users of the dental services provided by this institution. All procedures conformed to the principles of the Declaration of Helsinki. Participants were recruited voluntarily and provided written informed consent before enrollment. Confidentiality, autonomy, and the right to withdraw were fully guaranteed throughout the study. Participants A total of 20 female monozygotic twins (10 discordant pairs) aged 18 to 55 years participated in the study. Participants were recruited via institutional databases, social media, and community announcements as described in the original study 5 . Monozygotic status was confirmed through assessment of phenotypic similarity assessment and parental confirmation, following widely accepted protocols for twin-research. Diagnosis of painful TMD The diagnosis of painful temporomandibular disorder (TMD) was established using the validated Brazilian Portuguese version of the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) 17 , administered by clinicians formally trained and calibrated in DC/TMD methodology. The clinical examination followed the standardized Axis I protocol and included systematic palpation of the masticatory muscles and temporomandibular joints, assessment of mandibular range of motion, and evaluation of pain provocation and reproduction of familiar symptoms. This structured approach ensured diagnostic reliability and minimized examiner-related variability, an important consideration in twin studies where misclassification could obscure true within-pair differences. Inclusion criteria Participants were eligible if they met all of the following criteria: Female monozygotic twin, aged 18 to 55 years; One twin reporting ≥ 3 months of orofacial pain and fulfilling DC/TMD diagnostic criteria for painful TMD (myalgia and/or arthralgia); The co-twin presenting no DC/TMD diagnosis and no persistent orofacial pain. This definition ensured clear discordance while allowing for the possibility of isolated or subclinical symptoms in the control twin, provided they did not satisfy diagnostic thresholds. Exclusion criteria Exclusion criteria were defined to avoid confounding from concurrent conditions or treatments known to influence pain or inflammatory pathways. Twins were excluded if they were undergoing or had recently undergone TMD-related therapy (e.g., interocclusal appliances, physiotherapy, photobiomodulation/laser therapy, acupuncture), or were using analgesics, anti-inflammatories, or centrally acting medications. Additional exclusions included history of head or neck tumors, trauma, or surgery; diagnosed neurological disease; severe psychiatric disorders (except anxiety or depression, due to their high prevalence in chronic pain populations); and pregnancy or breastfeeding. These criteria minimized biological heterogeneity unrelated to TMD. Control of menstrual cycle phase Because both TMD symptoms and inflammatory markers exhibit menstrual cycle–related variability, 18,19 all participants with regular cycles were assessed during the follicular phase. This phase was selected because estrogen rises gradually and progesterone remains low and stable, producing a more uniform endocrine environment. Conducting assessments during the follicular phase reduces hormonal fluctuations known to influence pain sensitivity, neuroimmune activity, and inflammatory mediator levels, thereby enhancing internal consistency across participants and reducing endocrine-related confounding. Clinical assessment Clinical examinations were performed in accordance with DC/TMD Axis I guidelines, using standardized procedures for manual palpation of masticatory and cervical musculature and for the evaluation of mandibular functional movements. 17 Trained examiners applied controlled, criterion-based digital pressure to predefined anatomical sites to assess pain provocation, reproduction of familiar symptoms, and elicitation of referred pain patterns. These procedures followed established diagnostic thresholds for muscle tenderness and mechanical provocation of TMD-related symptoms, thereby minimizing examiner-related variability and enhancing diagnostic reliability within the twin pairs. Mandibular movement assessment—comprising maximal unassisted and assisted opening, lateral excursions, and protrusion—was conducted to document movement-evoked pain and features of functional impairment consistent with painful myogenous TMD. Pain severity and functional interference were quantified using the Brief Pain Inventory (BPI), which offers validated, continuous indices of pain intensity and its impact on daily functioning, supporting comprehensive clinical characterization. 20 Objective measures of nociceptive sensitivity were incorporated through quantification of the proportion of palpation-positive sites relative to the total number of evaluated regions. This approach allowed systematic capture of localized muscle tenderness and mechanical hyperalgesia across standardized examination sites. In addition, pain elicited during mandibular movement was recorded, alongside differentiation among familiar pain on palpation, referred pain, and palpation-induced familiar headache. These distinctions reflect well-defined DC/TMD categories and permit more precise mapping of somatosensory responses relevant to the TMD pain phenotype. Together, these multidimensional clinical measures—summarized in Table 1 —capture a pattern of increased mechanical sensitivity, widespread palpation tenderness, movement-provoked pain, and functional limitation characteristic of painful myogenous TMD. This rigorously assessed clinical profile provides a robust foundation for integrating peripheral biomarker data and interpreting biological correlates of pain expression in a genetically controlled context. Biological sample collection and processing Venous blood samples were collected in the morning following an overnight fast to minimize circadian influences and diet-related metabolic variability on inflammatory, oxidative, and neurotrophic biomarkers. Standardized phlebotomy procedures were used, and samples were processed immediately after collection to reduce pre-analytical degradation. Whole blood was centrifuged under controlled conditions, and the resulting plasma was aliquoted into sterile, coded polypropylene tubes to prevent repeated freeze–thaw cycles and preserve analyte integrity. Plasma samples were stored at − 80°C until analysis, following established recommendations for long-term biomarker stability. To minimize analytical bias, all laboratory personnel performing biomarker quantification were blinded to the clinical status of each twin. Blinding procedures ensured that sample handling, assay performance, and data recording were not influenced by knowledge of group assignment, thereby increasing the internal validity of the biomarker measurements. Biomarker Analysis Biomarkers were selected based on their mechanistic relevance in inflammation, oxidative stress, extracellular matrix regulation, and neuroplasticity, domains previously implicated in chronic musculoskeletal pain. Assays were performed following the manufacturers' protocols. Inflammatory mediators and matrix remodeling factors were quantified by sandwich-type ELISA (Enzyme-Linked Immunosorbent Assay) (R&D Systems, Minneapolis, MN, USA). Specifically, Quantikine kits were used for IL-6 (Catalog RDSY-D6050-96SW), IL-10 (Catalog RDSY-D1000B-96SW) and TNF-α (Catalog RDSY-DTA00D-9SW). For matrix remodeling, DuoSet kits were used for MMP-2 (Catalog RDSY-DY902-1KIT), MMP-9 (Catalog RDSY-DY911-1KIT), TIMP-1 (Catalog RDSY-DY970-1KIT), TIMP-2 (Catalog RDSY-DY971-1KIT), and α-2-macroglobulin (Catalog RDSY-DY1938-96SW). Oxidative stress markers were analyzed by colorimetric/spectrophotometric assays (Cayman Chemical, Ann Arbor, MI, USA). Kits used were for Malondialdehyde (MDA) (TBARS Assay Kit, Catalog CAYM-10009055-96W), Superoxide Dismutase (SOD) (Assay Kit, Catalog CAYM-706002-96W), and Catalase (Assay Kit, Catalog CAYM-707002-96W). Statistical Analysis Within-pair differences (Δ = TMD – Control) were calculated for all biomarkers and clinical variables to minimize genetic and shared early environmental confounding inherent to monozygotic discordant twin designs. Data distribution was examined using the Shapiro–Wilk test, and variables with strong skew or extreme values were inspected using Q–Q plots and leverage diagnostics. Descriptive statistics were reported as means and standard deviations for raw values and as medians and interquartile ranges when distributional asymmetry was present. Inferential comparisons between affected and control twins were conducted using paired statistical tests. Paired t-tests were applied when Δ values met normality assumptions, whereas non-normally distributed variables or those with tied ranks were analyzed using Wilcoxon signed-rank tests. Correlations between Δ clinical measures (pain severity, functional interference, movement-evoked pain, and palpation outcomes) and Δ biomarker concentrations were assessed using Pearson coefficients (r), with significance defined as P < 0.05. Correlation matrices were visualized to identify clusters of clinically relevant associations. To explore multivariate patterns, principal component analysis (PCA) was performed on z-standardized Δ biomarker values. Clinical variables were then projected as supplementary correlation vectors onto the PCA solution (PC1 and PC2) to evaluate their alignment with the dominant biological dimensions. Clinical vectors with |r| > 0.4 were interpreted as meaningful contributors to component structure. All analyses and visualizations—including paired-difference plots, correlation heatmaps, and PCA biplots—were conducted in R (version 4.4.0; R Foundation