Search Results (228)

Orthodontic tooth movement (OTM) is driven by force-induced alveolar bone remodeling, yet the molecular mechanisms by which periodontal ligament stem cells (PDLSCs) sense and transduce mechanical signals remain incompletely understood. Here, we identify the epigenetic regulator HELLS as a compressive force-responsive gene and investigate its role as a mechanosensitive mediator in human PDLSCs (hPDLSCs). Compressive force downregulated HELLS expression both in vitro and in a mouse OTM model. Functionally, siRNA-mediated HELLS knockdown impaired osteogenic differentiation, as evidenced by reduced Alizarin Red S staining and alkaline phosphatase activity, and induced global transcriptomic changes indicative of altered mechanotransduction pathways. Moreover, HELLS knockdown increased YAP and RANKL expression and potentiated osteoclast differentiation of co-cultured RAW264.7 cells. Finally, we identified E2F1 as a candidate transcription factor mediating the force-induced downregulation of HELLS. Collectively, these findings establish HELLS as a potential mechano-epigenetic regulator in hPDLSCs, and suggest that its force-induced downregulation may contribute to alveolar bone remodeling during OTM by simultaneously attenuating osteogenesis and enhancing pro-osteoclastogenic signaling via transcriptional reprogramming. Full article

Vascular calcification is an actively regulated process driven by vascular smooth muscle cell (VSMC) osteogenic reprogramming and promoted by oxidative stress and extracellular matrix remodeling. We investigated whether the novel histone deacetylase inhibitor YAK577 mitigates calcification by modulating an MMP14–NOX2/ROS-associated pathway in calcification medium (CM)-treated VSMCs and a vitamin D₃-induced arterial calcification model in 8-week-old male C57BL/6N mice. Calcification was assessed by Alizarin Red S/von Kossa staining and calcium quantification; osteogenic markers (BMP2, RUNX2, MSX2) and MMPs were examined by qRT-PCR and immunoblotting; intracellular ROS was measured by DHE staining with N-acetylcysteine as an antioxidant control; and MMP14 was manipulated by siRNA knockdown or plasmid overexpression. YAK577 was non-cytotoxic at effective concentrations and reduced CM-induced calcium deposition and osteogenic marker expression. YAK577 reduced MMP14 expression and suppressed CM-induced NOX2/p47phox activation and ROS accumulation, while GSK2795039 attenuated CM-induced DHE fluorescence. MMP14 silencing attenuated, whereas MMP14 overexpression enhanced, osteogenic signaling and increased NOX2. In vivo, YAK577 reduced vitamin D₃-induced aortic calcium burden, histological calcification, and the expression of MMP14, NOX2, and osteogenic markers. These data support a working model in which YAK577 alleviates vascular calcification, at least in part, by suppressing an MMP14-associated NOX2/p47phox–ROS axis. 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

Bone regeneration requires tight coordination between mesenchymal stem cells (MSCs), immune signaling, and extracellular matrix remodeling. Yet, how atypical immune receptors contribute to this process remains unclear. Here, we identify Toll-like receptor 10 (TLR10) as a key regulator of osteogenic differentiation in human adipose-derived MSCs. Herein, ASC/TERT1 MSCs were engineered to overexpress or silence TLR10 using lentiviral vectors, and osteogenic differentiation (0–14 days) was assessed by metabolic assays—RT-qPCR of COL1A2, ALPL and BGLAP—Alizarin Red S staining, and quantitative mass spectrometry. Enhancing TLR10 expression promoted osteogenic gene programs, extracellular matrix organization, metabolic adaptation, and robust matrix mineralization, whereas TLR10 suppression maintained proliferative states and impaired osteoblast maturation. Proteomic analyses revealed that TLR10 selectively activates osteogenic, ECM-remodeling, and vitamin D-responsive pathways, while restraining programs antagonistic to differentiation. Notably, active vitamin D induced TLR10 expression and partially restored osteogenesis in TLR10-deficient cells, indicating that TLR10 is associated with vitamin D-driven bone formation. Together, beyond its established role in innate immunity, TLR10 emerges as a vitamin D-responsive regulator of mesenchymal stem cell osteogenesis, highlighting a potential therapeutic axis to enhance bone regeneration and osteogenic outcomes. Full article

