Osteogenic differentiation requires sophisticated mitochondrial adaptation to meet bioenergetic demands, yet regulatory checkpoints governing this organelle reorganization remain poorly defined. Through single-cell RNA sequencing reanalysis and metabolic intervention, this study unveils a non-canonical signaling role for PFKM, traditionally recognized solely as a glycolytic enzyme, in orchestrating mitochondrial remodeling during bone formation. Beyond its established metabolic function, Pfkm suppression triggers distinctive donut-shaped mitochondria through a novel signaling cascade. Mechanistically, Pfkm knockdown expands mitochondria-endoplasmic reticulum contacts (MERCs), facilitating mitochondrial calcium influx. Concomitantly, elevated CD38 suppresses protein kinase A (PKA) activity, inducing DRP1 dephosphorylation at Serine 656. This signaling integration promotes DRP1 mitochondrial translocation, driving the characteristic donut architecture. This structural transformation initiates comprehensive mitochondrial quality control (MQC) encompassing enhanced biogenesis, selective mitophagy, and mitochondrial-derived vesicles (MDVs) secretion, collectively optimizing the osteogenic microenvironment and cellular mineralization capacity. In vivo validation demonstrates that AAV-mediated Pfkm knockdown accelerates bone repair in rat calvarial and femoral defect models. This work establishes PFKM as a dual-function regulator bridging metabolism and mitochondrial signaling, offering a potent therapeutic avenue for bone regeneration.
Tooth and maxillomandibular bone development is a synergistic process precisely and coordinately regulated by genetic programs, molecular signaling networks, and external environmental factors. Recent rapid progress in in situ biomechanical measurement techniques, cellular mechanical imaging, and molecular tracing technologies has greatly promoted research in developmental biomechanics. Accumulating evidence indicates that mechanical stimuli can be converted into biochemical signals via cellular mechanotransduction pathways, thereby modulating key cellular processes, including proliferation, differentiation, and apoptosis, and further regulating tissue morphogenesis and remodeling. In this review, we focus on the role of mechanical control in dental and jaw morphogenesis and remodeling. We first summarize the sources and spatiotemporal distribution features of mechanical stress during dentofacial development. Subsequently, we elaborate on the regulatory roles and underlying molecular mechanisms of mechanical signals in key dentofacial developmental processes, including crown development, root morphogenesis, tooth eruption and replacement, as well as maxillomandibular bone development and remodeling. Finally, we discuss the current research gaps in the field of mechanical stress regulation of dentofacial development and propose potential future research directions.
The two principal collagen receptors in bone, the collagen-binding β1 integrins and discoidin domain receptor 2 (DDR2), each have important roles in development, but their functions in bone regeneration are largely unexplored. Using a critical-size calvarial defect model, we assessed the relative roles of these two receptor classes in BMP2-induced regeneration using a conditional knockout approach where Itgb1 (encodes integrin β1) and/or Ddr2 were selectively inactivated in GLI1+ skeletal progenitor cells (SPCs). Inactivating either Itgb1 or Ddr2 partially inhibited calvarial regeneration, while inactivation of both receptors almost completely blocked bone healing. Responses were linked to reduced proliferation and migration of GLI1+ SPCs into defects and reduced endochondral and intramembranous bone formation. To examine the consequences of receptor inactivation at the cellular level, calvarial SPCs lacking Itgb1, Ddr2 or both receptors were generated. Loss of either receptor inhibited osteoblast differentiation, migration, cell spreading, focal adhesion formation, and nuclear localization of the mechanotransducer, YAP1. Importantly, inactivation of both receptors inhibited responses to a greater extent than was seen with individual knockouts. Interestingly, while inactivation of one allele of either Itgb1 (Itgb1fl/+) or Ddr2 (Ddr2fl/+) did not affect any of the above parameters, inactivation in double heterozygotes (Itgb1fl/+ ;Ddr2fl/+) was strongly inhibitory, which is indicative of a genetic interaction between Itgb1 and Ddr2. Lastly, immunofluorescence and immunoprecipitation analysis suggest that DDR2 and ITGB1 physically interact, providing a potential explanation for the observed functional cooperativity. These studies provide a mechanistic basis for bone regeneration strategies involving combined activation of integrin β1 and DDR2.