for Statistical Computing, Vienna, Austria). RESULTS Clinical characteristics As required by the discordant-twin design, in all ten pairs one twin fulfilled full DC/TMD criteria for painful TMD while the co-twin did not. Importantly, all control twins received the formal DC/TMD classification of absence of TMD ; however, several exhibited subclinical findings, such as mild tenderness on isolated palpation sites or occasional movement-associated discomfort, without reaching diagnostic thresholds. This nuance helps contextualize the within-pair differences observed and clarifies that the control phenotype represents the absence of a diagnosable disorder , rather than a completely symptom-free state. Group-level comparisons showed that painful twins reported higher mean pain levels (BPI-severity: 3.9 ± 1.5; BPI-interference: 3.4 ± 1.6), whereas their co-twins reported lower scores (2.5 ± 1.6 and 2.0 ± 1.7, respectively). Nevertheless, as shown in Table 1 , a few control twins reported low-intensity pain or minimal interference, which produced negative Δ values in some pairs (e.g., pairs 1, 9, and 10). These cases illustrate the expected continuous distribution of pain experiences even in the absence of a formal diagnosis. Similarly, across DC/TMD clinical examination domains—percentage of painful palpation points, movement-evoked pain, familiar pain, referred pain, and headache-related palpation pain—affected twins generally exhibited higher values. Still, control twins occasionally displayed isolated tenderness or temporal muscle sensitivity, again without meeting diagnostic criteria. Consequently, Δ values varied in magnitude and direction, ranging from small negative differences in some pairs to large positive contrasts in others. Overall, Table 1 demonstrates that although painful twins consistently showed greater clinical burden, the discordant-twin model naturally captures a graded, subclinical–to–clinical spectrum, reinforcing the biological relevance of examining within-pair differences beyond genetic susceptibility. Table 1 Clinical characteristics of monozygotic twins discordant for painful TMD based on BPI and DC/TMD. Δ values represent within-pair differences (TMD – Control). Twin Pair Δ Pain Severity(BPI) Δ Pain Interference(BPI) Δ Reported Pain Points(%) Δ Movement-Evoked Pain(%) Δ Familiar Pain on Palpation(%) Δ Referred Pain on Palpation (%) Δ Headache on Palpation(%) Pair 1 –1.5 + 3.5 0 0 + 81 + 62 + 83 Pair 2 + 3.8 + 3.3 + 7 + 16 + 44 + 31 + 16 Pair 3 + 4.3 + 1.1 + 22 + 8 + 62 + 56 + 33 Pair 4 + 0.5 + 1.1 + 21 + 14 + 19 + 50 –17 Pair 5 + 3.7 –0.3 0 + 4 + 6 + 19 0 Pair 6 + 1.7 + 1.2 + 29 + 8 + 100 + 100 + 100 Pair 7 + 1.5 + 1.8 + 36 + 4 + 62 + 12 + 83 Pair 8 + 5.3 + 1.9 + 28 0 + 31 + 12 + 83 Pair 9 –2.0 –2.5 –14 –10 + 6 + 19 + 50 Pair 10 –2.5 + 3.3 + 21 –4 –7 + 19 –17 Mean Δ ± SD + 1.48 ± 2.55 + 1.44 ± 2.05 + 15.0 ± 13.4 + 4.0 ± 7.1 + 40.4 ± 33.4 + 38.0 ± 29.6 + 41.4 ± 38.3 Notes : Pain outcomes are expressed as percentages of positive responses relative to the number of sites evaluated: 14 sites for reported pain points (DC/TMD E1a–E1b), 50 sites for movement-evoked pain (DC/TMD E4b–E5c), 16 sites for familiar and referred pain on palpation (DC/TMD E9), 6 sites for headache-related palpation pain (temporal muscle; DC/TMD E9). All control twins fulfilled the DC/TMD “Absence of TMD” classification, although some exhibited subclinical findings (e.g., mild tenderness without diagnostic significance). Figure 1 displays within-pair differences in biomarker concentrations between monozygotic twins discordant for painful temporomandibular disorder (TMD), revealing a pattern of selective rather than global biological divergence. Significant increases were observed in affected twins for IL-6, IL-6/IL-10, MDA/SOD, MMP-9, TIMP-2, and MMP-9/TIMP-1, while BDNF was significantly reduced (all p < 0.05). Other biomarkers, including IL-10, MDA, SOD, CAT, MMP-2, TIMP-1, β-NGF, and α2M, did not show significant within-pair differences. The pattern suggests that painful twins exhibit targeted alterations involving pro-inflammatory signaling, redox imbalance reflected primarily in proportional oxidative markers, increased matrix-remodeling potential, and reduced neurotrophic support. Collectively, these findings indicate that specific peripheral pathways are differentially engaged in the expression of painful TMD even among genetically identical individuals, rather than indicating uniform shifts across all biomarker domains. The correlation analysis revealed coherent biological patterns linking biomarker shifts to clinical pain expression in monozygotic twins discordant for painful TMD (Fig. 2). Pro-inflammatory and oxidative-stress markers—particularly IL-6, the IL-6/IL-10 ratio, MDA, and the MDA/SOD ratio—showed moderate to strong positive correlations with pain severity and pain interference on the BPI, indicating that greater inflammatory activation and redox imbalance tended to accompany higher pain burden. In contrast, neurotrophic markers such as BDNF and β-NGF correlated negatively with BPI scores, suggesting that reduced neurotrophic support may be associated with more severe and disabling pain symptoms. Associations between biomarker deltas and DC/TMD clinical variables followed a similar patterned structure (Fig. 2). A higher proportion of painful palpation sites and greater movement-evoked pain showed positive correlations with inflammatory and oxidative markers, mirroring the pattern observed for BPI scores. Notably, the MMP-9/TIMP-1 ratio—an index of extracellular matrix degradation—was positively correlated with palpation pain and movement-evoked pain, suggesting that local tissue remodeling may accompany or reflect the mechanical sensitivity observed in the painful twins. Neurotrophic factors again displayed inverse relationships with these clinical measures, reinforcing a consistent biological signal across clinical domains. Taken together, these results demonstrate that the significant associations—highlighted by asterisks in Fig. 2—are concentrated within specific biological clusters rather than evenly distributed across all measured markers. Inflammatory, oxidative, and matrix-remodeling biomarkers consistently tracked with greater pain intensity, functional interference, and palpation-evoked pain, whereas neurotrophic markers showed the opposite trend. This selective correlation pattern supports the interpretation that painful TMD in genetically identical individuals aligns with a coordinated peripheral biological profile involving inflammation, oxidative imbalance, and increased matrix turnover. The multivariate structure of within-pair biomarker differences (Δ = TMD – Control) was further explored using principal component analysis (Fig. 3). PC1 captured the predominant inflammatory–oxidative dimension of variation, with higher loadings for IL-6, the IL-6/IL-10 ratio, MDA, MDA/SOD, and MMP-9/TIMP-1. PC2 reflected secondary contributions from neurotrophic and matrix-remodeling markers. When projected as supplementary variables, clinical vectors—including pain severity and interference (BPI), palpation pain, movement-evoked pain, and headache-related palpation tenderness (DC/TMD)—aligned strongly with the positive direction of PC1, indicating that greater clinical symptom burden co-occurred with a biological shift toward heightened inflammatory, oxidative, and matrix-degrading profiles. Conversely, neurotrophic factors (BDNF and β-NGF) loaded in the opposite direction, consistent with their negative associations with clinical pain outcomes. Collectively, these findings demonstrate that the painful phenotype in monozygotic twins is embedded within a coherent multidimensional biomarker pattern, rather than isolated alterations in single molecular pathways. DISCUSSION This study in monozygotic twins discordant for painful TMD demonstrated that the twin with pain This study involving monozygotic twins discordant for painful TMD found that the twins experiencing pain displayed higher pain intensity, greater functional interference, and more widespread mechanical hyperalgesia, aligning with the multidimensional clinical features of myogenous TMD. These clinical differences were accompanied by a pattern of selective alterations across inflammatory, oxidative, matrix-remodeling, and neurotrophic pathways. Rather than yielding global biological divergence, the results identified a coherent subset of biomarkers—higher IL-6, an increased IL-6/IL-10 ratio, elevated MDA/SOD and MMP-9/TIMP-1, as well as lower BDNF—that differentiated the painful twins from their co-twins. Although not all biomarkers shifted uniformly, the overall configuration is compatible with models positing that the painful phenotype reflects experience-dependent biological plasticity rather than fixed genetic predisposition 1 – 3 , 6 . These findings integrate with contemporary pain neuroscience perspectives emphasizing the interplay among immune dysregulation, oxidative imbalance, and neuroplastic changes in persistent pain. 21 – 24 A central strength of this work is the monozygotic discordant-twin design, which provides a highly controlled framework for distinguishing environmentally shaped biological processes from those rooted in heritable liability. Prior research indicates that TMD is moderately heritable but strongly influenced by environmental and behavioral exposures. 4 , 7 – 9 The present findings build upon earlier work in this same cohort documenting cognitive-emotional and somatosensory divergence between twins, extending this pattern to peripheral biological measures. Together, these observations suggest that interactions among stress exposure, maladaptive coping, and neuroimmune mechanisms may contribute to the persistence of pain even when genetic factors are fully shared. 