Introduction: Conventional vital pulp therapy relies primarily on calcium silicate-based cements that induce reparative dentin formation without restoring the native neurovascular architecture of the pulp tissue. Advances in tissue engineering using stem cells and biodegradable scaffolds offer the potential for true pulp regeneration rather than mere preservation. Material and Methods: Human dental pulp stem cells were isolated from healthy third molars and encapsulated within gelatin methacryloyl hydrogel constructs. In this randomized laboratory trial, 60 standardized human tooth slices were allocated into three groups: negative control (empty), positive control (Biodentine™), and test group (human dental pulp stem cells-gelatin methacryloyl construct). Cell viability, proliferation, odontogenic differentiation, and angiogenic potential were assessed using Live/Dead staining, Cell counting Kit-8 colorimetric assay, and quantitative reverse transcription polymerase chain reaction analysis of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and vascular endothelial growth factor expression. Results: The quantitative reverse transcription polymerase chain reaction constructs demonstrated high cytocompatibility, with cell viability exceeding 94% at day 7. Proliferation was significantly greater in the test group compared with Biodentine at day 7 (p < 0.01). Odontogenic marker expression was comparable between the test and Biodentine groups, while vascular endothelial growth factor expression was markedly higher in the test group group, indicating superior angiogenic potential. Conclusion: Stem cell–laden gelatin methacryloyl constructs exhibit enhanced regenerative properties compared with conventional bioceramic materials in an ex vivo tooth slice model. These findings support the translational potential of hydrogel-based regenerative strategies as next-generation approaches for vital pulp therapy.

Organ-on-a-chip in oral medicine: Emerging approaches and applications.

For the genetic identification of skeletal remains, teeth can be used since they contain well-preserved DNA, which is present in various dental tissues, including dental pulp, dentin, and cementum. In forensics, the standard extraction method utilizes the whole tooth and destructive grinding. In our previous study, a highly effective non-destructive method was introduced to extract DNA from tooth cementum, bypassing whole tooth destruction. An analysis was performed on sixty-two canines obtained from adult skeletons from two archaeological cemeteries. After tooth cementum DNA extraction, teeth were stored in the freezer, and later used for destructive DNA extraction, grinding the entire tooth left after non-destructive extraction. The purpose of this study was to compare the preservation of DNA in tooth cementum and other tooth tissues. To extract the DNA from the stored teeth, they were pulverized, and DNA was obtained using the full demineralization method. Real-time PCR was employed to evaluate the quality and quantity of DNA, followed by STR typing. The amount of DNA, degradation rate, and success of STR typing were compared between DNA retrieved from tooth cementum and DNA extracted from the other tooth tissues. The results showed no successful STR typing in most stored teeth, and only five of them produced full or almost full profiles. Comparison to tooth root cementum, where 46 out of 62 teeth generated highly informative STR profiles, indicates that in the aged canines analysed, DNA preservation is strongest in tooth root cementum, while other tooth tissues generally yield little or no recoverable DNA. In five teeth that generated informative STR profiles, we assume that not all the tooth root cementum was decalcified using a non-destructive extraction method, and after grinding the whole tooth, some cementum tissue remained, contributing to successful genetic typing. In this study, the importance of tooth root cementum in archaeological samples is emphasized while exploring the forensic relevance of various tooth tissues. It highlights the notably poorer DNA preservation in dental pulp and dentin compared to cementum in the context of aged teeth.

Dental mesenchymal stem cell-derived exosomes for oral diseases: Classification, functionalization and clinical prospects.

