Journal of Dental Research

Our studies have established that loss-of-function mutations in the Wnt signaling antagonist SFRP4 (Secreted Frizzled Related Protein 4) cause Pyle disease, a rare skeletal disease characterized by limb deformity. Pyle disease can also present with a number of dental conditions, including tooth decay and taurodontism. Aside from the sparse clinical descriptions of the tooth anomalies seen in Pyle disease, the role of Sfrp4 in teeth has not been investigated. Here we show that in adult mouse incisors, Sfrp4 is expressed in the mesenchymal and epithelial compartments and their derivatives as well in the developing apex of the molar roots, pulp, odontoblasts, and periodontal ligament. We report that Sfrp4 deletion in mice leads to markedly shorter incisors, with reduced dentin mineral apposition and enamel volume. In addition, we find that Sfrp4 deletion leads to a reduction in the root length and bifurcation height of the developing molars, both features of taurodontism. Rodent incisors grow continuously during the animal’s lifetime, thanks to the maintenance and interactions of epithelial (EpSCs) and mesenchymal stem cells (MSCs) at their apex. As such, the mouse incisor is a powerful tool for studying adult stem cells, their interactions, and their regulation. Using this model, we found that Sfrp4 deletion leads to a significant decrease in Gli1⁺ MSCs and in Ki67 levels in mesenchymal transient amplifying cells and preameloblasts. Sfrp4-null incisors have reduced type I collagen levels and altered amelogenin and Mmp20 secretion accompanied by changes in the enamel maturation stages. When incisor growth is accelerated by preventing occlusion through tooth clipping, Sfrp4-null incisors grow significantly slower than wt incisors do. Our study suggests a key role for Sfrp4 in the fast-growing teeth (incisors) and developing molars, where there is a need to balance the maintenance and differentiation of the stem cell niche.

Untreated dental caries, a chronic disease linked to systemic health, is common among older adults due to a lack of dental insurance and affordable dental care. Minimally invasive interventions such as silver diamine fluoride (SDF) and atraumatic restorative treatments (ARTs) have been recommended by the World Health Organization to control caries where evidence is lacking in older adults. This cluster-randomized clinical trial tested the noninferiority of SDF vs ART in caries arrest (primary) and prevention (secondary) at 52 wk. Older adults ≥62 y living in 33 subsidized housing facilities were enrolled at baseline, with follow-up visits at 26 and 52 wk. Participants in the intervention arm received biannual topical application of 38% SDF, and the control arm received ART with glass ionomer cement (+2.5% sodium fluoride varnish). Caries arrest and new caries were determined through examinations conducted by hygienists utilizing criteria from the International Caries Detection and Assessment System. The noninferiority margin was 10% of the effectiveness of the control treatment. Among a total of 568 enrolled participants (intervention: 18 sites, n = 346; control: 15 sites, n = 222), 51% were female and 66% were Black; the mean ± SD age was 69.6 ± 6.8 y. Caries arrest at 52 wk was 88% in the intervention arm vs 92% in the control arm (difference, −0.04; 95% CI, −0.08 to 0.01). After adjusting for sociodemographics, a linear model with generalized estimating equations yielded an estimated treatment difference of −0.05 (95% CI, −0.09 to −0.01). The mean number of new decayed/carious lesions at 52 wk was 0.67 ± 1.72 for the intervention arm and 0.66 ± 1.71 for the control arm (difference, 0.01; 95% CI, −0.31 to 0.32). Results for both outcomes indicated noninferiority of 38% SDF at 52 wk. Findings support the use of a simple topical biannual 38% SDF treatment to control caries in clinical and public health settings, which can be delivered by dental and medical clinicians (ClinicalTrials.gov NCT03916926).

