Astrocytes in the spinal dorsal horn (SDH) exhibit diverse reactive phenotypes under neuropathic conditions, yet the mechanisms driving this diversity and its implications in chronic pain remain unclear. Here, we report that spared nerve injury (SNI) induces marked upregulation of both complement component 3 (C3⁺, A1-like) and S100 calcium-binding protein A10 (S100A10⁺, A2-like) astrocyte subpopulations in the SDH, with elevated microglial cytokines including interleukin-1α, tumor necrosis factor-α, and complement component 1q. Transcriptomic, immunohistochemical, and Western blot analyses reveal co-activation of multiple reactive astrocyte states over a unidirectional shift toward an A1-like phenotype. Fibroblast growth factor 8 (FGF8), a neuroprotective factor via FGFR3, mitigated microglia-induced C3⁺ astrocyte reactivity in vitro and suppressed spinal C3 expression and mechanical allodynia following intrathecal administration in SNI mice. These findings reveal a microglia-astrocyte signaling axis that promotes A1 reactivity and position FGF8 as a promising therapeutic candidate for neuropathic pain by modulating astrocyte heterogeneity.
Animals ; Astrocytes/drug effects* ; Neuralgia/pathology* ; Receptor, Fibroblast Growth Factor, Type 3/metabolism* ; Signal Transduction/physiology* ; Male ; Mice ; Microglia/drug effects* ; Fibroblast Growth Factor 8/pharmacology* ; Mice, Inbred C57BL ; Hyperalgesia/drug therapy* ; Spinal Cord/drug effects* ; Complement C3/metabolism* ; Spinal Cord Dorsal Horn/metabolism*

Neuropathic pain is frequently comorbidity with cognitive deficits. Neuralized1 (Neurl1)-mediated ubiquitination of CPEB3 in the hippocampus is critical in learning and memory. However, the role of Neurl1 in the cognitive impairment in neuropathic pain remains elusive. Herein, we found that lumbar 5 spinal nerve ligation (SNL) in male rat-induced neuropathic pain was followed by learning and memory deficits and LTP impairment in the hippocampus. The Neurl1 expression in the hippocampal CA1 was decreased after SNL. And this decrease paralleled the reduction of ubiquitinated-CPEB3 level and reduced production of GluA1 and GluA2. Overexpression of Neurl1 in the CA1 rescued cognitive deficits and LTP impairment, and reversed the reduction of ubiquitinated-CPEB3 level and the decrease of GluA1 and GluA2 production following SNL. Specific knockdown of Neurl1 or CPEB3 in bilateral hippocampal CA1 in naïve rats resulted in cognitive deficits and impairment of synaptic plasticity. The rescued cognitive function and synaptic plasticity by the treatment of overexpression of Neurl1 before SNL were counteracted by the knockdown of CPEB3 in the CA1. Collectively, the above results suggest that the downregulation of Neurl1 through reducing CPEB3 ubiquitination and, in turn, repressing GluA1 and GluA2 production and mediating synaptic plasticity impairment in hippocampal CA1 leads to the genesis of cognitive deficits in neuropathic pain.
Animals ; Male ; Neuralgia/metabolism* ; Rats ; Down-Regulation/physiology* ; Ubiquitination/physiology* ; Neuronal Plasticity/physiology* ; Rats, Sprague-Dawley ; CA1 Region, Hippocampal/metabolism* ; Cognitive Dysfunction/metabolism* ; RNA-Binding Proteins/metabolism* ; Receptors, AMPA/metabolism*

The increasing aging population and aging-associated diseases have become a global issue for decades. People over 65 show an increased prevalence and greater severity of periodontitis, which poses threats to overall health. Studies have demonstrated a significant association between aging and the dysfunction of neutrophils, critical cells in the early stages of periodontitis, and their crosstalk with macrophages and T and B lymphocytes to establish the periodontal lesion. Neutrophils differentiate and mature in the bone marrow before entering the circulation; during an infection, they are recruited to infected tissues guided by the signal from chemokines and cytokines to eliminate invading pathogens. Neutrophils are crucial in maintaining a balanced response between host and microbes to prevent periodontal diseases in periodontal tissues. The impacts of aging on neutrophils' chemotaxis, anti-microbial function, cell activation, and lifespan result in impaired neutrophil functions and excessive neutrophil activation, which could influence periodontitis course. We summarize the roles of neutrophils in periodontal diseases and the aging-related impacts on neutrophil functional responses. We also explore the underlying mechanisms that can contribute to periodontitis manifestation in aging. This review could help us better understand the pathogenesis of periodontitis, which could offer novel therapeutic targets for periodontitis.
Humans ; Neutrophils/immunology* ; Periodontitis/immunology* ; Aging/physiology*