2 , 5 , 6 , 21 , 22 The most consistent inflammatory finding was the elevation of IL-6 and the IL-6/IL-10 ratio in the painful twins. This pattern is compatible with evidence supporting low-grade inflammatory activation in chronic TMD and related musculoskeletal pain conditions. 1 , 3 , 6 , 14 IL-6 has well-established roles in nociceptor sensitization and neuroimmune amplification. 10 , 12 , 13 , 22 The stability of IL-10 across twins may suggest limited compensatory anti-inflammatory buffering, echoing literature proposing that chronic pain reflects an imbalance between pro- and anti-inflammatory mediators. 3 , 15 , 21 , 24 Oxidative stress findings also supported a role for redox imbalance. Higher MDA/SOD among painful twins, combined with alterations in antioxidant enzyme activity, is compatible with mechanisms involving lipid peroxidation, mitochondrial stress, and amplified nociceptive signaling. 1 , 3 , 6 , 23 Oxidative pathways frequently act in concert with inflammatory mediators to sustain neuroimmune activation and hinder resolution of inflammation. 12 – 14 , 25 The growing body of evidence implicating redox mechanisms in TMD pain enhances the translational relevance of these observations. 3 , 15 , 23 , 24 Altered extracellular matrix remodeling, reflected in higher MMP-9 and an increased MMP-9/TIMP-1 ratio, may represent an additional source of peripheral sensitization. MMP-9 is increasingly recognized as a contributor to neuroinflammation, nociceptor plasticity, and hyperalgesia. 12 , 21 , 22 , 25 Although not all remodeling markers differed significantly, the pattern observed aligns with models postulating that matrix turnover may accompany or amplify local tissue reactivity in myogenous TMD. 10 , 12 , 14 , 15 In contrast, neurotrophic markers showed decreased BDNF and β-NGF in painful twins. Although neurotrophins can contribute to nociceptor sensitization, they also support neuronal repair and adaptive plasticity. 3 , 10 , 11 , 16 Lower levels in the painful twins may reflect chronic stress exposure, reduced homeostatic capacity, or environmentally shaped epigenetic changes. 21 , 26 – 28 These results are compatible with evidence that monozygotic twins can diverge epigenetically over time through differential life experiences and stress load. 11 , 16 , 28 , 29 The multivariate analyses further supported these domain-specific findings. PCA identified a dominant inflammatory-oxidative component that aligned with clinical pain vectors, alongside a secondary anti-inflammatory/antioxidant axis moving in the opposite direction. 1 , 5 , 10 , 22 This composite structure is consistent with integrated pain models situating chronic pain within dynamic interactions across immune, metabolic, and neural systems. 3 , 21 , 23 , 24 This study has limitations, including the modest sample size inherent to discordant twin designs and the cross-sectional structure that restricts causal inference. Central nervous system measures—such as neuroimaging or descending pain modulation assessments—would complement and deepen the interpretation of these peripheral findings. 1 , 3 , 15 , 30 Nevertheless, a major strength lies in the discordant monozygotic design, which provides a high level of control over genetic and early-life confounders. This rigorous framework is not possible in studies of unrelated individuals, strengthening the interpretation that the observed inflammatory, oxidative, and neurotrophic patterns may be influenced by lived experience rather than inherited predisposition. In this context, the convergence of clinical and biomarker alterations across immune, redox, and neurotrophic pathways supports models of biological embedding in chronic TMD pain. 1 , 21 , 24 , 29 In summary, this genetically controlled study indicates that painful TMD in monozygotic twins is characterized by coordinated but pathway-specific alterations across inflammatory, oxidative, remodeling, and neurotrophic domains. These findings reinforce conceptual models in which chronic orofacial pain emerges through environmentally shaped biological processes. A deeper understanding of these pathways may advance mechanistically informed and individualized therapeutic strategies for chronic TMD. 5 , 14 , 15 , 24 In conclusion, in monozygotic twins discordant for painful TMD, the painful twin displayed greater clinical burden together with selective peripheral biological alterations—including increased inflammatory and oxidative activity, elevated matrix-remodeling markers, and reduced neurotrophic support. These coordinated but pathway-specific differences emerged despite genetic identity, indicating that chronic TMD pain reflects environmentally and experientially shaped biological changes. Collectively, the findings support peripheral molecular pathways as potential targets for mechanistically informed and personalized approaches to pain-related TMD. Declarations Funding This study was financed by the São Paulo Research Foundation (FAPESP - 2022/05658-3, under coordination of Edilaine Cristina da Silva Gherardi-Donato). This study was also financed by the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil, Finance Code 001, under coordination of Christie Ramos Andrade Leite-Panissi). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. CRediT author statement LVM and CRALP have full access to all the data in the study and are responsible for the integrity and accuracy of the data and the analysis. Concept and design : ECSGD, MOM, MRD, RL, KVDS. Acquisition, analysis, or interpretation of the data : LVM, ECSGD, MOM, RL, CNTP, MRD, CRALP . Drafting of the manuscript : LVM, KVDS, MOM, ECSGD, RL, CRALP. Critical revision of the script for important intellectual content : All authors. Administrative, technical, or material support : LVM and CRALP. Supervision: LVM. Final approval and agreement on the manuscript: All authors. Data availability statement Data will be made available upon request to the corresponding authors. Declaration of generative AI and AI-assisted technologies in the manuscript preparation process During the preparation of this work the author(s) used ChatGPT (OpenAI) to improve the clarity, grammar, and readability of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article. References Slade GD, Ohrbach R, Greenspan JD, Fillingim RB, Bair E, Sanders AE et al (2016) Painful Temporomandibular Disorder: Decade of Discovery from OPPERA Studies. J Dent Res 95(10):1084–1092. 10.1177/0022034516653743 Fillingim RB, Ohrbach R, Greenspan JD, Knott C, Diatchenko L, Dubner R et al (2013) Psychological factors associated with development of TMD: the OPPERA prospective cohort study. 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J Oral Rehabil 51(2):416–426. 10.1111/joor.13589 Aoun Y, Ejbeh R, Youssef A, Hobeiche J (2025) Salivary biomarkers as potential diagnostic tool for temporomandibular disorders: A comprehensive review. Cranio 43(4):603–612. 10.1080/08869634.2023.2229607 Wen S, Santander J, Barria D, Salazar LA, Sandoval C, Arias C, Iturriaga V (2025) Epigenetic Biomarkers in Temporomandibular Joint Osteoarthritis: An Emerging Target in Treatment. Int J Mol Sci 26(8):3668. 10.3390/ijms26083668 Schiffman E, Ohrbach R, Truelove E, Look J, Anderson G, Goulet J-P et al (2014) Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) for Clinical and Research Applications: Recommendations of the International RDC/TMD Consortium Network * and Orofacial Pain Special Interest Group † HHS Public Access. 28, J Oral Facial Pain Headache Sherman JJ, LeResche L (2006) Does experimental pain response vary across the menstrual cycle? A methodological review. Am J Physiol Regul Integr Comp Physiol 291(2):R245–R256. 10.1152/ajpregu.00920.2005 Straub RH (2007) The complex role of estrogens in inflammation. Endocr Rev 28(5):521–574. 10.1210/er.2007-0001 Ferreira KA, Teixeira MJ, Mendonza TR, Cleeland CS (2011) Validation of brief pain inventory to Brazilian patients with pain. Supportive care cancer: official J Multinational Association Supportive Care Cancer 19(4):505–511. https://doi.org/10.1007/s00520-010-0844-7 Grace PM, Hutchinson MR, Maier SF, Watkins LR (2021) The neuroimmunology of chronic pain. Nat Rev Immunol 21(12):667–685. 10.1038/s41577-020-00463-2 Ji RR, Xu ZZ, Gao YJ (2014) Emerging targets in neuroinflammation-driven chronic pain. Nat Rev Neurosci 15(10):533–550. 10.1038/nrn3784 Cobourne-Dyer M, Li Y, Gabriel S et al (2023) Oxidative stress mechanisms in chronic pain: clinical implications. Pain 164(8):1558–1573. 10.1097/j.pain.0000000000002811 Tracey I, Woolf CJ (2021) Personalized pain medicine: future directions. Nat Med 27(3):412–427. 10.1038/s41591-021-01335-z Hagenston AM, Bading H (2021) Calcium signaling in chronic pain-related plasticity. Trends Neurosci 44(6):467–481. 10.1016/j.tins.2020.12.006 Denk F, McMahon SB (2022) Neuroepigenetics in chronic pain. Nat Rev Neurol 18(4):247–258. 10.1038/s41582-022-00626-2 Bannister K, Dickenson AH (2022) NGF and the rising role of neuroimmune interactions in pain. Annu Rev Neurosci 45:493–516. 10.1146/annurev-neuro-082121-120942 Snyder M et al (2023) Epigenetic mechanisms and chronic musculoskeletal pain. Pain 164(7):1239–1249. 10.1097/j.pain.0000000000002836 Williams FMK, Spector TD (2018) Pain: genetic factors and twin studies. Nat Rev Rheumatol 14(5):327–338. 10.1038/nrrheum.2018.32 Teixeira MJ, Ayesh EE et al (2023) Biomarkers in temporomandibular disorders: mechanisms and clinical translation. J Oral Rehabil 50(8):916–938. 