Bone defects have become a leading cause of disability and mortality. The pro-inflammatory state and impaired vascularization are major factors hindering bone defect repair. However, current bone regeneration materials lack the ability to regulate the osteoimmune microenvironment and promote vascularized bone regeneration. In this study, we employed clinically used fat emulsion (FE), which is intravenously administered to provide nutrition and energy for patients, to investigate the effect of immunomodulation on promoting angiogenesis and osteogenesis. Results from RT-qPCR analysis and immunofluorescence staining demonstrated that FE exhibited potent anti-inflammatory effects by reducing the expression of the pro-inflammatory marker inducible nitric oxide synthase (iNOS) and upregulating the expression of the anti-inflammatory marker transforming growth factor-beta (TGF-β). Endothelial tube formation and scratch assays demonstrated that FE promoted angiogenesis and cell migration by releasing vascular endothelial growth factor (VEGF) within the inflammatory microenvironment. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining revealed that FE facilitated ALP activity and calcium nodule formation by releasing bone morphogenetic protein-2 (BMP-2) within the inflammatory microenvironment. These findings may prove promising and cost-effective for the clinical treatment of bone defects. Full article

In this study, a novel material was obtained by functionalizing shredded maize stalk (MS) with Alizarine Red S (ArS), a complexing agent that contains −OH and −C=O groups in its structure (MS-ArS). The obtained MS-ArS was employed in adsorption studies for Mn²⁺, Pb²⁺, Cu²⁺, Cr³⁺, Zn²⁺, and Fe³⁺ (Mⁿ⁺) removal from mixed aqueous matrices. Initially, complex formation between (Mⁿ⁺) and ArS in buffer solution at pH 4 and 10 was investigated using the UV-Vis spectrometric method. Continuous, the functionalization process of MS with ArS was tested at several pH values (2, 4, 6, 8, and 10) using a batch technique. It was observed that the best functionalization of MS with ArS was obtained at pH = 2. Subsequently, Mⁿ⁺ adsorption onto the MS-ArS mass was tested separately at pH 4 and 10. The study achieved that Mⁿ⁺ adsorption proved to be pH dependent. The results confirmed that at pH = 10, Mⁿ⁺ adsorption was increased, compared with pH = 4. MS-ArS has affinity for Mⁿ⁺ in the following order Fe³⁺ > Cu²⁺ > Zn²⁺ > Mn²⁺ > Pb²⁺ > Cr³⁺. Experimental data revealed remarkable desorption rates when 0.5 M HCl was used. After five adsorption/desorption cycles of Mⁿ⁺, the removal capability of MS-ArS was preserved. Overall, the potential of MS-ArS for effective Mⁿ⁺ removal/reuse makes it a sustainable polymer for wastewater treatment applications. Full article

Background/Objectives: Endodontic sealers can interact with periapical tissues through extrusion, yet the molecular mechanisms underlying their biological effects remain poorly defined. This study investigated how commonly used sealers influence mitogen-activated protein kinase (MAPK) signaling, cell viability, and osteogenic-associated responses in MC3T3-E1 pre-osteoblasts. Methods: Four commercial sealers, Calcium-silicate-based Bioceramic Sealer (EndoSequence^® BC Sealer, BC), Zinc oxide eugenol sealer (Kerr Pulp Canal Sealer, ZOE), Sealapex™, and AH26^®, were applied as standardized pellets, allowed to set, and cultured with MC3T3-E1 cells. Calcium deposition was assessed by Alizarin Red S (ARS) staining, and MAPK activation was evaluated by Western blotting. Due to excessive solubility (Sealapex™) or poor cell survival (AH26^®), mechanistic analyses were performed only for BC and ZOE. Osteogenic-associated gene expression was measured by qRT-PCR, and the functional role of MAPK signaling was assessed using ERK, JNK, and p38 inhibitors. Results: BC and Sealapex™ produced robust ARS staining, while ZOE and AH26^® produced minimal mineral-associated staining. Both BC and ZOE activated ERK, JNK, and p38, with ZOE inducing higher phosphorylation. However, BC maintained greater cell viability and increased Runx2 and Osx expression, whereas ZOE impaired early cell attachment and viability. MAPK inhibition in BC-treated cultures reduced osteogenic-associated gene expression and ARS staining, indicating MAPK involvement in BC-mediated responses. Conclusions: BC and ZOE elicit distinct MAPK activation patterns and cellular responses. Under the conditions tested, BC promoted a more favorable osteogenic-associated response, whereas ZOE compromised early cell viability. These mechanistic insights may help explain clinical differences in periapical tissue responses to sealer extrusion. Full article