The oral and maxillofacial region is a highly complex area composed of multiple tissue types and bears various critical functions of the human body. Diseases in this region pose significant diagnostic and management challenges; therefore, exploring new strategies for early diagnosis, targeted treatment, and tissue reconstruction is key to improving patient prognosis and quality of life. Extracellular vesicles are a group of heterogeneous lipid-bilayer membrane structures secreted by most cell types, including exosomes, microvesicles, and apoptotic bodies. Present in various body fluids and tissues, they act as messengers via the transfer of nucleic acids, proteins, and metabolites to recipient cells. To date, studies have revealed the different roles of extracellular vesicles in physiological or pathological processes, as well as applications in disease diagnosis, prognosis, and treatment. The importance and tissue specificity of the dental and maxillofacial tissues indicate that extracellular vesicles derived from this region are promising for further research. This paper reviews the published data on extracellular vesicles derived from cells, body fluids, and tissues in oral and maxillofacial regions, summarizes the latest advances in extracellular vesicles from extensive sources, and concludes with a focus on the current research progress and application prospects of engineered exosomes in oral science.
Humans ; Extracellular Vesicles/physiology* ; Mouth ; Exosomes/physiology*

Approximately 20% to 30% of the global workforce is engaged in shift work. As a significant cause of circadian disruption, shift work is closely associated with an increased risk for periodontitis. Nevertheless, how shift work-related circadian disruption functions in periodontitis remains unknown. Herein, we employed a simulated shift work model constructed by controlling the environmental light-dark cycles and revealed that shift work-related circadian disruption exacerbated the progression of experimental periodontitis. RNA sequencing and in vitro experiments indicated that downregulation of the core circadian protein brain and muscle ARNT-like protein 1 (BMAL1) and activation of the Gasdermin D (GSDMD)-mediated pyroptosis were involved in the pathogenesis of that. Mechanically, BMAL1 regulated GSDMD-mediated pyroptosis by suppressing NOD-like receptor protein 3 (NLRP3) inflammasome signaling through modulating nuclear receptor subfamily 1 group D member 1 (NR1D1), and inhibiting Gsdmd transcription via directly binding to the E-box elements in its promoter. GSDMD-mediated pyroptosis accelerated periodontitis progression, whereas downregulated BMAL1 under circadian disruption further aggravated periodontal destruction by increasing GSDMD activity. And restoring the level of BMAL1 by circadian recovery and SR8278 injection alleviated simulated shift work-exacerbated periodontitis via lessening GSDMD-mediated pyroptosis. These findings provide new evidence and potential interventional targets for circadian disruption-accelerated periodontitis.
Pyroptosis/physiology* ; ARNTL Transcription Factors/metabolism* ; Animals ; Periodontitis/etiology* ; Mice ; Phosphate-Binding Proteins/metabolism* ; Shift Work Schedule/adverse effects* ; Intracellular Signaling Peptides and Proteins/metabolism* ; Mice, Inbred C57BL ; Male ; Disease Models, Animal ; Gasdermins

Dental mesenchymal stem cells (DMSCs) are pivotal for tooth development and periodontal tissue health and play an important role in tissue engineering and regenerative medicine because of their multidirectional differentiation potential and self-renewal ability. The cellular microenvironment regulates the fate of stem cells and can be modified using various optimization techniques. These methods can influence the cellular microenvironment, activate disparate signaling pathways, and induce different biological effects. "Epigenetic regulation" refers to the process of influencing gene expression and regulating cell fate without altering DNA sequences, such as histone methylation. Histone methylation modifications regulate pivotal transcription factors governing DMSCs differentiation into osteo-/odontogenic lineages. The most important sites of histone methylation in tooth organization were found to be H3K4, H3K9, and H3K27. Histone methylation affects gene expression and regulates stem cell differentiation by maintaining a delicate balance between major trimethylation sites, generating distinct chromatin structures associated with specific downstream transcriptional states. Several crucial signaling pathways associated with osteogenic differentiation are susceptible to modulation via histone methylation modifications. A deeper understanding of the regulatory mechanisms governing histone methylation modifications in osteo-/odontogenic differentiation and immune-inflammatory responses of DMSCs will facilitate further investigation of the epigenetic regulation of histone methylation in DMSC-mediated tissue regeneration and inflammation. Here is a concise overview of the pivotal functions of epigenetic histone methylation at H3K4, H3K9, and H3K27 in the regulation of osteo-/odontogenic differentiation and renewal of DMSCs in both non-inflammatory and inflammatory microenvironments. This review summarizes the current research on these processes in the context of tissue regeneration and therapeutic interventions.
Mesenchymal Stem Cells/physiology* ; Humans ; Osteogenesis/genetics* ; Histones/metabolism* ; Cell Differentiation/physiology* ; Methylation ; Odontogenesis/genetics* ; Epigenesis, Genetic