Congenital anomalies often arise during critical developmental time windows, yet the underlying mechanisms remain unclear. Here, we identify a time-specific FGFR2-retinoic acid signaling axis regulating postnatal coronal suture development through dura mater-suture mesenchyme interactions. FGFR2 signaling gradually declines after birth, leading to reduced retinoic acid signaling by moderating the expression of Aldh1a3 in the dura mater and suture mesenchyme alongside that of Rbp1 in the dura mater, thereby preserving GLI1+ progenitors and restraining osteogenesis to maintain suture patency. Importantly, FGFR2 overactivation in Fgfr2IIIc mutant mice within an early time window, disrupts this physiological FGFR2-retinoic acid signaling decline by upregulating Rbp1 and Aldh1a3 through enhanced P38 signaling. This leads to increased retinoic acid synthesis, premature osteogenic differentiation of GLI1+ progenitors, and coronal suture craniosynostosis. Significantly, genetic restoration of retinoic acid signaling rescues craniosynostosis in Fgfr2IIIc mutant mice, confirming retinoic acid signaling as a key downstream effector of FGFR2 signaling. Notably, restoration of suture patency alone rescues neurocognitive dysfunctions in Fgfr2IIIc mutant mice despite Fgfr2 mutation in the brain, demonstrating that the neurocognitive impairments primarily arise from cranial structural constraints and elevated intracranial pressure rather than intrinsic neural defects. This critical postnatal time window in mice parallels the timing of FGFR2-related craniosynostosis onset in humans, underscoring the importance of this study in advancing our understanding of the molecular and cellular mechanisms in craniosynostosis. Our findings define a temporally regulated FGFR2-P38-retinoic acid signaling axis and highlight retinoic acid signaling as a promising therapeutic target in FGFR2 overactivation-related craniosynostosis.
Lymphocytes play a central role in adaptive immunity and represent the primary source of RANKL in periodontitis. Understanding their organization and activation within gingiva is therefore essential. This study investigates the role of tertiary lymphoid structures (TLSs), ectopic lymphoid aggregates that orchestrate local immune responses. Using multiplex immunohistochemistry staining, we found that TLSs form and mature with increasing inflammatory severity. These structures function as hubs for B cell activation and were associated with poorer surgical outcomes. Given the known role of fibroblasts in initiating TLS assembly, we explored the underlying mechanism. Using single-cell RNA sequencing and in vitro assays, we identified that oral pathogens activate the IFN-I–IRF7 axis in gingival fibroblasts, leading to overproduction of IFNβ and driving their differentiation into a CXCL13-producing subtype that facilitates TLS organization. Furthermore, in a mouse periodontitis model, we demonstrated that activated IgD+CD80+ B cells within TLSs promote osteoclastogenesis and bone resorption via RANKL secretion. Therapeutically, inhibition of IFN-I signaling disrupted this pathogenic cascade by suppressing TLS formation and subsequent RANKL production, thereby mitigating periodontitis bone loss. In conclusion, our findings establish TLSs as pathological hubs that coordinate B cell-mediated bone destruction in periodontitis and highlight the IFN-I signaling pathway as a promising target for therapeutic intervention.
The age-related decline in the pro-angiogenic capacity of mature dental pulp stem cells (DPSCs) severely limits pulp regeneration. We identify impaired glycolytic metabolism, driven by reduced glucose transporter type 1 (GLUT1) and hexokinase 2 (HK2) expression, as the key mechanism, as its inhibition diminished endothelial tube formation. To reverse this, we developed an aminolyzed highly branched poly(β-amino ester) (HBPA) as a vector for GLUT1/HK2 mRNA co-delivery, achieving >90% transfection efficiency with excellent biocompatibility. In vitro, conditioned medium from reprogrammed mature DPSCs resulted in a 2.0-fold increase in capillary length and a 2.3-fold increase in branch points, restoring angiogenic potential to levels equivalent to those of immature DPSCs. This efficacy translated robustly in vivo, where a tooth root slice model showed reprogrammed cells generated tissue with a vessel density of 10.2 vessels per mm2, 2.5-fold higher than that of untreated controls. Crucially, this level of vascularization was statistically indistinguishable from that achieved by the benchmark immature DPSCs. Our study demonstrates that HBPA-mediated metabolic reprogramming effectively rejuvenates mature DPSCs by restoring the “Metabolic-ECM-Angiogenesis Axis”, offering a translatable strategy for predictable, vascularized pulp regeneration.