Despite significant advances in dental pulp stem cell (DPSC)-based regeneration of the pulp-dentine complex, regulating the directed differentiation of these cells remains a key challenge. The present study investigated the role and underlying mechanism of the KDM4D-RPS5 complex in modulating the odontogenic differentiation of DPSCs, with the goal of providing insights to inform strategies for tooth tissue regeneration and repair. To assess the osteo/dentinogenic differentiation capacity of DPSCs, multiple techniques were employed, including alkaline phosphatase (ALP) activity assays, Alizarin Red S staining, quantitative calcium analysis, and detection of osteo/dentinogenic marker expression. Gene expression levels were quantified using quantitative real-time polymerase chain reaction and Western blot. Chromatin immunoprecipitation and co-immunoprecipitation (Co-IP) assays were performed to investigate the underlying molecular mechanisms. Mitochondrial morphology in DPSCs was observed via transmission electron microscopy (TEM), while the oxygen consumption rate was measured using a Seahorse XF Analyser, and mitochondrial membrane potential was assessed with a JC-10 assay. Finally, the invivo efficacy of odontogenic differentiation was validated through a subcutaneous transplantation assay in nude mice. We first demonstrated that KDM4D significantly promoted the osteo/dentinogenic differentiation of DPSCs. Furthermore, KDM4D bound directly to RPS5 via a specific structural domain to form a functional complex; disruption of this binding site abolished its capacity to drive differentiation. Mechanistically, ChIP assays revealed that the KDM4D-RPS5 complex epigenetically activated the downstream gene CNR1 by demethylating H3K9me2 at its promoter, thereby facilitating DPSC differentiation. Additionally, mitochondrial functional analysis showed that overexpression of KDM4D, RPS5, or CNR1 enhanced mitochondrial membrane potential and augmented energy metabolism, further supporting the differentiation process. KDM4D bound to RPS5 to form a protein complex, which regulated the demethylation of CNR1 H3K9me2 and further influenced the osteo/dentinogenic differentiation of DPSCs by promoting mitochondrial energy metabolism. These findings identify the KDM4D-RPS5-CNR1 axis as a promising therapeutic target for enhancing DPSC-based dental tissue regeneration.

Novel ready-to-use regenerative medicine with cryopreserved 3D nanostructured fiber scaffold and dental pulp stem cells for bone regeneration

Mammalian tooth development progresses through two principal stages-crown formation and root development-orchestrated by intricate interactions between the oral epithelium and neural crest-derived mesenchyme. After crown formation, Hertwig's epithelial root sheath (HERS) directs root development. In this phase, Gli1⁺ mesenchymal stem cells (MSCs) give rise to dental pulp, dentin, cementum, and the periodontal ligament (PDL). The root anchors the tooth to the alveolar bone via PDL fibers, forming a dynamic occlusal buffer that mediates mechanosensation and nutrient supply. Although previous work has shown that macrophages are abundant in the dental pulp and follicle, the functional importance of macrophages in tooth development has not been well characterized. Here, we investigated the spatiotemporal dynamics of macrophage populations (identified by CD68, F4/80, CD206, and other markers) in molars and surrounding tissues during postnatal root development in mice. Importantly, Macrophage depletion via clodronate liposomes resulted in shortened root, impaired PDL elongation and retarded alveolar bone shooting surrounding the root. Gli1⁺ MSCs exhibited increased proliferation but impaired osteo/odontogenic differentiation upon macrophage depletion. Single-cell RNA sequencing and in vitro co-culture experiments support a model in which macrophage-derived TGF-β acts on mesenchymal TGF-β receptors to direct MSC fate and thereby regulate root morphogenesis. Collectively, these findings establish macrophages as critical niche components that orchestrate tooth root development through immune-mesenchymal crosstalk.

Regenerative endodontics focuses on the restoration of the pulp-dentine complex by promoting odontogenic differentiation of stem cells of the apical papilla (SCAPs). Although SSUH2 has been implicated in developmental processes and dentine dysplasia type I (DD-I), the specific role of SSUH2 in regulating SCAPs differentiation remains unclear. In this study, we identified SSUH2 as a novel biomarker for SCAPs odontogenic differentiation and revealed its critical regulatory mechanism. Immunohistochemical staining of mouse mandibular first molar sections and immunofluorescence staining of human dental pulp tissues were performed to investigate the expression characteristics of SSUH2 during root development. SCAPs were treated with siRNA-mediated knockdown or lentivirus-mediated overexpression of SSUH2, followed by assessment of proliferation and odontogenic differentiation. Subcutaneous implantation of hydrogel-scaffold-root fragments loaded with SSUH2-overexpressing SCAPs in nude mice was performed to evaluate invivo odontogenic capacity. Mechanistically, transcriptome sequencing and bioinformatic analysis identified downstream signalling pathways. Co-immunoprecipitation, nuclear-cytoplasmic fractionation, and immunofluorescence confirmed the direct interaction between SSUH2 and FOXM1, promoting FOXM1 nuclear translocation and subsequent transcriptional upregulation of PDK1. Moreover, transmission electron microscopy, mitochondrial membrane potential assays and ROS detection collectively demonstrated that SSUH2 regulates mitochondrial function in SCAPs via the FOXM1/PDK1 axis. During the development of the first molar in the mouse's lower jaw, SSUH2 is enriched in the root tip papilla region and it is also enriched in the root tip papilla region of humans. SSUH2 positively regulated the odontogenic differentiation of SCAPs invitro. Invivo transplantation models showed that SSUH2 overexpression enhanced the odontogenic differentiation capacity of SCAPs and promoted dentine-pulp-like structure formation. Transcriptomic and functional analyses indicated that SSUH2 maintained mitochondrial function. SSUH2 directly interacted with FOXM1, promoted its nuclear translocation, and upregulated PDK1 expression. FOXM1 silencing abrogated SSUH2-mediated enhancement of both mitochondrial function and odontogenic differentiation in SCAPs. Our study is the first to reveal that SSUH2 promotes the odontogenic differentiation of SCAPs through the FOXM1/PDK1 axis by regulating mitochondrial function. These findings suggest that SSUH2 could serve as a potential therapeutic target for enhancing the odontogenic differentiation of SCAPs and provide a novel strategic direction for pulp-dentine complex regeneration.