Developing novel anabolic agents for bone regeneration remains a clinical priority. Polydeoxyribonucleotide (PDRN) exhibits tissue-regenerative properties, but its direct cellular effects on bone remodeling remain unclear. This in vitro study investigated PDRN’s effects on osteoblast (MC3T3-E1) and osteoclast (primary bone marrow-derived macrophages) differentiation. We evaluated metabolic activity, gene/protein expression, and specific differentiation markers using MTS, qRT-PCR, Western blotting, and functional assays (ALP, Alizarin Red S, TRAP, pit formation). In osteoblasts, PDRN dose-dependently modulated metabolic activity while upregulating the early transcription factor Runx2. PDRN significantly enhanced osteoblast differentiation, evidenced by increased ALP activity, elevated mineralized matrix deposition, and robust upregulation of osteocalcin and Runx2. Conversely, PDRN exhibited no direct effect on osteoclast precursor metabolic activity, differentiation, or resorptive function. These findings support a working hypothesis in which PDRN selectively promotes osteoblast differentiation without directly affecting osteoclastogenesis. While further pharmacological investigations are required to definitively elucidate the specific purinergic receptor mechanisms, our results highlight PDRN as a promising candidate anabolic agent for bone regeneration. Full article

Background: Bone remodeling depends on the dynamic balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Follistatin-like 1 (FSTL1) has been reported as an osteoclast-secreted protein that inhibits osteoclast differentiation, but its direct effects on osteoblast differentiation remain unclear. This study aimed to determine whether FSTL1 regulates osteoblast differentiation and mesenchymal stem cell migration and characterizes its role in osteoclast-osteoblast cellular cross-talk under in vitro conditions. Methods: Bone marrow-derived macrophages (BMMs) and stromal cells (BMSCs) from mice were used to induce osteoclast and osteoblast differentiation, respectively. Chemotaxis was assessed by Transwell migration, and osteoblast differentiation was evaluated in BMSC and MC3T3-E1 cells using staining, qRT-PCR, Western blotting, and proliferation assays. Results: FSTL1 significantly suppressed osteoclast differentiation and resorptive activity, confirmed by TRAP staining and pit assay, respectively. Expression of osteoclast markers such as NFATc1, TRAP, and DC-STAMP was reduced under FSTL1 treatment. In BMSCs, FSTL1 did not affect proliferation but significantly enhanced chemotaxis. Moreover, FSTL1 promoted osteogenic differentiation and mineralization, as demonstrated by increased ALP activity and Alizarin Red S staining. In MC3T3-E1 pre-osteoblasts, FSTL1 increased cell proliferation and mineralization by MTS and Alizarin Red staining. Key osteogenic markers, including Runx2 and osteocalcin, were also upregulated. Conclusions: Osteoclast-derived FSTL1 significantly suppresses osteoclastogenesis and promotes mesenchymal cell chemotaxis and osteogenic differentiation, indicating a role in regulating osteoclast–osteoblast cellular interactions in vitro. Targeting FSTL1 signaling may represent a promising therapeutic strategy for osteoporosis and other disorders of impaired bone remodeling. Full article

Background/Objectives: Cells derived from direct chemical reprogramming into osteoblasts represent a promising source for bone regeneration, but the efficiency needs improvement. Here, we systematically evaluated whether the natural compound psoralen (Psr) could enhance this process and explored its therapeutic potential and mechanism of action. Methods: Mouse embryonic fibroblasts (MEFs) were treated with a cocktail of forskolin and phenamil (FP), supplemented with Psr. In vitro differentiation was assessed by alkaline phosphatase and Alizarin Red S staining, reverse transcription quantitative PCR, immunofluorescence and Western blot. The bone-regenerative potential of the derived chemically induced osteoblast-like cells (ciOBs) was evaluated in critical-sized calvarial defects, femoral cortical defects and a subcutaneous ectopic implantation model, using micro-computed tomography and histology. Mechanistic insights of Psr were gained by analyzing the adenylyl cyclase 9 (ADCY9)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP response element-binding protein (CREB) axis using inhibitor SQ22536. Results: Psr acted synergistically with the FP cocktail to drive efficient osteogenic reprogramming of MEFs. At an optimal concentration of 25 μM, Psr enabled the most robust induction of early osteogenic markers and generation of mature, mineralizing ciOBs in vitro. In vivo, FP + Psr-induced ciOBs repaired critical-sized calvarial and femoral cortical defects and generated substantial, vascularized bone tissue in ectopic sites. Mechanistically, Psr co-treatment potently activated the ADCY9/cAMP/PKA/CREB pathway, and pharmacological inhibition of this pathway completely abolished the pro-osteogenic effects of Psr. Conclusions: Psr acts as a potent synergistic enhancer of direct chemical reprogramming, generating functional osteoblast-like cells with robust bone-regenerative capacity via activation of the ADCY9/cAMP/PKA/CREB pathway. 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