Porphyromonas gingivalis (P. gingivalis) invades host cells to evade antibiotics, driving the recurrence of periodontitis. However, current clinical treatment strategies remain ineffective in addressing this intracellular invasion by the bacteria. Here, we report a host-directed strategy that blocks bacterial invasion by targeting key host factors, including collagen I (using FT011), lipid rafts (using simvastatin), integrin β1 (using ATN-161), and FAK signaling (using YH-306). Inhibiting these targets significantly reduced P. gingivalis infection in human oral keratinocytes and periodontal ligament stem cells. Crucially, simvastatin restored the osteogenic ability of P. gingivalis-infected PDLSCs. These drugs subsequently functioned as effective antibiotic adjuvants. The combination of simvastatin or YH-306 with metronidazole, a standard-of-care antibiotic for periodontitis, exhibited potent synergistic effects, significantly eradicating intracellular bacteria at reduced antibiotic dosages. In a rat periodontitis model, simvastatin-metronidazole therapy yielded superior outcomes, significantly mitigating bacterial load, inflammation, and alveolar bone loss. The repurposing of clinically available drugs, such as simvastatin, as antibiotic adjuvants represents a promising, readily translatable therapeutic approach for periodontitis.
The proinflammatory (N1) polarization of bone marrow (BM) neutrophils, driven by central immune remodeling in response to peripheral inflammation, plays a critical role in propagating localized inflammatory conditions, such as periodontitis, to systemic levels. Although this process involves metabolic reprogramming, the specific underlying metabolic mechanisms of neutrophil N1 polarization within the periodontitis-modified BM niche remain poorly defined. Integrated transcriptomic and metabolomic analyses in this study revealed that periodontitis reprograms intracellular glutathione (GSH) metabolism in BM neutrophils, facilitating their N1 polarization. Central to this mechanism is the upregulation of Chac2, an enzyme that promotes GSH accumulation. This enhancement is accompanied by elevated GSH redox cycling, which supports sustained ROS production and NET formation, thereby amplifying inflammatory responses. We further identified type I interferon (IFN-I) signaling as a key upstream regulator that induces Chac2 expression and drives metabolic reprogramming. Importantly, the intraosseous delivery of AAV-delivered Chac2 shRNA in db/db mice with periodontitis markedly reduced neutrophil-aggravated systemic inflammatory comorbidity symptoms and improved glycemic control, underscoring the functional relevance of this pathway in diabetic comorbidity. Together, these findings thus delineate the IFN-I–Chac2–GSH axis as a core signaling mechanism regulating neutrophil N1 polarization in the BM niche, providing new insights into how periodontal inflammation reprograms immune functions at the systemic level. This study thus broadens the conceptual framework of neutrophil immunometabolism and proposes targeting the Chac2–GSH axis as a potential therapeutic strategy for systemic comorbidities associated with periodontitis.