Lymphatic vessels play a pivotal role in tissue homeostasis and repair, yet their function in periodontal healing remains poorly defined. In this study, we investigated the spatial dynamics and functional significance of lymphatics during periodontal wound repair using a mouse model of ligature-induced periodontitis. By combining Prox1-tdTomato transgenic reporter mice with advanced tissue clearing and light sheet microscopy, we generated high-resolution 3-dimensional images of gingival lymphatic networks under both physiological and repair conditions. Our observations revealed that lymphatic vessels undergo active and spatially organized reassembly during the healing phase. Notably, we found a region-specific increase in lymphatic branching and density in the marginal gingiva, suggesting localized lymphangiogenesis. Time course analysis confirmed that this lymphatic remodeling peaks during the granulation tissue formation phase. We further identified epithelial upregulation of Vegfc during the repair phase and demonstrated that local administration of vascular endothelial growth factor C (VEGF-C) C156S, a VEGFR-3–specific agonist, enhanced lymphatic vessel function and alveolar bone repair. To explore the underlying molecular mechanisms, we successfully established, for the first time, primary cultures of gingiva-derived lymphatic endothelial cells (LECs). Comparative transcriptomic analysis revealed distinct signatures of gingival LECs compared to lymph node or dermal LECs. These cells maintained typical LEC characteristics in vitro and responded robustly to VEGF-C stimulation. RNA sequencing and functional apoptosis assays revealed significant modulation of apoptosis-related genes, including downregulation of Ell3 and upregulation of Siah1b, suggesting a survival-promoting role for VEGF-C signaling in LECs. Collectively, our findings highlight a previously underappreciated regenerative role of lymphatic vessels in the periodontium and establish lymphangiogenic activation as a potential therapeutic strategy for enhancing periodontal repair. The successful culture of primary gingival LECs further provides a novel platform for mechanistic studies in oral lymphatic biology.

Oral epithelial dysplasia (OED) is characterized by abnormal structural and cellular changes in the oral mucosa epithelium. These changes are often detected histologically in potentially malignant disorders and are associated with an increased risk of malignant transformation into oral squamous cell carcinoma (OSCC). Currently, clinicians rely mainly on the pathologist’s histological gradings (binary or tertiary grading systems) to determine the follow-up protocol for patients with OED. However, both histological gradings are rather subjective and not highly reliable. Therefore, there is a need for biomarkers to distinguish high-risk OED cases from low-risk cases. Twelve oral tongue dysplasia samples were collected for multiplex immunostaining. The samples were derived from 7 patients who did not develop OSCC within 5 y of their OED diagnosis (low-risk group, LR) and 5 patients who developed OSCC (high-risk group, HR). Ten antibodies targeting epithelial cells, immune cells, a proliferation marker, and immune checkpoints were used (PanCK, CD4, CD8, CD11b, CD68, CD56, FoxP3, Ki67, PD-1, PD-L1). Uniform manifold approximation and projection generated from multiplex staining revealed distinct differences in the distribution of immune cells and immune checkpoint expression between the LR and HR groups. The HR group showed a higher number of CD8⁺ cytotoxic T cells and CD68⁺ macrophages than the LR group. Notably, both PanCK⁺ epithelial cells and CD8⁺ cytotoxic T cells exhibited increased proliferation in the HR group, as indicated by Ki67 expression. Additionally, PD-1 expression was significantly elevated in the HR group. The logistics regression model further confirmed the predictive value of individual markers and identified a multimarker model with superior predictive performance. Our multiplex immunostaining revealed significant differences in the immune landscape between LR and HR cases of OED dysplasia. CD8⁺ cytotoxic T cells, CD68⁺ macrophages, and PD-1⁺ cells may serve as potential biomarkers for stratifying HR from LR oral tongue dysplasia cases.