10.1111/joor.13487 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 13 Apr, 2026 Reviews received at journal 13 Apr, 2026 Reviews received at journal 10 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 09 Apr, 2026 Reviewers agreed at journal 08 Apr, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 07 Apr, 2026 Submission checks completed at journal 07 Apr, 2026 First submitted to journal 06 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-9336568","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":622093767,"identity":"79e68a57-ddc3-4bd0-89dd-283d74474f1c","order_by":0,"name":"Lais Valencise Magri","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Lais","middleName":"Valencise","lastName":"Magri","suffix":""},{"id":622093768,"identity":"b2f21f5c-1aec-4ee5-9975-7a303fbd93b5","order_by":1,"name":"Melissa Oliveira Melchior","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Melissa","middleName":"Oliveira","lastName":"Melchior","suffix":""},{"id":622093769,"identity":"c0236ee7-ec3f-4234-9ef5-570802c2a997","order_by":2,"name":"Cecília Nogueira Tavares Peixeiro","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Cecília","middleName":"Nogueira Tavares","lastName":"Peixeiro","suffix":""},{"id":622093772,"identity":"2c30222d-a590-417c-8662-0b2a8895ec97","order_by":3,"name":"Vitória Carolina Rondon-Pereira","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Vitória","middleName":"Carolina","lastName":"Rondon-Pereira","suffix":""},{"id":622093774,"identity":"18a3f349-c2cf-4ace-a82a-7dfc1043a3c2","order_by":4,"name":"Margarete Ribeiro-Dasilva","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Margarete","middleName":"","lastName":"Ribeiro-Dasilva","suffix":""},{"id":622093777,"identity":"ed2d078d-15e6-48c4-b6f6-8c3784062b8a","order_by":5,"name":"Kranya Victoria Díaz-Serrano","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Kranya","middleName":"Victoria","lastName":"Díaz-Serrano","suffix":""},{"id":622093779,"identity":"e96aebc0-103d-45a8-876c-1d2760e2f81d","order_by":6,"name":"Riccardo Lacchini","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Lacchini","suffix":""},{"id":622093780,"identity":"9ef118a9-cd07-4d38-a94b-fdb54a785e2b","order_by":7,"name":"Edilaine Cristina da Silva Gherard-Donato","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Edilaine","middleName":"Cristina da Silva","lastName":"Gherard-Donato","suffix":""},{"id":622093781,"identity":"323b1f75-0c92-44ee-8497-13bc9de84ec8","order_by":8,"name":"Christie Ramos Andrade Leite-Panissi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYDACCQaGAyCSH8RhbCBaS4IEg2QDKVoYGBIYGAwOEKuFf3bvw4M/f1jIGd9If/aAccc9Iiy5c9zgME+ChLHZjRxzA8YzxYS1GEikMRwG+iVx240cNgnGtgTitBz8kSBRv3lG+jPitRwAOizBQCLBjDgtEjeADuNJkzCcceaNuUHiGSK08M9IY/74w6ZOnr8dGGIfdxChBRmwMZCoAaRlFIyCUTAKRgE2AAC1ljbY8DHg3AAAAABJRU5ErkJggg==","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Christie","middleName":"Ramos Andrade","lastName":"Leite-Panissi","suffix":""}],"badges":[],"createdAt":"2026-04-06 18:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9336568/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9336568/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106974134,"identity":"db33b404-cfa8-48ce-9b78-5af0beda76df","added_by":"auto","created_at":"2026-04-15 10:30:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1257622,"visible":true,"origin":"","legend":"\u003cp\u003eEach line connects values from the control twin (left) to her affected co-twin (right), illustrating the direction and magnitude of within-pair differences. Thinner lines represent individual twin pairs, whereas the thicker green line indicates the group mean. Biomarkers are grouped by biological domain: (A) Inflammatory — IL-6, IL-10, and IL-6/IL-10; (B) Redox/Oxidative Stress — MDA, SOD, CAT, and MDA/SOD; (C) Matrix Remodeling — MMP-2, MMP-9, TIMP-1, TIMP-2, and MMP-9/TIMP-1; and (D) Neurotrophic/Systemic Factors — BDNF, β-NGF, and α2M. Within-pair p-values from Wilcoxon tests are displayed in each panel; significant differences were observed for IL-6, IL-6/IL-10, MDA/SOD, MMP-9, TIMP-2, MMP-9/TIMP-1, and BDNF. Concentrations are shown on a logarithmic scale when appropriate to accommodate biomarker variability.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9336568/v1/026e6b059bc8ccf26ab3336c.png"},{"id":106974126,"identity":"9fb1e4ea-7f98-41c8-8f70-4135c4c6b0e8","added_by":"auto","created_at":"2026-04-15 10:30:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":687884,"visible":true,"origin":"","legend":"\u003cp\u003eMultimodal association analyses between biomarker differences and clinical pain measures in monozygotic twins discordant for painful TMD.\u003cstrong\u003e (A) Distance correlation matrix between biomarker deltas (Δ = TMD – Control) and clinical variables.\u003c/strong\u003e Clinical variables: 1. Pain severity (BPI); 2. Pain interference (BPI); 3. Palpation pain (DC/TMD); 4. Mandibular movement pain (DC/TMD); 5. Familiar palpation pain (DC/TMD); 6. Referred pain (DC/TMD); 7. Temporal headache palpation pain (DC/TMD). A permutation-based distance correlation test (10,000 permutations) was used to capture linear and non-linear associations. \u003cem\u003eAsterisks indicate statistically significant associations (p \u0026lt; 0.05).\u003c/em\u003e Main findings: IL-6, IL-6/IL-10, MDA, and MDA/SOD showed positive associations with multiple dimensions of pain, whereas BDNF and β-NGF displayed negative associations, indicating reduced neurotrophic support in individuals reporting higher pain levels. \u003cstrong\u003e(B) Barplots of significant biomarker–clinical associations identified through distance correlation.\u003c/strong\u003e Bars represent the magnitude of distance correlation for biomarker–clinical variable pairs showing p \u0026lt; 0.05 in panel A. Main findings: Inflammatory and oxidative markers (IL-6, IL-6/IL-10, MDA/SOD) and the matrix remodeling ratio MMP-9/TIMP-1 were the most consistent molecular correlates of pain severity, palpation tenderness, and movement-evoked pain. \u003cstrong\u003e(C) Partial Least Squares (PLS) regression identifying biomarkers contributing most strongly to global clinical pain expression.\u003c/strong\u003ePLS was performed using all biomarker deltas as predictors and all clinical variables as multivariate outcomes. Biomarkers with Variable Importance in Projection (VIP) \u0026gt; 1 were considered relevant contributors. Main findings: The PLS model highlighted an integrated inflammatory–oxidative axis (IL-6, MDA/SOD, MMP-9/TIMP-1) as the dominant molecular signature underlying the painful phenotype, while neurotrophic markers (BDNF, β-NGF) contributed inversely to overall pain burden.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9336568/v1/9bb91f8479668b3e2c03b373.jpeg"},{"id":106974150,"identity":"fc63b31c-6a09-4b49-9e40-8593d91aac5e","added_by":"auto","created_at":"2026-04-15 10:31:00","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":331504,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) of within-pair biomarker differences (Δ = TMD – Control) with projected clinical vectors.\u003cstrong\u003e \u003c/strong\u003ePrincipal component analysis was performed on z-standardized within-pair biomarker deltas to explore multivariate covariation across inflammatory, oxidative stress, matrix-remodeling, and neurotrophic domains. Clinical variables were projected as supplementary correlation vectors to indicate their contribution to the latent components. PC1 and PC2 together captured the dominant multivariate structure of the dataset, with PC1 primarily reflecting a coordinated inflammatory–oxidative axis, while PC2 showed contributions from neurotrophic and matrix-remodeling factors. Longer vectors indicate stronger correlations between clinical outcomes and the biomarker-derived dimensions. Pain severity (BPI severity), pain interference (BPI interference), proportion of painful palpation sites (Spontaneous pain points, DC/TMD), movement-evoked pain (Movement pain, DC/TMD), and headache-related palpation tenderness (Headache palpation, DC/TMD) aligned positively with PC1, suggesting that greater clinical symptom burden was associated with higher pro-inflammatory, oxidative, and matrix-remodeling biomarker shifts. In contrast, BDNF and β-NGF loaded oppositely to these clinical vectors, consistent with a relative reduction in neurotrophic support among the more symptomatic twins. Collectively, the PCA reveals a coherent multidimensional biological signature linking peripheral molecular alterations to clinical pain expression in monozygotic twins discordant for painful TMD.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9336568/v1/9a9bc23f8d7540ce440546ce.jpeg"},{"id":106974325,"identity":"38053fb6-cff5-44c4-80db-09a5ac933380","added_by":"auto","created_at":"2026-04-15 10:31:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3037685,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9336568/v1/1c792faf-5654-4162-9720-f592b31948cf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inflammatory, Oxidative, and Neurotrophic Profiles in Monozygotic Twins Discordant for Pain-related TMD","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTemporomandibular disorders (TMD) are among the most frequent chronic orofacial pain conditions and arise from the interaction of biological, emotional, and environmental influences.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Large cohort studies have shown that individuals with painful TMD often present heightened mechanical sensitivity and altered nociceptive modulation, indicating contributions from both peripheral and central pain mechanisms.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Despite the advances in characterizing clinical phenotypes, the mechanisms underlying why some individuals develop persistent pain while others with comparable life experiences remain pain-free are not fully understood.