Peri-implantitis (PI) is a multifactorial chronic inflammatory disease characterized by persistent mucosal inflammation and progressive marginal bone loss, yet the tissue architecture and cellular programs that sustain chronic inflammation remain incompletely defined. Here, we integrate single-cell and spatial transcriptomic analyses with targeted validation to map the PI microenvironment and its regulatory circuits. PI lesions exhibit marked immune enrichment, including expansion of CXCR4⁺ aged neutrophils with enhanced survival signaling consistent with apoptosis delay. Spatial and communication analyses reveal a PI-specific stromal–myeloid organization in which fibroblast-rich regions are closely associated with myeloid hotspots and display heightened CXCL- and CSF-related signaling. Within the fibroblast compartment, inflammatory myofibroblasts emerge as a prominent predicted source within a CXCL/CSF-enriched secretory program, with CXCL6 and CSF3 highlighted as candidate mediators of neutrophil recruitment and survival. Functionally, blockade of the CXCR2 axis reduces neutrophil infiltration and mitigates peri-implant bone resorption. Upstream regulatory analyses nominate STAT4 as a candidate upstream regulator of the inflammatory stromal program; pharmacologic perturbation with lisofylline reduces CXCL6/CSF3 expression, weakens neutrophil chemotaxis, partially restores apoptosis sensitivity, and alleviates inflammation and bone loss in vivo. Together, these findings support a stromal–neutrophil circuit that sustains chronic inflammation in PI and highlight stromal inflammatory programs and neutrophil recruitment/survival pathways as potential therapeutic entry points.
Tooth autotransplantation requires recipient socket preparation that matches donor root morphology while preserving surrounding bone. We developed an autonomous multi-axis robotic system that executes nonlinear, surface-conforming milling trajectories to create a geometry-matched socket and compared it with a static tooth-supported guide in forty 3D-printed mandibular models representing single-rooted and double-rooted anatomies. Recipient sockets were planned by offsetting the donor root surface by 0.5 mm and eliminating insertion axis undercuts. The robot executed the planned milling path with a depth-stop Lindemann bur, whereas the guide workflow used guided pilot drilling followed by freehand refinement. Robot assistance reduced deep positional errors and improved agreement between planned and prepared socket geometry, with the most pronounced benefit in double-rooted models, while overall preparation time was comparable between approaches. These findings support further clinical validation to confirm that autonomous robotic, surface-conforming osteotomy can improve full-depth geometric fidelity and reduce unnecessary bone removal in technique-sensitive autotransplantation procedures.
Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease with limited therapeutic options. Stem cell-based tissue engineering, particularly utilizing human periodontal ligament stem cells (hPDLSCs), represents a promising approach for cartilage regeneration. However, we have previously demonstrated that chronic inflammation and hypoxic stress in the TMJOA microenvironment markedly accelerate cellular senescence in hPDLSCs, severely impairing their regenerative potential. Here, we identify the YTHDC1–m⁶A–GADD45B axis as a critical regulator of senescence and chondrogenic differentiation in hPDLSCs. We show that YTHDC1, an m⁶A reader protein, is downregulated under inflammatory and senescent conditions. Functional studies reveal that YTHDC1 overexpression attenuates senescence and enhances chondrogenesis, whereas its knockdown exacerbates senescence and suppresses differentiation. Mechanistically, YTHDC1 recognizes m⁶A modifications on GADD45B mRNA and promotes its decay, leading to inhibition of the p53/p21 signaling pathway. Mutation of the m⁶A site in GADD45B abolishes the regulatory effects of YTHDC1. In rats with TMJOA, transplantation of YTHDC1–overexpressing hPDLSCs ameliorated disease phenotypes, an effect reversed by co-expression of wild-type GADD45B. Our findings reveal a novel epitranscriptomic mechanism that regulates hPDLSCs senescence and subsequently affects chondrogenic differentiation, and highlight the therapeutic potential of targeting the YTHDC1-GADD45B-p53/p21 axis.
Condylar hyperplasia (CH), characterized by progressive facial deviation, occlusion disorders, and temporomandibular joint dysfunction, often requires combined temporomandibular joint (TMJ)-orthognathic interventions to reconstruct joint and occlusal functions as well as rebuilding harmonious facial expressions. Managing the dentomaxillofacial deformities secondary to CH involves hyperplastic condylar resection, orthognathic surgery, facial contour surgery and orthodontic treatment. Based on the activity of condylar hypertrophy, the severity of dentomaxillofacial deformity and malocclusion, an individualized treatment plan should be formulated to reconstruct joint functions, correct deformities and recover occlusal relationships. This expert consensus, informed by the latest clinical research and practical experience, addresses clinical considerations for surgical treatment strategies for patients with different CH types, delineating indications, objectives, procedures, and principles with the aim of providing clear and practical guidance for clinical practitioners.