X-inactivation is the process of dosage compensation to balance X-linked gene expression levels between females (XX) and males (XY). One of the 2 X-chromosomes is randomly selected for inactivation in each cell during embryogenesis and remains inactive throughout life. Therefore, females have 2 types of cells: those with paternal X-chromosome activation and those with maternal X-chromosome activation. However, it remains unclear how X-inactivation is involved in palate and tooth development. Ofd1 is located on the X-chromosome. We found cleft palate in all heterozygous Ofd1 mutant mice (Ofd1^fl/WT;Wnt1Cre[HET]), even though some cells should activate the normal X-chromosome due to random X-inactivation. Cells with Ofd1 mutant X chromosome activation (Ofd1 mutant cells) accumulated at the tip of the palatal shelves due to cell segregation caused by a lack of EFNB1 expression, resulting in a cleft palate in all Ofd1^fl/WT;Wnt1Cre(HET) mice. Unlike the palate, Ofd1^fl/WT;Wnt1Cre(HET) mice showed multiple tooth phenotypes, including extra and absent incisors, while a normal number of incisors was also found. Accumulation of Ofd1 mutant cells also occurred in the tooth mesenchyme, and these clusters of Ofd1 mutant cells failed to initiate incisor tooth formation due to a lack of Wnt signaling. In contrast to palate development, the location of Ofd1 mutant cell clusters differed between Ofd1^fl/WT;Wnt1Cre(HET) mice, likely leading to diversity of tooth phenotypes. Thus, X-inactivation exhibits different involvement between palate and tooth development.

Multilevel modeling (MLM) is increasingly applied in oral health research to reflect the hierarchical structure of dental data, in which teeth are nested within individuals and individuals are embedded within social and environmental contexts. By partitioning both within- and between-individual variability, MLM improves estimate precision, reduces bias, and strengthens causal interpretation, thereby enhancing the evidence base for preventive strategies, clinical practice, and public health policy. This review synthesizes recent methodological developments and ongoing challenges in the application of MLM to oral health research, with particular emphasis on causal inference. Key issues include reciprocal relationships between individual- and contextual-level factors, establishing temporality, collinearity among contextual variables, population mobility, and the selection of appropriate geographic or institutional levels. We also highlight methodological advances, such as integrating propensity score weighting within multilevel frameworks, which offer promising approaches to improving causal assessment in observational studies. Accumulating evidence demonstrates the importance of contextual determinants, such as school and neighborhood environments, in shaping oral health outcomes across the life course. However, causal inference regarding these distal exposures remains underdeveloped. Persistent threats to validity include measurement error in contextual indicators, unmeasured multilevel confounding, and limited consideration of cross-level interactions. Using a school-based example, we illustrate how food insecurity mediates the association between household income and active dental caries, highlighting the value of analytical approaches that jointly address individual- and contextual-level determinants. Overall, MLM provides a robust framework for clarifying causal pathways, improving analytical rigor, and guiding interventions that target both behavioral and structural drivers of oral health inequalities.

Metabolic syndrome (MetS) and periodontitis (PD) are major interconnected health burdens, and the inflammatory interplay between them remains elusive. Neutrophils, central to periodontal immunity, can undergo functional alterations contributing to disease exacerbation. This study investigates the role of Krüppel-like factor 4 (KLF4) in regulating neutrophil aging during the exacerbation of PD by MetS. The analysis of clinical human samples and murine models revealed that MetS enhances the infiltration of senescent neutrophils into periodontal tissues. These neutrophils exhibit a heightened proinflammatory phenotype, including increased secretion of senescence-associated secretory phenotype factors, enhanced formation of neutrophil extracellular traps, and a metabolic reprogramming toward a glycolytic shift. Bioinformatics identified KLF4 as a key downregulated gene in comorbid MetS and PD. Adoptive transfer of MetS-derived or Klf4-deficient neutrophils into mice with periodontitis aggravated periodontal inflammation and alveolar bone destruction. In vitro, KLF4 knockdown in human neutrophil-like cell lines promoted metabolic reprogramming and proinflammatory phenotype. Through virtual docking and molecular dynamics simulations, we identified rosiglitazone as a candidate compound predicted to stabilize KLF4. This compound alleviated periodontal damage and improved insulin resistance in mice by mitigating the neutrophil aging phenotype and suppressing proinflammatory responses. Critically, all of these therapeutic effects were abolished in Klf4-deficient models. These results indicate that KLF4 is associated with the regulation of neutrophil aging through which MetS exacerbates periodontitis, highlighting KLF4 activation as a promising therapeutic strategy.

