Search Results (505)

Dental pulp tissue contains mesenchymal stem/progenitor cells that possess high proliferative potential for self-renewal. They are neural-crest derived cells and exhibit multi-lineage differentiation properties. These progenitor stem cells are now recognized as being vital to the dentin regeneration process following injury. Understanding the molecular mechanisms that mediate the differentiation of adult stem cells into odontoblasts and their use in the repair of the dentin–pulp complex is of significant interest in regenerative dental medicine. Dentin Phosphophoryn (DPP), synthesized and processed predominantly by the odontoblasts, functions both as a structural and signaling protein. We had previously demonstrated that DPP activates NF-κB and promotes Wnt5a expression in dental pulp stem cells. In this context, we observed that DPP can activate TAZ, a biologically potent transcriptional coactivator which serves as a downstream element of the NF-κB signaling cascade. Furthermore, binding of NF-κB p65 subunit to the TAZ promoter was facilitated by DPP stimulation, and their interaction was confirmed by ChIP analysis. In addition, DPP-dependent activation of the TAZ/TEAD reporter was confirmed by luciferase activity in DPSCs. Co-immunoprecipitation analysis confirmed the in vivo interaction between TAZ and β-catenin with DPP stimulation. This regulatory complex facilitated TAZ to bind to the conserved TEAD binding motifs of key gene targets involved in odontogenic differentiation such as RUNX2, OSX, OCN, ALP, BMP4, and WNT5A. Some of these genes also contain binding sites for the TCF/LEF transcription factors that interact with the Wnt effector, β-catenin. Activation of TAZ and β-catenin resulted in the upregulation of odontoblast gene expression and reduced expression in the presence of the TAZ–TEAD protein complex inhibitor. Using mandibles of DSPP KO and WT mice, we confirmed reduced TAZ and β-catenin protein levels in the dental pulp cells and in the odontoblasts of DSPP KO mice when compared with WT. Thus, DPP, an extracellular matrix protein, provides biological cues to activate the TAZ signaling pathway that can stimulate the terminal differentiation of DPSCs into functional odontoblasts. Full article

Background: Impaired angiogenesis and persistent inflammation are hallmarks of chronic diabetic wounds. Extracellular vesicles derived from dental pulp stem cells (DPSC-EVs) represent a promising cell-free therapy for tissue repair; however, their clinical translation is hindered by suboptimal yields and attenuated bioactivity associated with conventional two-dimensional (2D) culture. This study investigated whether a biomimetic three-dimensional (3D) fibrin/gelatin hydrogel system could optimize the therapeutic potency of DPSC-EVs for diabetic wound healing. Methods: DPSCs were encapsulated within 3D fibrin/gelatin scaffolds, followed by comprehensive characterization of cell viability and morphology. 3D-EVs and 2D-EVs were isolated via ultracentrifugation and validated by transmission electron microscopy and nanoparticle tracking analysis. The pro-angiogenic capacity of 3D-EVs was evaluated using human umbilical vein endothelial cells (HUVECs) under high-glucose (HG) stress. Additionally, the immunomodulatory effects were assessed by monitoring macrophage polarization in lipopolysaccharide-stimulated RAW 264.7 cells. The therapeutic efficacy was further validated in vivo using a streptozotocin (STZ)-induced diabetic mouse model with full-thickness cutaneous wounds. Results: The 3D fibrin/gelatin hydrogel provided a supportive microenvironment that significantly augmented the secretory productivity of DPSCs. Compared to 2D-EVs, 3D-EVs exhibited superior functional resilience in restoring HUVEC migration and tube formation under HG-induced oxidative stress. Furthermore, 3D-EVs effectively orchestrated the macrophage transition from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 phenotype, thereby modulating the immune microenvironment. In vivo, topical administration of 3D-EVs markedly accelerated wound closure, promoted re-epithelialization, and enhanced microvascular density and collagen maturation in diabetic mice. Conclusions: Our findings demonstrate that the 3D fibrin/gelatin culture system effectively primes the therapeutic profile of DPSC-EVs. These engineered vesicles accelerate diabetic wound healing by synergistically promoting angiogenesis and resolving chronic inflammation, offering a robust and potent cell-free strategy for the management of chronic diabetic ulcers. Full article