Mesenchymal stem cells (MSCs) hold significant promise for applications in regenerative medicine, yet their therapeutic potential is often limited by replicative senescence. Identifying effective strategies to reverse replicative senescence in MSCs and elucidating the underlying molecular mechanisms are essential steps in advancing their clinical use. Here, this study demonstrated that the pluripotency regulator octamer-binding transcription factor 4 (OCT4) promoted odontogenic differentiation by activating period circadian regulator 1 (PER1) in replicative senescent stem cells from apical papilla (SCAP) spheres. Specifically, OCT4 overexpression significantly alleviated cell cycle arrest, reduced senescence-associated β-galactosidase activity, and downregulated the expression of senescence-related markers, including CDKN2A/P16, CDKN1A/P21, and TP53/P53. Moreover, this approach markedly enhanced the proliferation and odontogenic differentiation potential of SCAP spheres in vitro and promoted the formation of regenerative pulp-like tissue in vivo. Mechanistically, we demonstrated that OCT4 transcriptionally activated PER1 through direct binding to its promoter, thereby restoring the odontogenic differentiation capacity of replicative senescent SCAP. Collectively, our findings establish the OCT4-PER1 axis as a critical regulatory pathway that counteracts replicative senescence in SCAP. These insights suggest new therapeutic strategies targeting senescence-associated signaling pathways to enhance MSC-based regenerative outcomes.
Amelogenesis imperfecta is a hereditary enamel defect arising from dental epithelium dysfunction. Although keratinocyte differentiation factor 1 (KDF1) acts as an intracellular regulator in epithelial cells, the underlying disease mechanism of the patient-derived KDF1 missense mutation in amelogenesis remains unclear. Here, we show that a patient-derived KDF1 mutation (c.908 G > C, p.R303P) causes enamel defects by disrupting cell adhesion and Hippo-YAP signaling. Immunohistochemistry revealed strong KDF1 expression throughout dental epithelium development, particularly at the cell membrane. Kdf1 mutation knock-in heterozygotes and homozygotes displayed graded defective enamel with reduced thickness, inadequate mineralization and disorganized microstructure. This phenotype correlated with a gradual reduction in enamel matrix proteins and proteases across genotypes. Bulk RNA sequencing of ameloblasts suggested marked changes in adhesion-related genes and the Hippo-YAP pathway. We characterized cellular consequences of this variant using both LS8 and ALC cell lines, which appeared abnormalities including accelerated proliferation, undermined differentiation, weakened adhesion, and enhanced migration. In vivo and in vitro findings supported a model wherein the KDF1 mutation impaired intercellular and cell-matrix adhesion in ameloblasts. As a result, ameloblast differentiation was hampered through excessive nuclear yes-associated protein (YAP) accumulation and overactivation of downstream proliferative genes. Pharmacological blockade of YAP and TEA domain family member 1 (TEAD1) interaction rescued the mutant phenotypes. Taken together, Kdf1 mutation compromised murine amelogenesis through adhesion defects and subsequent Hippo-YAP dysregulation.
Osteoblasts orchestrate the infiltration and crystallization of mineral precursors within collagen fibrils. Certain osteoblast-secreted mineralization-inducing proteins further stimulate bone formation. In this study, scRNA-seq analysis of murine skull and long bone revealed a striking expression pattern of carbonic anhydrase III (Car3) in osteoblasts. We uncovered a pivotal role for CAR3 in osteoblast lineage cells, revealing its critical function in skeletal development and homeostasis. Conditional ablation of Car3 in Prx1-lineage cells resulted in osteopenia and markedly impaired osteoblast activity, underscoring its functional role. Mechanistically, the primary transcription factor RUNX2 directly regulated Car3 expression, mediating its spatiotemporal expression during development. Notably, CAR3 promoted collagen intrafibrillar mineralization by forming a ternary complex with COL1A1 and bone sialoprotein (BSP), thereby facilitating mineral deposition. Furthermore, CAR3-functionalized scaffolds significantly improved bone repair and regeneration by promoting both matrix mineralization and recruitment of Prx1-lineage cells. These findings establish CAR3 as a critical coordinator of osteoblast differentiation and collagen interfibrillar mineralization, positioning it as a central mediator for maintaining skeletal integrity and enabling regeneration.