Patients with inflammatory bowel disease (IBD) have a higher prevalence of apical periodontitis (AP), which is often associated with accelerated jawbone destruction. However, the mechanisms underlying this exacerbation remain unclear. In this study, we established a colitis+AP model and demonstrated that colitis exacerbates alveolar bone destruction. The upregulation of genes related to inflammation and neutrophils was also observed in the colitis+AP model compared with the AP-only model. In addition, colitis-induced local neutrophil infiltration was observed in the alveolar bone, which was further amplified by bacterial infection originating from the root canal. To address the exacerbated bone destruction in colitis+AP, we used a novel laser-induced cavitation system to locally deliver the immunosuppressant tacrolimus into the jawbone. This targeted approach effectively suppressed alveolar bone loss in the colitis+AP model. Our findings highlight the importance of the inflammatory interplay between IBD and AP. Systemic inflammation caused by IBD weakens local neutrophil function, leading to increased bone destruction and treatment-resistant AP. Furthermore, laser-activated local administration of tacrolimus significantly ameliorated bone destruction in AP associated with colitis.

Vision-language models (VLMs) have demonstrated significant potential in medical image analysis, yet their application in intraoral photography remains largely underexplored due to the lack of fine-grained, annotated datasets and comprehensive benchmarks. To address this, we present MetaDent, a comprehensive resource that includes 1) a novel and large-scale dentistry image dataset collected from clinical, public, and web sources; 2) a semistructured annotation framework designed to capture the hierarchical and clinically nuanced nature of dental photography; and 3) comprehensive benchmark suites for evaluating state-of-the-art VLMs on clinical image understanding. Our labeling approach combines a high-level image summary with point-by-point, free-text descriptions of abnormalities. This method enables rich, scalable, and task-agnostic representations. We curated 60,669 dental images from diverse sources and annotated a representative subset of 2,588 images using this meta-labeling scheme. Leveraging large language models (LLMs), we derive standardized benchmarks: approximately 15,000 visual question answering (VQA) pairs and an 18-class multilabel classification dataset, which we validated with human review and error analysis to justify that the LLM-driven transition reliably preserves fidelity and semantic accuracy. We then evaluate state-of-the-art VLMs across VQA, classification, and image captioning tasks. Quantitative results reveal that even the most advanced models struggle with a fine-grained understanding of intraoral scenes, achieving moderate accuracy (e.g., less than 70% in VQA) and producing inconsistent or incomplete descriptions in image captioning. These findings underscore the gap between general-purpose VLMs and the demands of specialized models, highlighting the need for domain-adapted training and more sophisticated evaluation protocols to assist professional dental practice and community oral health efforts. We publicly release our dataset, annotations, and tools to foster reproducible research and accelerate the development of vision-language systems for dental applications.

Tooth loss has been associated with increased mortality risk in older adults. This study examined heterogeneity in the association between tooth loss and mortality among older Japanese adults. Data were obtained from 69,265 individuals aged ≥65 y who participated in the 2013 wave of the Japan Gerontological Evaluation Study. Questionnaire survey responses were linked to mortality records from municipal registries for 9 y. The average treatment effect (ATE) of tooth loss (having <20 natural teeth) on all-cause mortality was estimated using a linear probability model with an inverse probability weighting estimator. Heterogeneity in the association was assessed by estimating conditional average treatment effects (CATEs) using the causal forest machine learning algorithm. A set of 44 covariates, including demographic, economic, social, health, and community-related factors, was incorporated as potential drivers of heterogeneity. During the follow-up period, 26.4% of participants with <20 teeth and 14.4% of those with ≥20 teeth died. Tooth loss was significantly associated with increased mortality risk by 3.2 percentage points after adjustment for covariates (average treatment effect = 0.032; 95% confidence interval, 0.023–0.040). A statistically significant heterogeneity in the estimated effects was observed (median CATE = 0.028; interquartile range = 0.010). Greater effects were identified among subgroups characterized by men, poor health status, and lower socioeconomic conditions. Heart disease, sex, and depression were the most influential contributors to this heterogeneity. Tooth loss was associated with mortality risk, with significant variation in the magnitude across population subgroups. Tooth loss may serve as an informative indicator of elevated mortality risk among older adults, particularly among vulnerable subgroups.