The Wnt/β-catenin signaling pathway regulates key cellular processes, including proliferation, migration, differentiation, apoptosis and tissue homeostasis, and plays a pivotal role in tooth development and post-developmental dental physiology. In mineralized tissues such as bone and dentin, the Wnt signaling is critically involved in reparative and regenerative mechanisms. The Wnt signaling in the dentin–pulp complex is tightly controlled by extracellular modulators and receptor availability, and its balance appears crucial for an appropriate response. Hydraulic calcium silicate-based cements (HCSBCs) are widely used in endodontics due to their bioactivity and favorable biological properties. Increasing data indicate that HCSBCs promote odontogenic responses and reparative dentinogenesis through the recruitment and activation of dental stem cells (DSCs), possibly via the Wnt/β-catenin signaling pathway modulation. Therefore, the aim of the present narrative review was to summarize current knowledge on the role of the Wnt signaling in oral tissues and its interaction with HCSBCs. It is hypothesized that these materials may enhance pathway activation through the release of ionic products, growth factors and inflammatory mediators, thereby supporting biologically driven reparative processes. Understanding these mechanisms may guide the development of next-generation biomaterials designed to optimize the intrinsic regenerative potential of the dentin–pulp complex. Full article

The aim of this study was to evaluate the biological effects of bioactive glass-containing self-adhesive resin cements (SARCs) on human dental pulp stem cells (DPSCs), focusing on cytocompatibility, odontogenic differentiation, and mineralization. Experimental SARCs containing 0–5 wt% BAG (BG0–BG5) were compared with two commercially available SARCs, RelyX U200 and TheraCem. Eluates were prepared and applied to DPSCs for the methylthiazol tetrazolium (MTT) assay, quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, and Alizarin Red S (ARS) staining. The result showed there were no significant differences in cell viability across all groups (p > 0.05), indicating that the addition of BAG did not affect cell viability, while the early odontogenic differentiation markers, such as RUNX2, ALP, and COL1A1, showed no clear trend among the groups. However, late-stage markers (DMP-1 and DSPP) were significantly higher in the BG2–BG5 groups relative to the OM group (p < 0.05). IF staining revealed intense signals in the BG2–BG5 groups (p < 0.05) and also ARS staining showed a time-dependent increase in mineral deposition. Within the limitations of this study, BAG-containing SARCs do not negatively impact cytocompatibility and promote late-stage odontogenic differentiation and mineral deposition. Full article

Objectives: The primary objective of vital pulp therapy (VPT) is to induce the formation of tertiary dentin bridge through the application of bioactive materials to maintain pulp vitality. This study aimed to evaluate the odontogenic potential of Moringa oleifera ethanolic and aqueous extracts as sustainable herbal pulp capping materials in comparison to mineral trioxide aggregate (MTA) and a bioceramic putty. The evaluation was conducted in vitro on human dental pulp stem cells (DPSCs) and in vivo using a rat model of direct pulp capping. Methods: Moringa leaf extracts were prepared and the cytotoxicity assessed using MTT assay to measure the IC50, and their odontogenic potential was further evaluated in vitro by measuring alkaline phosphatase (ALP) activity and performing Alizarin Red staining for mineralization in comparison to MTA. For the in vivo study, the exposed rat pulps were capped with the extracts, MTA, the bioceramic putty, or a plain collagen sponge (control). Histological analysis was performed to evaluate inflammation and tertiary dentin bridge formation. Results: The ethanolic, aqueous extracts and MTA showed low cytotoxicity, though the ethanolic extract had significantly enhanced ALP expression (p < 0.05) and mineralized nodule formation (p < 0.05) compared to the other groups. In the in vivo study, the plain collagen sponges failed, resulting in necrosis. Conversely, MTA and the ethanolic extract exhibited the best outcomes, inducing the lowest inflammatory response (mainly score 1 and 2) and the successful formation of an irregular dentin bridge by odontoblast-like cells, whereas the aqueous extract and bioceramic putty showed intermediate results, with higher inflammation levels. Conclusions: Ethanolic Moringa oleifera extract is a highly biocompatible material with potent odontogenic induction capabilities, primarily driven by its flavonoid content. It is a promising, novel, and cost-effective material for use in the VPT as an effective alternative to MTA. Full article

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease characterized by progressive cartilage destruction, and chondrocyte apoptosis plays a critical role in TMJOA progression. As chondrocytes reside in an avascular microenvironment inside the cartilage matrix, energy production via glycolysis is crucial for their survival. This study investigated the role of the key glycolytic enzyme Hexokinase 2 (HK2) in TMJOA pathogenesis and the therapeutic potential of dental pulp stem cell-derived extracellular vesicles (DPSC-EVs). In a rat experimental TMJOA model induced by monosodium iodoacetate (MIA) intra-articular injection, we observed a significantly decreased expression of HK2 along with cartilage matrix degradation. In the in vitro study, MIA induced chondrocyte apoptosis with caspase-3 activation, accompanied by impaired glycolytic function. Intervention with DPSC-EVs effectively rescued the expression of HK2 within chondrocytes, leading to a notable restoration of cellular glycolysis. Consequently, DPSC-EV treatment markedly attenuated the progression of TMJOA by reducing chondrocyte apoptosis and improved cartilage integrity. Our findings demonstrated that DPSC-EVs represent a promising cell-free therapeutic strategy for TMJOA, exerting their protective effects by targeting HK2, thereby preserving chondrocyte viability and attenuating osteoarthritis development. Full article