Supramolecular hydrogels hold significant potential in drug delivery and tissue engineering, with standing out for their unique properties. Despite their promise, predicting nucleoside bioactivity remains challenging. This study aims to predict the biological activity of nucleosides to guide the rational synthesis of hydrogels. Specifically, nine predictive models and databases for various biological activities were built with feature-selected machine learning methods including decision trees, logistic regression, random forest, and extreme gradient boosting. Then, the Molecular Bioactivity Specificity Index (MBSI) was introduced to gauge the primary bioactivity of nucleoside derivatives, and the Composite Molecular Attribute Score (CMAS) was devised to measure the overall performance of nucleoside derivatives. Subsequently, screening strategies for bioactive nucleoside hydrogels were established, and two candidate hydrogels (GMP and dGMP) with high hydrogel-forming ability, biocompatibility, and antibacterial activity were identified. Finally, two hydrogels were validated for antibacterial treatment of periodontitis. This study highlights the feasibility of ML-based strategies and MBSI/CMAS in rationally designing bioactive nucleoside hydrogels for biomedical applications. The discovery of GMP and dGMP hydrogels and their successful validation in periodontitis models highlight the potential of this strategy for developing targeted therapies for oral diseases.
Organ defects involving hierarchical tissue structures remain a major challenge due to limited regenerative capacity in adulthood. To break this bottleneck, regenerative medicine is undergoing a paradigm shift from simulating the healing process of mature organs to reactivating re-development potential, namely developmental engineering strategy. In this study, we propose a novel paradigm based on developmental niche-empowered stem cell-derived apoptotic extracellular vesicles (DevNiche-ApoEVs), which integrates cues from both parental stem cells and their environmental niches. Using the periodontium as a classical hierarchical model, we identified developmental M2-phenotype macrophages (DevM2φ) as a key niche component that induces a developmental metabolic profile in stem cells, characterized by enhanced energy metabolism, mitochondrial homeostasis, and dominance of oxidative phosphorylation. We subsequently empowered ApoEVs with DevM2φ-mediated developmental niche to generate DevNiche-ApoEVs capable of delivering mitochondrial complex I and recapitulating the developmental metabolic profile. In vitro studies confirmed DevNiche-ApoEVs reactivated the developmental potential of adult periodontal ligament cells (PDLCs), while complex I inhibition abrogated this effect. Consistently, DevNiche-ApoEVs promoted re-development-based hierarchical periodontal regeneration by recapitulating critical developmental events in vivo. This study highlights the pivotal role of the developmental niche in hierarchical tissue regeneration and provides a promising DevNiche-ApoEVs-focused developmental engineering strategy, which offers both a solid theoretical foundation and an effective translational solution to overcome the longstanding bottleneck in adult tissue regeneration.