Alveolar bone regions with low innate thickness are prone to dehiscence, potentially causing harmful gingival recession. In situ alveolar bone thickening via local bioagent administration has recently emerged as a promising research area. Parathyroid hormone-related protein (PTHrP), encoded by the Pthlh gene, plays a pivotal role in skeletal development and bone remodeling. In this study, we developed a recombinant adeno-associated virus (rAAV) vector overexpressing Pthlh (rAAV9-Pthlh). Using a split-mouth design, we administered rAAV9-Pthlh via a single submucosal injection into the right maxillary buccal gingiva of rats. Micro-computed tomography scans, both in vivo and ex vivo, revealed a significant increase in alveolar bone thickness on the PTHLH sides 6 wk post-transfection. This enhanced alveolar bone formation was accompanied by marked periosteal thickening, with elevated expression of PTHrP, parathyroid hormone 1 receptor (PTH1R), OCN, and COL1A1. RNA sequencing of the buccal bone tissue showed upregulation of osteogenesis-related pathways along with increased mitophagy activity. Immunofluorescence confirmed enhanced colocalization of mitochondrial and lysosomal markers in the PTHLH group, primarily in the periosteum. Overexpressing Pthlh in primary rat periosteal stem cells via lentiviral transduction promoted cell proliferation and migration and elevated expression of COL1A1, as well as mitophagy markers BNIP3 and LC3-II. These effects were mitigated by Mdivi-1, an inhibitor of mitochondrial fission, suggesting potential involvement of mitophagy-dependent mitochondrial homeostasis maintenance. Finally, we established a rat model of repeated maxillary expansion and validated the effects of prophylactic rAAV9-Pthlh injection to protect against alveolar crest loss in this scenario. In conclusion, local rAAV9-Pthlh injection enhances in situ alveolar bone formation, holding potential for preventing dehiscence in aging or following orthodontic interventions. Additionally, overexpression of PTHrP in the alveolar periosteum has reiterated its well-documented role in expanding the pool of chondroskeletal progenitors, a process vital for the development of long bones and the mandibular condyle.

Valid diagnostic criteria for classifying pain-related temporomandibular disorders (TMDs) exist and can be applied with high interexaminer reliability to patients. However, adoption in most clinical settings, generalist and specialist, remains limited due to the examination complexity and time constraints in clinics. To address this limitation, we created a simplified examination protocol, and we test it here against reference standards based on the Diagnostic Criteria for TMD (DC/TMD). DC/TMD assessments include multiple provocation tests, 5 range-of-motion tasks, and 40 palpation points, as well as imaging for definitive joint diagnoses. We tested the simplified protocol using data from 2 multicenter studies: the TMJ Impact Project (n = 401) and OPPERA (n = 547). Examiners were trained and assessed annually for reliability, supporting generalizability. Diagnostic validity was assessed by area under the curve, sensitivity, specificity, and likelihood ratios (positive and negative). By using 2-s palpation at the full muscle (bilateral temporalis, masseter) and TMJ lateral pole, results showed high diagnostic performance for identifying painful TMD: area under the curve = 0.93, sensitivity = 0.88, and specificity = 0.98. Targeted single-band palpation within each muscle yielded comparable results. Adding other joint pain provocation procedures to the index test did not improve diagnostic accuracy. For acute closed lock, other disc-based TMDs, and degenerative joint disease, sensitivity remained ≤0.12 while specificity was ≥0.92. For subluxation, sensitivity/specificity was 0.81/0.97. Based on the likelihood ratios, clinical decision-making guidelines were developed for brief DC diagnoses. In conclusion, a simplified and shortened version of the DC/TMD protocol demonstrates excellent validity for identifying painful TMDs and is feasible for clinical practice. This approach reduces examination time while maintaining diagnostic accuracy, potentially improving patient care across broader clinical settings.