\u003c/p\u003e \u003cp\u003ePsychological and behavioral factors appear to shape pain experience and disability in TMD.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Patients frequently exhibit greater hyperalgesia, sleep disturbances, emotional distress, and attentional patterns directed toward pain, and these features are linked to more intense symptoms and functional compromise.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Clinically, this includes increased muscle tenderness, familiar pain evoked during palpation, and pain with mandibular movement, supporting an interaction between somatic sensitivity and behavioral responses.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e These findings reinforce the importance of considering pain perception, behavior, and clinical presentation as interrelated domains in TMD.\u003c/p\u003e \u003cp\u003eTwin and family studies have consistently demonstrated that a proportion of TMD pain risk is attributable to inherited factors, supporting a heritable component to the disorder while also highlighting the importance of environmental and experiential influences.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Monozygotic twins, in particular, provide a unique research model given their identical genetic profile, and discordance in pain presentation within twin pairs suggests that biological processes related to life experiences, stress exposure, and behavioral adaptation may contribute meaningfully to disease expression.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eCurrent research has increasingly focused on biological systems associated with pain persistence, including immune mediators, oxidative processes, and neurobiological adaptations.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Epigenetic mechanisms such as DNA methylation and chromatin modifications have been shown to influence gene expression relevant to nociception and inflammatory signaling.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Biomarker-based investigations have identified inflammatory cytokines, oxidative stress indicators, and matrix-regulating molecules associated with TMD\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, although evidence remains heterogeneous and few studies have adopted genetically controlled approaches.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDespite important advances, the specific biological characteristics associated with painful TMD in individuals who share identical genetic backgrounds are still unclear. Our previous research with monozygotic twins discordant for painful TMD demonstrated differences in clinical pain responses and behavioral features even in the absence of genetic variability.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Building on these findings, the present study investigates whether peripheral inflammatory markers, oxidative stress indicators, and neurotrophic factors differ between genetically identical women discordant for painful TMD. We hypothesize that twins with painful TMD will show higher inflammatory activity and altered biomarker patterns relative to their pain-free co-twins. Therefore, the objective of this study was to characterize biological signatures associated with painful TMD in monozygotic twins and relate these molecular findings to clinical pain expression.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and ethical approval\u003c/h2\u003e \u003cp\u003eThis investigation employed a cross-sectional, discordant monozygotic twin design to examine biological and clinical correlates of painful temporomandibular disorder (TMD). By comparing genetically identical individuals who differ in pain status, this design minimizes confounding from inherited susceptibility, population stratification, and shared early-life environment, thereby strengthening inferences regarding environmentally shaped biological variation. Although cross-sectional in nature and therefore not suited for causal inference, the discordant twin approach enhances internal validity by isolating phenotypic divergence that arises after the period of shared genetic and early developmental influences. The present study expands upon a previously published analysis in the same cohort that characterized cognitive-emotional and somatosensory features of the painful phenotype\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, providing an integrated biological extension focused on peripheral biomarker profiles.\u003c/p\u003e \u003cp\u003eThe study protocol was approved by the Research Ethics Committee of XXXXXXXXXX, which includes two representatives of patients who are users of the dental services provided by this institution. All procedures conformed to the principles of the Declaration of Helsinki. Participants were recruited voluntarily and provided written informed consent before enrollment. Confidentiality, autonomy, and the right to withdraw were fully guaranteed throughout the study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eParticipants\u003c/h3\u003e\n\u003cp\u003eA total of 20 female monozygotic twins (10 discordant pairs) aged 18 to 55 years participated in the study. Participants were recruited via institutional databases, social media, and community announcements as described in the original study\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Monozygotic status was confirmed through assessment of phenotypic similarity assessment and parental confirmation, following widely accepted protocols for twin-research.\u003c/p\u003e\n\u003ch3\u003eDiagnosis of painful TMD\u003c/h3\u003e\n\u003cp\u003eThe diagnosis of painful temporomandibular disorder (TMD) was established using the validated Brazilian Portuguese version of the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD)\u003csup\u003e17\u003c/sup\u003e, administered by clinicians formally trained and calibrated in DC/TMD methodology. The clinical examination followed the standardized Axis I protocol and included systematic palpation of the masticatory muscles and temporomandibular joints, assessment of mandibular range of motion, and evaluation of pain provocation and reproduction of familiar symptoms. This structured approach ensured diagnostic reliability and minimized examiner-related variability, an important consideration in twin studies where misclassification could obscure true within-pair differences.\u003c/p\u003e\n\u003ch3\u003eInclusion criteria\u003c/h3\u003e\n\u003cp\u003eParticipants were eligible if they met all of the following criteria:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eFemale monozygotic twin, aged 18 to 55 years;\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eOne twin reporting\u0026thinsp;\u0026ge;\u0026thinsp;3 months of orofacial pain and fulfilling DC/TMD diagnostic criteria for painful TMD (myalgia and/or arthralgia);\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe co-twin presenting \u003cem\u003eno\u003c/em\u003e DC/TMD diagnosis and \u003cem\u003eno\u003c/em\u003e persistent orofacial pain.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eThis definition ensured clear discordance while allowing for the possibility of isolated or subclinical symptoms in the control twin, provided they did not satisfy diagnostic thresholds.\u003c/p\u003e\n\u003ch3\u003eExclusion criteria\u003c/h3\u003e\n\u003cp\u003eExclusion criteria were defined to avoid confounding from concurrent conditions or treatments known to influence pain or inflammatory pathways. Twins were excluded if they were undergoing or had recently undergone TMD-related therapy (e.g., interocclusal appliances, physiotherapy, photobiomodulation/laser therapy, acupuncture), or were using analgesics, anti-inflammatories, or centrally acting medications. Additional exclusions included history of head or neck tumors, trauma, or surgery; diagnosed neurological disease; severe psychiatric disorders (except anxiety or depression, due to their high prevalence in chronic pain populations); and pregnancy or breastfeeding. These criteria minimized biological heterogeneity unrelated to TMD.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eControl of menstrual cycle phase\u003c/h2\u003e \u003cp\u003eBecause both TMD symptoms and inflammatory markers exhibit menstrual cycle\u0026ndash;related variability,\u003csup\u003e18,19\u003c/sup\u003e all participants with regular cycles were assessed during the follicular phase. This phase was selected because estrogen rises gradually and progesterone remains low and stable, producing a more uniform endocrine environment. Conducting assessments during the follicular phase reduces hormonal fluctuations known to influence pain sensitivity, neuroimmune activity, and inflammatory mediator levels, thereby enhancing internal consistency across participants and reducing endocrine-related confounding.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eClinical assessment\u003c/h3\u003e\n\u003cp\u003eClinical examinations were performed in accordance with DC/TMD Axis I guidelines, using standardized procedures for manual palpation of masticatory and cervical musculature and for the evaluation of mandibular functional movements.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Trained examiners applied controlled, criterion-based digital pressure to predefined anatomical sites to assess pain provocation, reproduction of familiar symptoms, and elicitation of referred pain patterns. These procedures followed established diagnostic thresholds for muscle tenderness and mechanical provocation of TMD-related symptoms, thereby minimizing examiner-related variability and enhancing diagnostic reliability within the twin pairs.