The periodontal ligament (PDL) plays a crucial role in transmitting and dispersing occlusal force, acting as mechanoreceptor for muscle activity during chewing, as well as mediating orthodontic tooth movement. It transforms mechanical stimuli into biological signals, influencing alveolar bone remodeling. Recent research has delved deeper into the biological and mechanical aspects of PDL, emphasizing the importance of understanding its structure and mechanical properties comprehensively. This review focuses on the latest findings concerning both macro- and micro- structural aspects of the PDL, highlighting its mechanical characteristics and factors that influence them. Moreover, it explores the mechanotransduction mechanisms of PDL cells under mechanical forces. Structure-mechanics-mechanotransduction interplay in PDL has been integrated ultimately. By providing an up-to-date overview of our understanding on PDL at various scales, this study lays the foundation for further exploration into PDL-related biomechanics and mechanobiology.
Periodontal Ligament/cytology* ; Humans ; Biomechanical Phenomena ; Mechanotransduction, Cellular/physiology* ; Stress, Mechanical

Tissue interactions play a crucial role in tooth development. Notably, extracellular vesicle-mediated interactions between the mandible and tooth germ are considered essential. Here, we revealed that mandible extracellular vesicles could modulate the proliferation and differentiation of dental mesenchymal cells by regulating the histone demethylase KDM2B. Further investigation showed that mandible derived extracellular vesicles could deliver miR-206 to KDM2B, thereby regulating tooth development. An animal study demonstrated that the miR-206/KDM2B pathway affected tooth morphogenesis and mineralization after eight weeks of subcutaneous transplantation in nude mice. In conclusion, this study suggested that the mandible played a critical role in tooth morphogenesis and mineralization, which could be a potential therapeutic target for abnormal tooth development and an alternative model for tooth regeneration.
Animals ; Extracellular Vesicles/metabolism* ; Jumonji Domain-Containing Histone Demethylases/metabolism* ; Swine ; MicroRNAs/metabolism* ; Mandible ; Mice, Nude ; Odontogenesis/physiology* ; Swine, Miniature ; Mice ; Cell Differentiation ; Cell Proliferation

Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications. In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner. However, existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes, thereby hindering a comprehensive understanding of their interactions. Here, we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria, respectively. Using a key oral pathobiont, Fusobacterium nucleatum, as the primary test bed, we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes. Compared to conventional models, this system enhanced bacterial invasion, elevated intracellular bacterial loads, and elicited more robust host pro-inflammatory responses, including increased secretion of CXCL10, IL-6, and IL-8. In addition, the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens. Finally, we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont, Porphyromonas gingivalis. These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics, offering a physiologically relevant approach to advance oral and systemic health research.
Coculture Techniques/methods* ; Humans ; Fusobacterium nucleatum/physiology* ; Gingiva/microbiology* ; Keratinocytes/microbiology* ; Host Microbial Interactions ; Mouth/microbiology* ; Host-Pathogen Interactions ; Epithelial Cells/microbiology* ; Cells, Cultured ; Porphyromonas gingivalis

Piezo2, a mechanosensitive ion channel, serves as a crucial mechanotransducer in dental primary afferent (DPA) neurons and is potentially involved in hypersensitivity to mild mechanical irritations observed in dental patients. Given Piezo2's widespread expression across diverse subpopulations of DPA neurons, this study aimed to characterize the mechanosensory properties of Piezo2-expressing DPA neurons with a focus on distinct features of voltage-gated sodium channels (VGSCs) and neuropeptide profiles. Using whole-cell patch-clamp recordings, we observed mechanically activated action potentials (APs) and classified AP waveforms based on the presence or absence of a hump during the repolarization phase. Single-cell reverse transcription polymerase chain reaction combined with patch-clamp recordings revealed specific associations between AP waveforms and molecular properties, including tetrodotoxin-resistant VGSCs (Na_V1.8 and Na_V1.9) and TRPV1 expression. Reanalysis of the transcriptomic dataset of DPA neurons identified correlations between neuropeptides-including two CGRP isoforms (α-CGRP and β-CGRP), Substance P, and Galanin-and the expression of Na_V1.8 and Na_V1.9, which were linked to defined AP subtypes. These molecular associations were further validated in Piezo2⁺ DPA neurons using fluorescence in situ hybridization. Together, these findings highlight the electrophysiological and neurochemical heterogeneity of Piezo2-expressing DPA neurons and their specialized roles in distinct mechanosensory signal transmission.
Ion Channels/physiology* ; Mechanotransduction, Cellular/physiology* ; Animals ; Neurons, Afferent/metabolism* ; Patch-Clamp Techniques ; Mice ; TRPV Cation Channels/metabolism* ; Action Potentials ; Rats