The demand for rapid, reliable, and esthetic ceramic restorations continues to drive innovation in dental materials and chairside manufacturing technology. However, conventional yttria-stabilized zirconia presents an inherent trade-off between mechanical and optical properties, with moderate fracture toughness ≤5 MPa·m^1/2. This study investigates the feasibility of chairside speed-sintering of a 4.5 mol% CaO-stabilized tetragonal zirconia polycrystal (4.5Ca-TZP), focusing on its microstructure, mechanical reliability, translucency, and aging resistance. Nano-sized 4.5Ca-TZP powders were compacted and speed-sintered within 60 min at 1,250 °C to 1,350 °C. Four sintering groups were evaluated (n = 20/group for density; n = 1 for grain size; n = 10 for hardness; n = 3 for Rietveld refinement; n = 5 for fracture toughness, translucency, and aging; n = 30 for biaxial strength and Weibull analysis) with statistical differences set at α = 0.05. Speed-sintering gave rise to fully dense ceramics (≥99% relative density) with a homogeneous, fine microstructure (<200 nm) and negligible monoclinic content (≤2 vol%). The ceramics demonstrated excellent mechanical reliability, exhibiting a characteristic strength of ≥1.1 GPa and a Weibull modulus ≥11. Hardness slightly decreased with increasing temperature, whereas translucency reached its maximum (≈22) at 1,250 °C. Indentation testing revealed the absence of radial cracks in specimens sintered above 1,300 °C. No tetragonal-to-monoclinic transformation occurred after 20 h of hydrothermal aging at 134 °C, demonstrating aging resistance. Compared with conventionally sintered 4.5Ca-TZP, speed-sintered ceramics showed comparable translucency and strength with enhanced mechanical reliability but significantly lower fracture toughness of >5 MPa·m^1/2. Overall, this work demonstrates that 4.5Ca-TZP can be speed-sintered to achieve fully dense zirconia with performance exceeding that of conventional 3Y-TZP, providing a tougher alternative zirconia restoration. A computer-aided design/computer-aided manufacturing–milled crown fabricated as a proof of concept showed isotropic shrinkage, supporting its potential for chairside application, while further investigation of long-term resistance, large-scale manufacturing, and dimension limitations is required to confirm clinical applicability.

Electronic nicotine delivery systems (ENDS), including e-cigarettes, are increasingly marketed as safer alternatives to combustible tobacco, yet their effects on oral health remain underexplored. Although the role of ENDS in creating dysbiotic oral bacterial communities is documented, effects on the oral mycobiome remain underexplored. This study compared the subgingival fungal communities of 123 periodontally and systemically healthy e-cigarette–only users, smokers, dual users, former smokers, and never-smokers using whole-genome shotgun sequencing for functional profiling. Taxonomic assignment using Kraken 2 and the PlusPF database identified 98 fungal taxa, and functional annotation with the Kyoto Encyclopedia of Genes and Genomes identified 2,960 fungal genes. Cross-domain bacterial–fungal interactions were interrogated using a correlation threshold of |r| ≥ 0.7 and P ≤ 0.001. E-cigarette users demonstrated a significantly higher α-diversity than smokers and never-smokers did (P < 0.001; P < 0.005) and a mycobiome enriched with Candida albicans, Aspergillus oryzae, and Schizosaccharomyces pombe. Functional profiling revealed enrichment of genes encoding or DNA repair, xenobiotic degradation, membrane transport, and stress response. The mycobiome of dual users and former smokers using e-cigarettes did not differ from that of e-cigarette users. Cross-kingdom networks identified 5- to 10-fold higher bacterial–fungal connectivity in e-cigarette users, with fungi capable of enhanced stress tolerance, DNA repair capacity, and metabolic adaptability acting as network anchors. Our data support an association between e-cigarette use and remodeling of the oral mycobiome and microbiome, driven by enhanced polymicrobial interactions and increased functional complexity, suggesting that assumptions regarding the biological neutrality of e-cigarette aerosols warrant further investigation.