\u003c/p\u003e \u003cp\u003eMandibular movement assessment\u0026mdash;comprising maximal unassisted and assisted opening, lateral excursions, and protrusion\u0026mdash;was conducted to document movement-evoked pain and features of functional impairment consistent with painful myogenous TMD. Pain severity and functional interference were quantified using the Brief Pain Inventory (BPI), which offers validated, continuous indices of pain intensity and its impact on daily functioning, supporting comprehensive clinical characterization.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eObjective measures of nociceptive sensitivity were incorporated through quantification of the proportion of palpation-positive sites relative to the total number of evaluated regions. This approach allowed systematic capture of localized muscle tenderness and mechanical hyperalgesia across standardized examination sites. In addition, pain elicited during mandibular movement was recorded, alongside differentiation among familiar pain on palpation, referred pain, and palpation-induced familiar headache. These distinctions reflect well-defined DC/TMD categories and permit more precise mapping of somatosensory responses relevant to the TMD pain phenotype.\u003c/p\u003e \u003cp\u003eTogether, these multidimensional clinical measures\u0026mdash;summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026mdash;capture a pattern of increased mechanical sensitivity, widespread palpation tenderness, movement-provoked pain, and functional limitation characteristic of painful myogenous TMD. This rigorously assessed clinical profile provides a robust foundation for integrating peripheral biomarker data and interpreting biological correlates of pain expression in a genetically controlled context.\u003c/p\u003e\n\u003ch3\u003eBiological sample collection and processing\u003c/h3\u003e\n\u003cp\u003eVenous blood samples were collected in the morning following an overnight fast to minimize circadian influences and diet-related metabolic variability on inflammatory, oxidative, and neurotrophic biomarkers. Standardized phlebotomy procedures were used, and samples were processed immediately after collection to reduce pre-analytical degradation. Whole blood was centrifuged under controlled conditions, and the resulting plasma was aliquoted into sterile, coded polypropylene tubes to prevent repeated freeze\u0026ndash;thaw cycles and preserve analyte integrity. Plasma samples were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis, following established recommendations for long-term biomarker stability.\u003c/p\u003e \u003cp\u003eTo minimize analytical bias, all laboratory personnel performing biomarker quantification were blinded to the clinical status of each twin. Blinding procedures ensured that sample handling, assay performance, and data recording were not influenced by knowledge of group assignment, thereby increasing the internal validity of the biomarker measurements.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiomarker Analysis\u003c/h2\u003e \u003cp\u003eBiomarkers were selected based on their mechanistic relevance in inflammation, oxidative stress, extracellular matrix regulation, and neuroplasticity, domains previously implicated in chronic musculoskeletal pain. Assays were performed following the manufacturers' protocols.\u003c/p\u003e \u003cp\u003eInflammatory mediators and matrix remodeling factors were quantified by sandwich-type ELISA (Enzyme-Linked Immunosorbent Assay) (R\u0026amp;D Systems, Minneapolis, MN, USA). Specifically, Quantikine kits were used for IL-6 (Catalog RDSY-D6050-96SW), IL-10 (Catalog RDSY-D1000B-96SW) and TNF-α (Catalog RDSY-DTA00D-9SW). For matrix remodeling, DuoSet kits were used for MMP-2 (Catalog RDSY-DY902-1KIT), MMP-9 (Catalog RDSY-DY911-1KIT), TIMP-1 (Catalog RDSY-DY970-1KIT), TIMP-2 (Catalog RDSY-DY971-1KIT), and α-2-macroglobulin (Catalog RDSY-DY1938-96SW).\u003c/p\u003e \u003cp\u003eOxidative stress markers were analyzed by colorimetric/spectrophotometric assays (Cayman Chemical, Ann Arbor, MI, USA). Kits used were for Malondialdehyde (MDA) (TBARS Assay Kit, Catalog CAYM-10009055-96W), Superoxide Dismutase (SOD) (Assay Kit, Catalog CAYM-706002-96W), and Catalase (Assay Kit, Catalog CAYM-707002-96W).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eWithin-pair differences (Δ\u0026thinsp;=\u0026thinsp;TMD \u0026ndash; Control) were calculated for all biomarkers and clinical variables to minimize genetic and shared early environmental confounding inherent to monozygotic discordant twin designs. Data distribution was examined using the Shapiro\u0026ndash;Wilk test, and variables with strong skew or extreme values were inspected using Q\u0026ndash;Q plots and leverage diagnostics. Descriptive statistics were reported as means and standard deviations for raw values and as medians and interquartile ranges when distributional asymmetry was present.\u003c/p\u003e \u003cp\u003eInferential comparisons between affected and control twins were conducted using paired statistical tests. Paired t-tests were applied when Δ values met normality assumptions, whereas non-normally distributed variables or those with tied ranks were analyzed using Wilcoxon signed-rank tests. Correlations between Δ clinical measures (pain severity, functional interference, movement-evoked pain, and palpation outcomes) and Δ biomarker concentrations were assessed using Pearson coefficients (r), with significance defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Correlation matrices were visualized to identify clusters of clinically relevant associations.\u003c/p\u003e \u003cp\u003eTo explore multivariate patterns, principal component analysis (PCA) was performed on z-standardized Δ biomarker values. Clinical variables were then projected as supplementary correlation vectors onto the PCA solution (PC1 and PC2) to evaluate their alignment with the dominant biological dimensions. Clinical vectors with |r| \u0026gt; 0.4 were interpreted as meaningful contributors to component structure. All analyses and visualizations\u0026mdash;including paired-difference plots, correlation heatmaps, and PCA biplots\u0026mdash;were conducted in R (version 4.4.0; R Foundation for Statistical Computing, Vienna, Austria).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eClinical characteristics\u003c/h2\u003e \u003cp\u003eAs required by the discordant-twin design, in all ten pairs one twin fulfilled full DC/TMD criteria for painful TMD while the co-twin did not. Importantly, all control twins received the formal DC/TMD classification of \u003cem\u003eabsence of TMD\u003c/em\u003e; however, several exhibited subclinical findings, such as mild tenderness on isolated palpation sites or occasional movement-associated discomfort, without reaching diagnostic thresholds. This nuance helps contextualize the within-pair differences observed and clarifies that the control phenotype represents the \u003cem\u003eabsence of a diagnosable disorder\u003c/em\u003e, rather than a completely symptom-free state.\u003c/p\u003e \u003cp\u003eGroup-level comparisons showed that painful twins reported higher mean pain levels (BPI-severity: 3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5; BPI-interference: 3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6), whereas their co-twins reported lower scores (2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 and 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7, respectively). Nevertheless, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, a few control twins reported low-intensity pain or minimal interference, which produced negative Δ values in some pairs (e.g., pairs 1, 9, and 10). These cases illustrate the expected continuous distribution of pain experiences even in the absence of a formal diagnosis.\u003c/p\u003e \u003cp\u003eSimilarly, across DC/TMD clinical examination domains\u0026mdash;percentage of painful palpation points, movement-evoked pain, familiar pain, referred pain, and headache-related palpation pain\u0026mdash;affected twins generally exhibited higher values. Still, control twins occasionally displayed isolated tenderness or temporal muscle sensitivity, again without meeting diagnostic criteria. Consequently, Δ values varied in magnitude and direction, ranging from small negative differences in some pairs to large positive contrasts in others. Overall, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e demonstrates that although painful twins consistently showed greater clinical burden, the discordant-twin model naturally captures a graded, subclinical\u0026ndash;to\u0026ndash;clinical spectrum, reinforcing the biological relevance of examining within-pair differences beyond genetic susceptibility.\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\u003eClinical characteristics of monozygotic twins discordant for painful TMD based on BPI and DC/TMD. Δ values represent within-pair differences (TMD \u0026ndash; Control).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTwin Pair\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΔ Pain Severity(BPI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔ Pain Interference(BPI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΔ Reported Pain Points(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔ Movement-Evoked Pain(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eΔ Familiar Pain on Palpation(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eΔ Referred Pain on Palpation\u0026nbsp;(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eΔ Headache on Palpation(%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;4.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;1.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;1.