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

Corilagin is a hydrolyzable ellagitannin and naturally occurring polyphenolic compound widely distributed in medicinal plants. It is also present in longan (Dimocarpus longan), known as lumyai in Thailand, a subtropical fruit extensively cultivated across China and Southeast Asia. Corilagin has been reported to exhibit strong antioxidant, anti-inflammatory, hepatoprotective, and anticancer activities through modulation of multiple cellular signaling pathways. However, despite these well-established pharmacological properties, its potential role in regulating bone marrow mesenchymal stem cell (BM-MSC) differentiation has not been fully explored in biomedical applications. In this study, we investigated the effects of corilagin on BM-MSC viability, protein-binding interactions, and lineage-specific differentiation toward osteogenic and chondrogenic pathways. Cytotoxicity assessment using human synovial SW-982 cells demonstrated that corilagin maintained cell viability at concentrations ranging from 1.56 to 50 µg/mL within 48 h, whereas prolonged exposure resulted in a time-dependent reduction in viability. In BM-MSCs, corilagin significantly enhanced osteogenic and chondrogenic differentiation in a dose-dependent manner, as evidenced by increased mineral deposition and cartilage matrix formation, as revealed by Alizarin Red S, Toluidine Blue, and Alcian Blue staining. Quantitative analyses further showed the upregulation of key lineage-specific genes, including Runx2 and osteopontin (OPN) for osteogenesis and Sox9 and aggrecan for chondrogenesis. Protein-binding assays confirmed the molecular interaction capacity of corilagin, supporting its biological activity. Overall, these findings demonstrate that corilagin promotes MSC-mediated osteogenic and chondrogenic differentiation while maintaining acceptable cytocompatibility, highlighting its potential as a natural small-molecule candidate for bone and cartilage tissue engineering and other biomedical fields with regenerative medicine applications. Full article

This study aimed to develop a sustainable route for processing biogenic calcium carbonate from Perna perna mussel shell waste and converting it into hydroxyapatite (HA), as well as to evaluate its potential for bone and dental tissue engineering applications. Mussel shells were decarbonized (400 °C), milled, and converted to HA via wet chemical precipitation using a nominal Ca/P molar ratio of 1.67 during synthesis followed by thermal treatment (900 °C). Comprehensive characterization included SEM, FTIR, XRD, Raman spectroscopy, XRF, TGA, and BET analysis. Biological evaluation involved cytotoxicity assays (MTT), antimicrobial testing, and odontogenic differentiation studies (Alizarin Red) using SHEDs. Statistical analysis by one-way ANOVA and Tukey post hoc tests (α = 0.05). SEM revealed a microstructured morphology composed of agglomerates, favorable for biomedical applications. FTIR and XRD confirmed the conversion of CaCO₃ to hydroxyapatite, while thermal analysis demonstrated the material’s stability. The HA exhibited secondary minor phase (13%) β-TCP form of calcium phosphate (Ca_2.997H_0.006(PO₄)₂), high crystallinity (about 80%), and nanoscale crystallite size (85 nm, 2.5–5.0 m²/g), despite forming larger agglomerates in suspension. The material showed favorable physicochemical properties (neutral pH, −18.5 mV zeta potential), but no inhibition was detected in antimicrobial testing. In vitro assays showed excellent cytocompatibility (viability > 70% at 12.5 µg/mL) and significant osteogenic potential (high mineralization vs. controls, p < 0.05). Mussel shell-derived HA presents a sustainable, clinically relevant biomaterial with ideal properties for bone regeneration. The study establishes a complete waste-to-biomaterial pipeline while addressing key requirements for dental and orthopedic applications. Full article

Background: Bone fracture is a partial or complete break in the continuity of a bone, which poses a significant healthcare burden. It is important to discover a novel method to stimulate and speed-up the healing of bone fractures. Aim: This study aimed to investigate the effects and mechanisms of alternating current (AC) in promoting bone fracture healing. Methods: A rabbit bone fracture model was used. X-ray and Micro-CT evaluated fracture healing, while HE staining and immunohistochemistry assessed morphological changes. In vitro, pre-osteoblastic cells were tested with alizarin red S staining and alkaline phosphatase (ALP) activity. RNA-seq analysis explored potential mechanisms. Results: X-ray evaluation showed that alternating current stimulation (ACS) promoted bone formation and shaping by day 14 post-treatment. Micro-CT results revealed significant new bone formation as early as day 3 and day 7 (p < 0.05). HE staining indicated more trabecular bone formation in the ACS group compared to the model group at days 7 and 14. Immunohistochemistry showed higher expression of BMP-2 and VEGF in the ACS group by day 7. In vitro, ACS enhanced osteogenic differentiation, increasing calcified nodule formation and ALP activity. Gene expression analysis demonstrated significant changes in key osteogenic genes, confirmed by multiple immunohistochemical staining. Conclusions: ACS may be a novel method for treating bone fractures more rapidly, significantly relieving the patient’s burden, particularly in the early stages of bone healing. Full article