Mandibular incisors are the most frequently congenitally absent teeth in Asian populations, affecting mastication, speech, aesthetics, and oral and maxillofacial functions over the long term. However, the genetic etiology of mandibular incisor agenesis (MIA) remains obscure. In this study, we conducted the first genome-wide association study on MIA in a Chinese population and identified an enhancer variant, rs71514987, associated with an increased risk of MIA (odds ratio, 2.18; 95% CI, 1.83 to 2.58; P = 3.62 × 10^-19). Functional characterization indicated that the rs71514987 C allele reduced the binding affinity of GATA4 to the OSR2 promoter, leading to increased OSR2 expression via remote regulation. Notably, elevated OSR2 levels promoted cell proliferation but diminished cell differentiation, migration, and apoptosis capabilities in stem cells from human exfoliated deciduous teeth, as well as abnormal development of pharyngeal teeth in zebrafish models. Mechanistic experiments further revealed that OSR2 bound to the promoter region of CXCR4, promoting its transcription level in the GPCR pathway, thereby influencing odontogenic differentiation during tooth development. Our findings provide novel insights into the molecular etiology of MIA.

Graphical Abstract

Given its unique composition and physicochemical stability, dentin obtained from teeth extracted for clinical reasons represents a promising source for the development of functional biomaterials. Photopolymerizable compounds based on demineralized dentin modified with acrylamide groups were developed, with the aim of formulating ester-free adhesive primers. Dentin powder obtained from the crown and root was demineralized and reacted with methacrylic anhydride (MA). Following reaction, dialysis, and lyophilization, the resulting granular and sticky materials were evaluated for their degree of functionalization (methacrylation) using the 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay and infrared (attenuated total reflectance Fourier transform infrared [ATR-FTIR]) spectrometry. The compounds were subsequently suspended in phosphate-buffered saline and collagen-methacrylamide, supplemented with lithium acylphosphinate, injected into custom molds, and photopolymerized using a dental-curing unit, producing 3-dimensional constructs. Morphology, water sorption, solubility, degree of swelling, flexural modulus, biodegradability, and biocompatibility were assessed. TNBS analysis indicated a lower degree of functionalization in methacrylated crown dentin (CD-MA, 41%) than in methacrylated root dentin (RD-MA, 55%), consistent with ATR-FTIR results. CD-MA exhibited greater solubility and swelling, as well as a lower flexural modulus (1.8 ± 0.2 MPa), compared with RD-MA (2.2 ± 0.3 MPa). However, CD-MA and RD-MA demonstrated comparable water sorption, biodegradability, and >95% metabolic activity and cell viability in OD-21 and MDPC-23 cell lines. As a proof of concept, CD-MA, which exhibited the higher yield, was selected for evaluation as an adhesive primer and compared with a commercial methacrylate-based etch-and-rinse adhesive. Specimens bonded with the experimental CD-MA primer showed significantly lower bond strength at the initial evaluation (24 h) than the control group. Nonetheless, after 6 mo of storage in simulated body fluid, the experimental group maintained stable bond strength, whereas the control group exhibited an approximately 30% reduction. Taken together, these findings demonstrate that dentin is a viable raw material for the development of methacrylamide-based dental primers, which exhibit greater bond strength stability than a conventional methacrylate-based adhesive system.