Objectives: pH-responsive zeolite imidazolate framework-8 (ZIF-8) enables selective release of 5-fluorouracil (5-FU) within the acidic tumor microenvironment. However, the direct effects of ZIF-8 itself on cancer cells or surrounding tissues remain unclear. Since oral cancer involves interactions between epithelial tumor cells and stromal cells, comparing the effects of ZIF-8 on epithelial cancer cells and orofacial mesenchymal stem/stromal cells (OMSCs) is critical to understanding its broader biological impact. Methods: The effects of ZIF-8 on SCC7 epithelial cancer cells and OMSCs, including periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs), were evaluated using RNA sequencing, nuclear staining, live/dead assays, and immunocytochemistry. Cells were treated with 0, 1, 10, or 100 μg/mL ZIF-8. Based on nuclear staining results, live/dead viability assays were conducted on SCC7 and DPSCs treated with 0 or 100 μg/mL ZIF-8. Apoptosis-related markers (Bax, caspase-3, caspase-6, and caspase-10) were assessed following exposure to 100 μg/mL ZIF-8. Results: Transcriptomic analysis revealed that ZIF-8 not only facilitates selective 5-FU release but also directly induces apoptosis in SCC7 cells compared with 5-FU alone. At 100 μg/mL ZIF-8, SCC7 viability was significantly reduced, whereas OMSC viability was preserved. Nonviable SCC7 cells increased markedly compared with controls, while DPSCs showed no significant change. Apoptosis-related signaling was also elevated in SCC7 cells compared with DPSCs. Conclusions: ZIF-8 at 100 μg/mL selectively inhibits SCC7 growth while sparing OMSC viability and apoptosis. Full article

The immune microenvironment critically influences bone healing, particularly in the oral cavity where inflammation and microbial biofilms can compromise regeneration. Plant-derived extracellular vesicles (PDEVs) offer a biocompatible means to modulate immune responses, and apple-derived extracellular vesicles (ADEVs) have shown antioxidant and anti-inflammatory activity, although their osteoregenerative potential remains unclear. Here, we investigate the indirect effects of ADEVs on bone regeneration by assessing how their immunomodulatory action on macrophages influences the osteogenic commitment of human dental pulp stem cells (DPSCs). ADEVs were isolated, characterized, and applied to THP-1-derived macrophages to evaluate polarization via morphology and immunofluorescence for M1 (iNOS) and M2 (ARG1) markers. Then, the extracellular vesicles (EVs) from untreated and ADEV-treated macrophages were isolated and applied to DPSCs. All EVs were efficiently internalized by both macrophages and DPSCs. Treated macrophages shifted toward an M2-like phenotype, and macrophage-derived EVs (MDEVs) promoted stem cell morphological features consistent with osteogenic activation. These findings suggest that ADEVs promote osteoregeneration indirectly by influencing macrophage polarization and modifying the osteoactive cargo of MDEVs, thereby supporting their potential in cell-free, immunomodulatory approaches for oral bone regeneration. Full article

Background: Advanced therapy medicinal products (ATMPs) require strict control of critical process parameters (CPPs) to ensure manufacturing efficiency. The relative impact of donor systemic factors, such as vitamin D status, versus technical process parameters on dental pulp-derived stem cell (DPSC) production remains unclear. Methods: In this prospective observational study, 250 adults undergoing extraction of impacted mandibular third molars were included. Dental pulp was processed under a standardized SOP using different preparation methods and enzyme conditions. Primary endpoints were serum 25(OH)D concentration and cell yield; secondary endpoints included number of passages and cryovials. Results: Mean 25(OH)D concentration was 30.1 ± 14.5 ng/mL and was higher in supplemented individuals (38.2 ± 14.0 vs. 25.6 ± 12.7 ng/mL; p < 0.0001) but was not associated with cell yield (ρ = 0.14, p = 0.168) or passages (ρ = 0.07, p = 0.406). In contrast, process parameters showed strong effects: scissor preparation resulted in a substantially higher yield than mechanical methods (median 5.00 vs. 1.00 million cells; p = 3.6 × 10⁻¹³), and type II collagenase was independently associated with a higher yield (+2.04 million cells; p = 0.026). The number of passages was the strongest predictor of yield (β = 2.28 million per passage; p < 10⁻²⁶). Post-thaw viability remained high (mean 90.1% and range 81–98%). Conclusions: Manufacturing efficiency of DPSCs is primarily determined by critical process parameters, particularly preparation method, enzyme selection, and passage control, whereas donor vitamin D status did not significantly influence outcomes under the studied SOP. These findings highlight process standardization as the key driver of reproducible ATMP manufacturing. Full article