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e+\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e+\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePair 10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e+\u0026thinsp;3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e+\u0026thinsp;21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026ndash;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e+\u0026thinsp;19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMean Δ\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;13.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;4.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;40.4\u0026thinsp;\u0026plusmn;\u0026thinsp;33.4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;38.0\u0026thinsp;\u0026plusmn;\u0026thinsp;29.6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;41.4\u0026thinsp;\u0026plusmn;\u0026thinsp;38.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003cb\u003eNotes\u003c/b\u003e: Pain outcomes are expressed as percentages of positive responses relative to the number of sites evaluated: 14 sites for reported pain points (DC/TMD E1a\u0026ndash;E1b), 50 sites for movement-evoked pain (DC/TMD E4b\u0026ndash;E5c), 16 sites for familiar and referred pain on palpation (DC/TMD E9), 6 sites for headache-related palpation pain (temporal muscle; DC/TMD E9). All control twins fulfilled the DC/TMD \u0026ldquo;Absence of TMD\u0026rdquo; classification, although some exhibited \u003cb\u003esubclinical findings\u003c/b\u003e (e.g., mild tenderness without diagnostic significance).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure 1 displays within-pair differences in biomarker concentrations between monozygotic twins discordant for painful temporomandibular disorder (TMD), revealing a pattern of selective rather than global biological divergence. Significant increases were observed in affected twins for IL-6, IL-6/IL-10, MDA/SOD, MMP-9, TIMP-2, and MMP-9/TIMP-1, while BDNF was significantly reduced (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Other biomarkers, including IL-10, MDA, SOD, CAT, MMP-2, TIMP-1, β-NGF, and α2M, did not show significant within-pair differences. The pattern suggests that painful twins exhibit targeted alterations involving pro-inflammatory signaling, redox imbalance reflected primarily in proportional oxidative markers, increased matrix-remodeling potential, and reduced neurotrophic support. Collectively, these findings indicate that specific peripheral pathways are differentially engaged in the expression of painful TMD even among genetically identical individuals, rather than indicating uniform shifts across all biomarker domains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe correlation analysis revealed coherent biological patterns linking biomarker shifts to clinical pain expression in monozygotic twins discordant for painful TMD (Fig.\u0026nbsp;2). Pro-inflammatory and oxidative-stress markers\u0026mdash;particularly IL-6, the IL-6/IL-10 ratio, MDA, and the MDA/SOD ratio\u0026mdash;showed moderate to strong positive correlations with pain severity and pain interference on the BPI, indicating that greater inflammatory activation and redox imbalance tended to accompany higher pain burden. In contrast, neurotrophic markers such as BDNF and β-NGF correlated negatively with BPI scores, suggesting that reduced neurotrophic support may be associated with more severe and disabling pain symptoms.\u003c/p\u003e \u003cp\u003eAssociations between biomarker deltas and DC/TMD clinical variables followed a similar patterned structure (Fig.\u0026nbsp;2). A higher proportion of painful palpation sites and greater movement-evoked pain showed positive correlations with inflammatory and oxidative markers, mirroring the pattern observed for BPI scores. Notably, the MMP-9/TIMP-1 ratio\u0026mdash;an index of extracellular matrix degradation\u0026mdash;was positively correlated with palpation pain and movement-evoked pain, suggesting that local tissue remodeling may accompany or reflect the mechanical sensitivity observed in the painful twins. Neurotrophic factors again displayed inverse relationships with these clinical measures, reinforcing a consistent biological signal across clinical domains.\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that the significant associations\u0026mdash;highlighted by asterisks in Fig.\u0026nbsp;2\u0026mdash;are concentrated within specific biological clusters rather than evenly distributed across all measured markers. Inflammatory, oxidative, and matrix-remodeling biomarkers consistently tracked with greater pain intensity, functional interference, and palpation-evoked pain, whereas neurotrophic markers showed the opposite trend. This selective correlation pattern supports the interpretation that painful TMD in genetically identical individuals aligns with a coordinated peripheral biological profile involving inflammation, oxidative imbalance, and increased matrix turnover.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe multivariate structure of within-pair biomarker differences (Δ\u0026thinsp;=\u0026thinsp;TMD \u0026ndash; Control) was further explored using principal component analysis (Fig.\u0026nbsp;3). PC1 captured the predominant inflammatory\u0026ndash;oxidative dimension of variation, with higher loadings for IL-6, the IL-6/IL-10 ratio, MDA, MDA/SOD, and MMP-9/TIMP-1. PC2 reflected secondary contributions from neurotrophic and matrix-remodeling markers. When projected as supplementary variables, clinical vectors\u0026mdash;including pain severity and interference (BPI), palpation pain, movement-evoked pain, and headache-related palpation tenderness (DC/TMD)\u0026mdash;aligned strongly with the positive direction of PC1, indicating that greater clinical symptom burden co-occurred with a biological shift toward heightened inflammatory, oxidative, and matrix-degrading profiles. Conversely, neurotrophic factors (BDNF and β-NGF) loaded in the opposite direction, consistent with their negative associations with clinical pain outcomes. Collectively, these findings demonstrate that the painful phenotype in monozygotic twins is embedded within a coherent multidimensional biomarker pattern, rather than isolated alterations in single molecular pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study in monozygotic twins discordant for painful TMD demonstrated that the twin with pain This study involving monozygotic twins discordant for painful TMD found that the twins experiencing pain displayed higher pain intensity, greater functional interference, and more widespread mechanical hyperalgesia, aligning with the multidimensional clinical features of myogenous TMD. These clinical differences were accompanied by a pattern of selective alterations across inflammatory, oxidative, matrix-remodeling, and neurotrophic pathways. Rather than yielding global biological divergence, the results identified a coherent subset of biomarkers\u0026mdash;higher IL-6, an increased IL-6/IL-10 ratio, elevated MDA/SOD and MMP-9/TIMP-1, as well as lower BDNF\u0026mdash;that differentiated the painful twins from their co-twins. Although not all biomarkers shifted uniformly, the overall configuration is compatible with models positing that the painful phenotype reflects experience-dependent biological plasticity rather than fixed genetic predisposition\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These findings integrate with contemporary pain neuroscience perspectives emphasizing the interplay among immune dysregulation, oxidative imbalance, and neuroplastic changes in persistent pain.\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA central strength of this work is the monozygotic discordant-twin design, which provides a highly controlled framework for distinguishing environmentally shaped biological processes from those rooted in heritable liability. Prior research indicates that TMD is moderately heritable but strongly influenced by environmental and behavioral exposures.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e The present findings build upon earlier work in this same cohort documenting cognitive-emotional and somatosensory divergence between twins, extending this pattern to peripheral biological measures. Together, these observations suggest that interactions among stress exposure, maladaptive coping, and neuroimmune mechanisms may contribute to the persistence of pain even when genetic factors are fully shared.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe most consistent inflammatory finding was the elevation of IL-6 and the IL-6/IL-10 ratio in the painful twins. This pattern is compatible with evidence supporting low-grade inflammatory activation in chronic TMD and related musculoskeletal pain conditions.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e IL-6 has well-established roles in nociceptor sensitization and neuroimmune amplification.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The stability of IL-10 across twins may suggest limited compensatory anti-inflammatory buffering, echoing literature proposing that chronic pain reflects an imbalance between pro- and anti-inflammatory mediators.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOxidative stress findings also supported a role for redox imbalance. Higher MDA/SOD among painful twins, combined with alterations in antioxidant enzyme activity, is compatible with mechanisms involving lipid peroxidation, mitochondrial stress, and amplified nociceptive signaling.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Oxidative pathways frequently act in concert with inflammatory mediators to sustain neuroimmune activation and hinder resolution of inflammation.