Periodontitis is the most prevalent chronic inflammatory condition affecting oral health and is associated with long treatment duration. It is triggered by microbial plaque, which leads to localized and diffuse inflammation, ultimately causing progressive and irreversible damage to the alveolar bone and connective tissue. Therefore, early and effective treatment strategies should prioritize both antimicrobial and anti-inflammatory interventions. Herein, we report a multifunctional DNA nanodrug delivery platform based on tetrahedral framework nucleic acids (tFNAs), which effectively delivers curcumin and defensin to periodontal tissues. This platform exhibits a triple therapeutic effect by eliminating periodontal pathogenic bacteria, reducing inflammatory infiltration in periodontal tissues, and inhibiting bone resorption and degradation. Experimental results showed that curcumin was uniformly loaded onto the framework nucleic acid via groove binding, while defensin was anchored via chemical conjugation, forming the curcumin–defensin–tFNA (Cur-de-tFNA) complex. Due to its structural advantages, this nanodrug platform demonstrates exceptional cellular uptake efficiency and biosafety, significantly enhancing the bioavailability of curcumin and the antimicrobial activity of defensin. Moreover, as the platform degrades into nucleic acids, it presents one of the cleanest nanodrug delivery platforms currently available. As anticipated, the complex demonstrated potent antimicrobial activity, modulated the TLR4 pathway, improved the local microenvironment, promoted the expression of osteogenic proteins, and alleviated local tissue inflammation in a rat model of periodontitis, effectively reducing alveolar bone resorption. We believe that this study offers meaningful insights for multi-targeted combination therapies for periodontitis and provides new directions for the management of bacterial infection-induced local inflammation and bone resorption-related diseases.
Tobacco use, alcohol consumption, and infection with human papilloma virus (HPV) are well-established risk factors for head and neck squamous cell carcinomas (HNSCC). However, the incidence of oral cancer, particularly in the mobile tongue, has been rising in non-smoker/non-drinker and HPV-negative patients, suggesting the emergence of a new clinical entity. To understand in molecular terms this subtype of oral cavity squamous cell carcinomas (OCSCC) with no-identified risk factor (NIRF), we analyzed the available public head and neck cancer multi-omics data. We identified mutational signatures that stratified 253 OCSCC and 94 laryngeal cancer cases, used as tobacco-only-related controls, according to their clinico-pathological characteristics. We show that tobacco, depending on the anatomical site, triggers distinct mutational processes and further demonstrate that the single-base-substitution (SBS) signature SBS16 in OCSCC is associated with tobacco smoking, reflecting the combined effects of smoking and drinking. Importantly, we identified a tongue cancer-enriched NIRF OCSCC subgroup exhibiting significantly increased endogenous clock-like mutagenesis, while another NIRF subgroup manifested with elevated apolipoprotein B mRNA editing enzyme catalytic polypeptide-like (APOBEC)-associated mutagenesis. Both NIRF OCSCC subgroups harbored specific cancer driver mutations and distinct methylation patterns, which differed from those observed in OCSCC linked to traditional HNSCC risk factors, reflecting unique molecular programs underlying disease development. Specifically, NIRF-OSCC exhibited pronounced immune evasion strategies and antimicrobial transcriptomic responses. Our study presents the first molecular and genomic characterization of the emerging NIRF OCSCC subtype likely driven by increased endogenous mutagenesis and responses to microbial insults. These findings warrant future detailed investigations into etiology and have implications for clinical management and cancer prevention.
Pain is the most common symptom of temporomandibular joint (TMJ) disorders, which present significant clinical challenges due to their complexity and limited treatment options. Our previous study demonstrates that gut microbiome-derived butyrate is critical for the modulation of TMJ pain. In this study, we investigated its underlying mechanisms, and we found that oral administration of tributyrin, a prodrug of butyrate, not only significantly alleviated TMJ pain but also reversed the reduction in histone acetylation in the spinal trigeminal nucleus caudalis (Sp5C) under the TMJ pain condition. Using single-cell multi-omics sequencing, we profiled gene expression and chromatin accessibility in the Sp5C cells at the single-cell resolution. Bioinformatics analysis revealed that TMJ pain disrupted both the expression and chromatin accessibility of Nop14, Matk, Idh3b, Ndst2, and Tomm6 across four cell types in the Sp5C, and these alterations were reversed by tributyrin treatment. Specifically, Nop14 exhibited increased chromatin accessibility at its promoter region under TMJ pain condition, and knockdown of Nop14 in the Sp5C restored histone acetylation and alleviated TMJ pain. Together, our findings reveal cell-type–specific gene regulation that underlies butyrate-mediated epigenetic regulation of TMJ pain, which suggesting that targeting gut microbiome metabolites could develop a non-opioid novel therapy for TMJ disorders.