Heterozygous mutations of the gene encoding caspase-8 protease occur in 10% of human head and neck squamous cell carcinomas (HNSCCs). Cell line studies indicate that these mutations block apoptosis induced by death ligands. However, the in vivo role of caspase-8 mutations in the development of HNSCC and their impact on response to immune checkpoint blockade have not been determined. We generated mice with heterozygous, epithelium-specific knock-in of a representative, HNSCC-associated caspase-8 mutation (D305G). The impact of the caspase-8 mutation was assessed following treatment with the carcinogen 4-nitroquinoline-1-oxide (4NQO) in drinking water. Treatment of the D305G caspase-8 mutant mice with 4NQO resulted in a greater number of tongue tumors per mouse and a higher percentage of advanced-stage invasive carcinomas than was observed in 4NQO-treated mice with wild-type caspase-8, and tumors from the mutant mice were more resistant to anti–PD-1. We also engineered the murine oral cancer cell line MOC1 for heterozygous expression of caspase-8 mutations. Tumors generated from these engineered cells in syngeneic, immunocompetent mice demonstrated reduced responsiveness to anti–PD-1, relative to tumors with wild-type caspase-8. Further, the caspase-8 mutant tumors displayed reduced intratumoral and splenic CD8⁺ T cells and impaired recruitment of monocytes and dendritic cells during PD-1 blockade. Collectively, these findings demonstrate that caspase-8 mutation promotes carcinogen-induced HNSCC development and resistance to anti–PD-1. Investigation of caspase-8 mutations as potential biomarkers of poor response to immunotherapy in patients with HNSCC is warranted.

Dynamic immune responses are evident during the development of pulpitis. However, the cellular and molecular characterization of B cells in this context has long been neglected. This study investigates pulpitis through the lens of B-cell involvement, combining single-cell RNA sequencing with histopathological analysis to generate a comprehensive cellular atlas of healthy and inflamed dental pulp. Our findings reveal a near absence of B/plasma cells in healthy pulp but a marked increase during inflammation. These cells display active antigen presentation and immunoglobulin secretion. In the mouse pulpitis model, B-cell depletion alleviated pulpal inflammation, highlighting the proinflammatory role of B cells. Subclustering analysis identified 4 transcriptionally distinct B-cell subsets, which were validated at the protein level through multiplex immunofluorescence and flow cytometry. Among these, transitional B cells exhibited a noticeable breakdown of tolerance, suggesting a propensity for autoreactivity. Elevated autoantibodies were detected in both tissue samples and isolated plasma cells. Finally, Mendelian randomization analyses and serological assays in mice indicated potential local–systemic interactions in pulpitis. These findings reveal that B cells may contribute to both the local pathology and systemic immunological characteristics of pulpitis, providing a novel perspective on its pathogenesis through potential autoimmune mechanisms.

With aging, the morphology and function of the parotid glands are impaired, and the current mechanism is unknown. The integrity of mitochondria-associated membranes (MAMs), the structure connecting mitochondria and the endoplasmic reticulum (ER), is compromised during aging. This study investigated the effects of aging on MAMs and ER stress in the parotid glands of mice. Here, aged mice presented abnormalities in gland morphology and mitochondrial morphology and reduced MAMs integrity. Protein kinase R-like endoplasmic reticulum kinase (PERK) signaling is the primary mediator of ER stress, which is activated in the parotid glands of aged mice. Furthermore, aging-induced MFN2 downregulation disrupts mitochondrial dynamics. In addition, aging reduces MAMs function by blocking the MFN2–PERK interaction. Treatment with 4-phenylbutyric acid (4-PBA) improved MAMs integrity, inhibited the PERK pathway, and reduced apoptosis. Like 4-PBA, GSK2606414, a pharmacological antagonist of PERK, regulates ER stress and MAMs. Collectively, our data highlight disruption of the MFN2–PERK axis-mediated ER–mitochondrion connection as a cause of aging-induced parotid gland dysfunction.