Pulp necrosis remains a significant clinical challenge in dentistry, as current therapeutic approaches fail to achieve functional pulp regeneration. Extracellular vesicles (EVs), as crucial mediators of intercellular communication, offer new opportunities for regenerative strategies. In this study, we focus on CD24⁺ human dental papilla cells (CD24⁺ hDPCs), a functionally defined subpopulation previously characterized as having superior regenerative potential, and evaluate the regenerative potential of their derived EVs (CD24⁺ EVs) in pulp-like tissue regeneration. CD24⁺ EVs significantly enhanced the proliferation, migration, and osteo/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and markedly promoted endothelial tube formation in vitro. In a treated dentin matrix (TDM)-based ectopic regeneration model, CD24⁺ EVs increased cellular accumulation within the regenerated tissue and robust angiogenesis, inducing the formation of well-organized, highly vascularized pulp-like tissue with dense cellular architecture and positive DSPP expression. Together, these findings suggest that CD24⁺ EVs concurrently enhance cell migration, odontogenic differentiation, and angiogenesis, and support a promising cell-assisted EV strategy grounded in functionally defined cellular subpopulations for pulp-like tissue regeneration. Full article

Autogenous grafts remain the gold standard for repairing extensive maxillofacial bone defects, but their associated morbidity motivates the search for alternative strategies in tissue bioengineering. Deciduous dental pulp stem cells (DDPSCs) represent a promising cell source due to their accessibility, multipotency, and osteogenic potential, while nanostructured carbonated hydroxyapatite (cHA) microspheres exhibit biochemical similarity to bone mineral and favorable bioabsorption. This study investigated the osteogenic response induced by the association of DDPSCs with cHA in a rat calvaria critical-size defect model. DDPSCs were expanded, seeded onto cHA microspheres, and characterized in vitro prior to bilateral implantation in 12 Wistar rats, with each animal receiving cHA + DDPSC on the right defect and acellular cHA on the left. After 60 and 90 days, histological and histomorphometric analyses revealed new bone formation in both groups, predominantly from the defect margins toward the center. At 60 days, no significant difference in newly formed bone was observed between groups (p = 0.249). At 90 days, the DDPSC + cHA group demonstrated significantly greater bone formation compared with acellular cHA (median 40.70 vs. 11.10 histomorphometric points; p = 0.028) and significant reduction in connective tissue (p = 0.028). Complete scaffold resorption was observed in all DDPSC-treated defects at 90 days, whereas residual biomaterial persisted in the cHA group (p = 0.015), indicating progressive cHA resorption over time. These findings suggest that combining DDPSCs with cHA enhances bone regeneration and that this synthetic, bioabsorbable scaffold represents a promising strategy for future applications in bone tissue engineering. Full article

Background/Objectives: This study aimed to compare the biological effects of two amelogenin-derived peptides—the leucine-rich amelogenin peptide (LRAP) and a synthetic peptide (SP)—on human dental pulp stem cells (hDPSCs) and human bone marrow–derived mesenchymal stem cells (hBMSCs). The investigation focused on cell viability, osteogenic differentiation, mineralization, gene expression, and β-catenin expression. Methods: hDPSCs and hBMSCs were cultured in osteogenic medium and treated with LRAP and SP at 1, 5, 10, 50, and 100 ng/mL. Cytotoxicity was assessed by MTT assay, while osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene expression of RUNX2, COL1A1, OCN, MEPE, and DMP1 was quantified by qPCR. β-catenin localization was analyzed by immunofluorescence. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (p < 0.05). Results: Both peptides exhibited good biocompatibility with hBMSCs, while high concentrations (≥50 ng/mL) reduced hDPSC viability. In both cell types, LRAP and SP increased ALP activity and mineral deposition in a concentration-dependent manner, with the greatest effects at 10 ng/mL. LRAP significantly upregulated osteogenic (RUNX2, COL1A1, OCN) and odontogenic (MEPE, DMP1) gene expression in hDPSCs. Immunofluorescence revealed nuclear β-catenin translocation in hDPSCs and membrane-associated accumulation in hBMSCs, indicating activation of canonical and non-canonical pathways, respectively. Conclusions: LRAP and SP promote osteogenic differentiation through distinct cell-type-specific signaling mechanisms, highlighting their potential as biomimetic agents for mineralized tissue regeneration. Full article