Berberine pretreatment enhances the homing and anti-inflammatory efficacy of dental pulp mesenchymal stem cells in TNBS-induced inflammatory bowel disease via activating the CXCR4/SDF-1 signaling pathway.

Inflammation critically determines dental pulp regenerative outcomes, with dental pulp stem cells (DPSCs) orchestrating tissue homeostasis through differentiation, self-renewal and immunomodulation processes dynamically regulated by Wnt/β-catenin and NF-κB signaling crosstalk. Given the rising therapeutic potential of Wnt-targeted interventions in dental tissue engineering, elucidating these molecular interactions under pathological conditions is essential for developing regenerative therapeutics capable of simultaneously promoting reparative dentinogenesis while resolving inflammatory insults. This perspective review aims to: (1) critically evaluate existing literature on lipopolysaccharide (LPS)-mediated modulation of dental pulp stem cell (DPSC) fate, addressing inconsistencies in LPS concentrations, bacterial sources and inflammatory models; (2) identify methodological gaps in current standardisation and elucidate molecular mechanisms governing Wnt/NF-κB signaling crosstalk in DPSCs under acute versus chronic inflammatory conditions; and (3) assess the therapeutic potential of GSK3β inhibitors and exosome-based interventions for dentine-pulp regeneration. A comprehensive literature search was conducted across PubMed/MEDLINE, Scopus and Web of Science Core Collection for publications through November 2025. Search strategies combined four thematic domains: (1) cell populations ("dental pulp stem cell" OR "DPSC"); (2) signaling pathways ("Wnt" OR "β-catenin" AND "NF-κB" OR "crosstalk"); (3) biological processes ("odontogenic differentiation" OR "immunomodulation" OR "macrophage polarization"); (4) inflammatory context ("pulpitis" OR "inflammation" OR "LPS"). Articles were screened for relevance to Wnt/NF-κB interactions in dental pulp regeneration under inflammatory conditions. Evidence demonstrates context-dependent Wnt/NF-κB crosstalk in DPSC fate specification. Low-dose LPS (< 1 μg/mL) stimulates reparative responses through coordinated Wnt/NF-κB activation, whereas sustained high-dose exposure (> 1 μg/mL) suppresses Wnt signaling via NF-κB-driven DKK1 upregulation, attenuating differentiation capacity. While no direct evidence links Wnt/NF-κB crosstalk to DPSC self-renewal, both pathways independently maintain stemness. Critically, DPSCs and macrophages exhibit reciprocal regenerative interactions: DPSC-derived Wnt3a polarises macrophages toward the anti-inflammatory M2 phenotype, while M2-secreted Wnt7b enhances DPSC odontogenic differentiation by suppressing NF-κB expression. However, standardised inflammation models remain lacking, hindering comprehensive elucidation of context-dependent mechanisms. Developing such models would clarify how inflammation temporally and spatially influences regenerative outcomes across clinical scenarios.

Transdentinal cytotoxicity of resin-based cements on odontoblast-like (MDPC-23) and human dental pulp cells.