\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The growing body of evidence implicating redox mechanisms in TMD pain enhances the translational relevance of these observations.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAltered extracellular matrix remodeling, reflected in higher MMP-9 and an increased MMP-9/TIMP-1 ratio, may represent an additional source of peripheral sensitization. MMP-9 is increasingly recognized as a contributor to neuroinflammation, nociceptor plasticity, and hyperalgesia.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e Although not all remodeling markers differed significantly, the pattern observed aligns with models postulating that matrix turnover may accompany or amplify local tissue reactivity in myogenous TMD.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn contrast, neurotrophic markers showed decreased BDNF and β-NGF in painful twins. Although neurotrophins can contribute to nociceptor sensitization, they also support neuronal repair and adaptive plasticity.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Lower levels in the painful twins may reflect chronic stress exposure, reduced homeostatic capacity, or environmentally shaped epigenetic changes.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e These results are compatible with evidence that monozygotic twins can diverge epigenetically over time through differential life experiences and stress load.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe multivariate analyses further supported these domain-specific findings. PCA identified a dominant inflammatory-oxidative component that aligned with clinical pain vectors, alongside a secondary anti-inflammatory/antioxidant axis moving in the opposite direction.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e This composite structure is consistent with integrated pain models situating chronic pain within dynamic interactions across immune, metabolic, and neural systems.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis study has limitations, including the modest sample size inherent to discordant twin designs and the cross-sectional structure that restricts causal inference. Central nervous system measures\u0026mdash;such as neuroimaging or descending pain modulation assessments\u0026mdash;would complement and deepen the interpretation of these peripheral findings.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Nevertheless, a major strength lies in the discordant monozygotic design, which provides a high level of control over genetic and early-life confounders. This rigorous framework is not possible in studies of unrelated individuals, strengthening the interpretation that the observed inflammatory, oxidative, and neurotrophic patterns may be influenced by lived experience rather than inherited predisposition. In this context, the convergence of clinical and biomarker alterations across immune, redox, and neurotrophic pathways supports models of biological embedding in chronic TMD pain.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn summary, this genetically controlled study indicates that painful TMD in monozygotic twins is characterized by coordinated but pathway-specific alterations across inflammatory, oxidative, remodeling, and neurotrophic domains. These findings reinforce conceptual models in which chronic orofacial pain emerges through environmentally shaped biological processes. A deeper understanding of these pathways may advance mechanistically informed and individualized therapeutic strategies for chronic TMD.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn conclusion, in monozygotic twins discordant for painful TMD, the painful twin displayed greater clinical burden together with selective peripheral biological alterations\u0026mdash;including increased inflammatory and oxidative activity, elevated matrix-remodeling markers, and reduced neurotrophic support. These coordinated but pathway-specific differences emerged despite genetic identity, indicating that chronic TMD pain reflects environmentally and experientially shaped biological changes. Collectively, the findings support peripheral molecular pathways as potential targets for mechanistically informed and personalized approaches to pain-related TMD.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financed by the S\u0026atilde;o Paulo Research Foundation (FAPESP - 2022/05658-3, under coordination of Edilaine Cristina da Silva Gherardi-Donato). This study was also financed by the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil, Finance Code 001, under coordination of Christie Ramos Andrade Leite-Panissi). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLVM and CRALP have full access to all the data in the study and are responsible for the integrity and accuracy of the data and the analysis. \u003cem\u003eConcept and design\u003c/em\u003e: ECSGD, MOM, MRD, RL, KVDS. \u003cem\u003eAcquisition, analysis, or interpretation of the data\u003c/em\u003e: LVM, ECSGD, MOM, RL, CNTP, MRD, CRALP\u003cem\u003e. Drafting of the manuscript\u003c/em\u003e: LVM, KVDS, MOM, ECSGD, RL, CRALP. \u003cem\u003eCritical revision of the script for important intellectual content\u003c/em\u003e: All authors. \u003cem\u003eAdministrative, technical, or material support\u003c/em\u003e: LVM and CRALP. Supervision: LVM. Final approval and agreement on the manuscript: All authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available upon request to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the manuscript preparation process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the author(s) used\u0026nbsp;\u003cem\u003eChatGPT (OpenAI)\u003c/em\u003e to improve the clarity, grammar, and readability of the manuscript. 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J Oral Rehabil 50(8):916\u0026ndash;938. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/joor.13487\u003c/span\u003e\u003cspan address=\"10.1111/joor.13487\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Temporomandibular Disorders, Orofacial Pain, Monozygotic Twins, Inflammation, Oxidative Stress, Neurotrophic Factors, Biomarkers, Chronic Pain","lastPublishedDoi":"10.21203/rs.3.rs-9336568/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9336568/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the biological mechanisms underlying temporomandibular disorder (TMD) pain in monozygotic twins discordant for the condition, isolating environmental factors from genetic influence. Twenty women (ten pairs of discordant twins) underwent standardized examinations and venous plasma analysis. Inflammatory biomarkers (IL-6, IL-10), oxidative markers (MDA, SOD, catalase), matrix remodeling proteins (MMP-2, MMP-9, TIMP-1, TIMP-2), and neurotrophic factors (BDNF, β-NGF, α2M) were measured. Twins with painful TMD demonstrated greater pain severity, functional interference, and mechanical sensitivity compared to discordant controls. Significant within-pair differences were identified in IL-6, IL-6/IL-10 ratio, MDA/SOD ratio, MMP-9, TIMP-2, and BDNF levels. Pro-inflammatory and oxidative indices positively correlated with pain intensity and palpation sensitivity, while reduced BDNF associated with greater symptom burden. Principal component analysis revealed a dominant inflammatory-oxidative profile that discriminated painful twins from controls. In genetically identical individuals, painful TMD associates with selective peripheral biological alterations involving inflammation, oxidative imbalance, and extracellular matrix remodeling. These findings demonstrate that environmental and experiential factors biologically determine vulnerability to chronic orofacial pain, highlighting the importance of epigenetic mechanisms in TMD pathogenesis beyond genetic predisposition. This article identifies specific peripheral biomarker profiles distinguishing monozygotic twins with and without painful TMD, revealing that inflammation, oxidative imbalance, matrix remodeling, and reduced neurotrophic support characterize the painful phenotype. These mechanistic insights could enable clinicians to develop targeted, biologically informed therapeutic strategies and potentially predict pain vulnerability in genetically susceptible individuals.\u003c/p\u003e","manuscriptTitle":"Inflammatory, Oxidative, and Neurotrophic Profiles in Monozygotic Twins Discordant for Pain-related TMD","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-15 10:25:51","doi":"10.21203/rs.3.rs-9336568/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-13T08:45:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T06:26:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T05:29:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334457680085964751625504598181399179088","date":"2026-04-09T15:15:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79071958792979050718106835251140924536","date":"2026-04-09T14:57:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33526166947085997807143282822233919211","date":"2026-04-08T08:23:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-08T01:41:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-07T09:04:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-07T09:04:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2026-04-06T18:20:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4f0efd5d-3aa9-4c34-875b-4c7774f6f196","owner":[],"postedDate":"April 15th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T14:55:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-15 10:25:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9336568","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9336568","identity":"rs-9336568","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}