The toxicity of micro- and nanoplastics in aquatic life is well documented, yet limited information is available on their effects in humans; moreover, most in vitro nanotoxicology studies rely on cancer cells. This study examined the effects of pristine and aged polystyrene micro- and nanoparticles on human dental pulp stem cells. While both particle sizes were internalized by the cells, primarily through endocytosis, they did not affect cell viability. In contrast, leachates from particles, aged for one month in culture medium, significantly reduced viability, indicating that toxicity arises from degradation byproducts rather than the particles themselves. Atomic force microscopy confirmed surface changes in aged plastics. Both particle sizes disorganized the cytoskeleton, leading to reduced actomyosin cortex integrity. Gene expression analysis revealed that leachates and aged particles activated inflammatory pathways, markedly increasing IL-8 and TGF-β1 expression, while also decreasing SOD levels associated with oxidative stress. No notable effects were observed on genes related to stemness or senescence. These results suggest that, while pristine micro- and nanoplastics may be relatively inert, their degradation products pose greater toxicological risks to human health. The findings highlight the importance of considering leachate toxicity in plastic pollution studies and demonstrate the value of stem cell-based models for evaluating the cellular and molecular impacts of environmental contaminants on human health. Full article

Metformin is a promising small molecule for dentin regeneration, but an effective local delivery system for pulp applications has been underexplored. This study encapsulated metformin in poly(lactic-co-glycolic acid) (PLGA) microspheres and incorporated them into calcium phosphate–chitosan cement (CPCC) as a direct pulp-capping material (DPC). Metformin-PLGA microspheres were prepared by double emulsion and mixed with CPCC at a concentration of 0% to 20% by weight. Microsphere morphology, encapsulation efficiency, chemical composition, and physico-mechanical properties were characterized, and compatibility with human dental pulp stem cells (hDPSCs) was evaluated by live/dead assay and SEM. The microspheres were spherical (5.43 ± 0.17 µm) with (51 ± 3.69%) encapsulation efficiency, and FTIR confirmed metformin incorporation. The 15% Met-PLGA-CPCC group showed flexural strength (15.22 ± 1.98 MPa), elastic modulus (4.60 ± 0.73 GPa), and work of fracture (104.96 ± 12.48 J/m²) comparable to or higher than CPCC and MTA, while all Met-PLGA-CPCC groups had shorter setting times ranging from 18 min to 27 min than CPCC (39.15 ± 2.10 min) and MTA (123 ± 4.2 min). Metformin release increased proportionally with Met-PLGA content. hDPSCs exhibited good attachment and high viability on all materials over the evaluated period. In conclusion, Met-PLGA-CPCC provides fast-setting and favorable physico-mechanical properties, sustained metformin delivery, and excellent hDPSC compatibility. These properties support its potential as a bioactive direct pulp-capping material and as a versatile platform for regenerative applications. Full article

Hydraulic calcium silicate cements (HCSCs) are contemporary materials in vital pulp therapy (VPT) and regenerative endodontic therapy (RET) due to their favorable effects on pulpal and periodontal cells, including cell differentiation and hard tissue formation. Recent studies also indicated the involvement of several S100A proteins in inflammatory, differentiation, and mineralization processes of the pulp. The aim of the present study was to investigate the effects of HCSCs on S100A gene expression in human dental pulp stem cells (hDPSCs). Human DPSCs were isolated and characterized by multi-lineage stem-cell markers and differentiation protocols. In stimulation experiments hDPSCs were exposed to ProRoot^®MTA, Biodentine^®, IL-1β, and dexamethasone. Cell viability was determined by XTT assay. IL-6 and IL-8 mRNA expression was measured to analyze proinflammatory response. In addition, odontogenic differentiation and biomineralization assays were conducted (DSPP- and ALP-mRNA expression, ALP activity, and Alizarin Red staining). Differential expression of 13 S100A genes was examined using qPCR. Low concentrations of HCSCs enhanced the proliferation of hDPSCs, whereas higher concentrations exhibited cytotoxic effects. HCSCs induced a pro-inflammatory response and led to odontogenic differentiation and biomineralization. This was accompanied by significant alterations in the expression levels of various S100A genes. ProRoot^®MTA and Biodentine^® significantly affect the expression of several S100A genes in hDPSCs, supporting their role in inflammation, differentiation, and mineralization. These findings indicate a link between the effects of HCSCs on human pulp cells during VPT or RET and S100A proteins. Full article