Maxillofacial bone defects remain a complex clinical challenge due to their complex anatomy and limited regenerative capacity. In this study, a bioactive chitosan (CS) scaffold co-incorporated with nicotinamide mononucleotide (NMN) and naringin (Ng) was designed to stimulate osteogenesis and cellular metabolism for enhanced bone regeneration. Fourier-transform infrared analysis verified successful molecular integration of NMN and Ng within the chitosan framework, while scanning electron microscopy showed a uniformly porous surface favorable for stem-cell adhesion. Dental pulp stem cells (DPSCs) cultured on CS/NMN/Ng scaffolds exhibited enhanced cell proliferation, mitochondrial activity, and osteogenic differentiation compared with unmodified chitosan. At the cellular level, calcium deposition was markedly increased; molecular analysis confirmed up-regulated expression of Runx2, alkaline phosphatase (ALP), and type-I collagen. The bone regenerative capacity was further validated in a rat mandibular defect model, where micro-computed tomography and histological staining demonstrated near-complete closure of the defect with increased bone volume fraction and trabecular density after 8 weeks. Overall, the NMN–naringin-functionalized chitosan scaffold provides a metabolically active, osteoinductive microenvironment that promotes bone regeneration, indicating a translational potential for maxillofacial and craniofacial bone tissue engineering applications.

Craniofacial tissue regeneration remains a pivotal challenge in oral and regenerative medicine. Mesenchymal stem/stromal cells (MSCs) are central effector cells in this process, and their functions are regulated by a sophisticated, multidimensional network. This article provides a comprehensive overview of the regulatory mechanisms governing MSCs in craniofacial regeneration. We highlight the interactive roles of metabolism, epigenetics, and immunity in precisely controlling MSC stemness, lineage-specific differentiation, and immunomodulatory capabilities. Key regulatory dimensions are explored in detail. Metabolic reprogramming, such as serine one-carbon metabolism and mitochondrial dynamics under hyperosmotic stress, couples energy production with epigenetic modifications to dictate MSC fate. The gasotransmitter hydrogen sulfide (H2S) exerts tissue-specific effects, modulating immunoregulation via the Fas/FasL axis in gingival MSCs and promoting odontogenic differentiation in dental pulp stem cells (DPSCs) via the transient receptor potential action channel subfamily vanilloid member 1 (TRPV1)/β-catenin pathway. Epigenetic mechanisms, including DNA demethylation by ten-eleven translocation (TET) enzymes and chromatin remodeling by special AT-rich sequence-binding protein 2 (SATB2), finely tune MSC homeostasis and differentiation potential. Crucially, MSCs do not function in isolation. Their bidirectional crosstalk with immune cells, mediated by exosomes and soluble factors, is essential for bone homeostasis. Mechanical overloading can trigger MSCs to promote T helper 17 (Th17) cell polarization via metabolic reprogramming, exacerbating bone destruction. Conversely, H2S-modified exosomes from M2 macrophages can enhance MSC osteogenesis, demonstrating a synergistic metabolic-immune axis for bone regeneration. Exosomes themselves serve as versatile therapeutic carriers, capable of delivering miRNAs (e.g., miR-125a/b) or functional mitochondrial DNA to modulate immunity or repair cellular metabolism. The clinical translation of MSCs holds great promise for treating conditions like periodontitis and temporomandibular joint disorders. Advances in engineered exosomes and biomaterial carriers (e.g., hydrogels) offer strategies for targeted delivery and enhanced efficacy. Future research must focus on developing tissue-specific delivery systems, refining exosome engineering for precise cargo loading, and leveraging multi-omics technologies to decipher the complex stem cell niche. This progression from empi-rical application to rationally designed, precision therapies will be critical for addressing clinical challenges in craniofacial reconstruction.

This study investigated the radiopacity, elemental composition, cytotoxicity, and cytokine responses of contemporary calcium silicate-based cements containing different radiopacifiers. Four cement materials (NeoMTA2, NeoPUTTY, TheraCal PT, and One-Fil PT) were evaluated. Radiopacity was measured using digital radiography with a 10-step aluminum wedge and expressed in mm Al in accordance with ISO 6876; among three calibration models compared, the quadratic provided the best fit. Elemental composition was analyzed by SEM/EDX. Cytotoxicity was assessed on human dental pulp cells using the MTT assay, and IL-6 and IL-10 levels were quantified by ELISA. One-Fil PT (6.61 mm Al) and NeoPUTTY (6.09 mm Al) showed the highest radiopacity, whereas TheraCal PT (1.61 mm Al) did not meet ISO standards. SEM/EDX revealed tantalum in NeoMTA2 and NeoPUTTY, and zirconium in One-Fil PT and TheraCal PT. NeoPUTTY and NeoMTA2 demonstrated superior cell viability, while One-Fil PT showed the lowest. TheraCal PT and One-Fil PT increased IL-6 expression, whereas NeoPUTTY and NeoMTA2 promoted higher IL-10 levels. Within the limitations of this 24-h in vitro assessment, NeoMTA2 and NeoPUTTY exhibited more favorable short-term cytocompatibility and inflammatory profiles together with adequate radiopacity. These findings require confirmation through long-term in vivo and clinical studies.

Dose-dependent effects of metformin on proliferation and odontogenic differentiation of dental pulp stem cells.

Experimental study of mRNA from human dental pulp tissue for late postmortem interval estimation.

MiR-224-5p has been proven to play an important role in regulating cell differentiation. This study aimed to clarify the regulatory role and mechanism of miR-224-5p in the osteogenic differentiation of human dental pulp stem cells (hDPSCs), thereby laying a theoretical foundation for subsequent jaw defect repair. Human dental pulp stem cells (hDPSCs) were isolated, cultured, and sorted from healthy dental pulp tissues. We performed integrated bioinformatics analysis to screen and identify the potential targets and pathways of miR-224-5p involved in the osteogenic induction of hDPSCs. Subsequently, in vitro experiments were conducted. Plasmid transfection was used to regulate the overexpression and knockdown of miR-224-5p in hDPSCs, and the expression of osteogenesis-related proteins was detected. Furthermore, luciferase reporter assays and Western blot assays were used to confirm the direct targets of miR-224-5p, and rescue experiments were performed to verify the underlying mechanism. The results demonstrated that overexpression of miR-224-5p inhibited the osteogenic differentiation of DPSCs, as reflected by the significantly decreased expression of osteogenic markers (OCN, Runx2, and ALP). In contrast, inhibition of miR-224-5p promoted the osteogenic differentiation of DPSCs. Bioinformatics analysis and dual-luciferase reporter gene assays indicated that miR-224-5p specifically targets the 3' untranslated region of the PTEN gene. Rescue experiments further confirmed that miR-224-5p regulates this process by modulating the PTEN/PI3K/AKT pathway. Inhibition of miR-224-5p promotes osteogenesis in DPSCs by targeting the PTEN/PI3K/AKT signaling axis. These findings provide reliable evidence for the fabrication of three-dimensional tissue-engineered structures and further repair of maxillofacial bone defects.

Mechanical Signaling in the Microenvironment Regulates the Differentiation of Dental Pulp Stem Cells: A Novel Strategy For Pulp-Dentin Complex Regeneration.

Isolation and Characterization of Dental Pulp Stem Cell-Derived Exosomes Across Cell Passages

This study evaluated the histopathological response of the dental pulp following direct pulp capping (DPC) using Mineral Trioxide Aggregate Repair High Plasticity (MTA Repair HP) supplemented with green-synthesized selenium nanoparticles (SeNPs). The objective was to determine how SeNP concentration influences the biological behavior of MTA HP and to identify the dose that best supports pulp healing compared with MTA HP alone. Twelve male dogs with 132 teeth were included. Twelve teeth served as negative controls, while 120 teeth were assigned to five groups: MTA HP alone or MTA HP combined with 0.5%, 1%, 1.5%, or 2% (w/w) SeNPs (n = 24 per group). Standardized pulp exposures were created and treated according to group allocation. Samples were collected at 7, 14, 30, and 60 days (n = 6 per interval) for histopathological assessment of inflammation, necrosis, and reparative dentine formation. The group treated with 1% SeNP had the best results. They displayed very little inflammation, no signs of tissue necrosis, and started forming reparative dentine as early as day 14. While inflammation diminished over time in all the groups, the statistical analysis showed that the different SeNP levels made a difference in how much inflammation occurred, how much tissue necrosis there was, and the quality of the new dentine. Adding SeNP to MTA HP depended on the amount used. The 1% version presented the best biocompatibility and assisted pulp tissue heal better. On the other hand, higher amounts triggered more inflammation. Overall, these results recommend that 1% SeNP-enhanced MTA HP could be a choice for direct pulp capping, offering better healing and clinical results.