Search Results (83)

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

Magnolia kobus (M. kobus) has long been used to treat nasal congestion, allergic rhinitis, and sinusitis. In the current study, we demonstrate the effects and underlying mechanisms of M. kobus flower water extract (ME) and ME-derived constituent magnolin on in vitro osteoblastogenic and anti-osteoclastogenic responses. Treatment with ME or magnolin markedly enhanced the osteoblast differentiation and mineralization in MC3T3-E1 pre-osteoblasts. This osteoblastogenic activity of ME or magnolin was closely associated with upregulation of osteoblast-specific molecules, including RUNX2, DLX5, OSX, alkaline phosphatase, collagen type I, and osteopontin, as well as the activation of mitogen-activated protein kinase (MAPK) signaling pathways. Concurrently, magnolin inhibited osteoclast differentiation through inactivating MAPK pathways and downregulating NFATc1, c-Fos, tartrate-resistant acid phosphatase, and cathepsin K in RANKL-treated RAW264.7 cells. These observations suggest that ME and magnolin have pharmacological potential for the treatment and prevention of metabolic bone disorders, including osteoporosis. Full article

Mechanical stimulation critically regulates mesenchymal stem cell (MSC) differentiation, yet its effects in three-dimensional (3D) environments remain poorly defined. Here, we developed a custom dynamic stretcher integrating poly(dimethylsiloxane) (PDMS) chambers to apply cyclic strain to human MSCs encapsulated in Fmoc-diphenylalanine (Fmoc-FF) peptide hydrogels—a fully synthetic, tunable extracellular matrix mimic. Finite element modeling verified uniform strain transmission across the hydrogel. Dynamic stretching at 0.5 Hz and 10% strain induced pronounced cytoskeletal alignment, enhanced actin stress fiber formation (coherency index $\approx 0.85$), and significantly increased proliferation compared to static or high-frequency (2.5 Hz, 1%) conditions (coherency index $\approx 0.6$). Quantitative image analysis confirmed strain-dependent increases in coherency index and F-actin intensity, indicating enhanced mechanotransductive remodeling. Biochemical assays and qRT–PCR revealed 2–3-fold upregulation of osteogenic markers—RUNX2, ALP, COL1A1, OSX, BMP, ON, and IBSP—under optimal strain. These results demonstrate that low-frequency, high-strain mechanical loading in 3D peptide hydrogels activates RhoA/ROCK and YAP/TAZ pathways, driving osteogenic differentiation. The integrated experimental–computational approach provides a robust platform for studying mechanobiological regulation and advancing mechanically tunable biomaterials for bone tissue engineering. Full article

Two-dimensional cell cultures are crucial research tools, and they have been widely used, although they are not completely representative of biological processes in vivo due to the lack of tissue architecture and complexity. Recent advances in organoid technology have addressed these limitations and are revolutionizing the tools available for in vitro culture. Although there are no unified protocols for generating organoids, they can be obtained with various techniques, leading to cell aggregation by promoting cell adhesion. This work aims to generate and characterise organoid models of dental pulp from dental pulp stem cells (DPSCs), a type of mesenchymal stem/stromal cells known for their high regenerative potential and ease of accessibility, to establish a model for translational studies. The organoids were subjected to osteogenic differentiation conditions. Cell viability was evaluated using a CCK-8 assay, while osteogenic morphology and mineralization were confirmed by Alizarin red analysis, Raman microspectroscopy, and by immunofluorescence for the lineage markers expression. The Alizarin red analysis indicated a higher presence of calcium phosphate deposits in the differentiated organoids than in the control group (CTR). These results were confirmed by spectral profiles obtained using Raman microspectroscopy, which were attributable to a hydroxyapatite-based biomaterial. Immunofluorescence analysis also revealed increased expression of odonto/osteogenic markers (RUNX and OSX), alongside reduced expression of stemness markers. In conclusion, the organoids appeared to have successfully differentiated into an osteogenic lineage, forming a mineralized matrix containing hydroxyapatite and showing increased expression of relevant lineage markers. Full article

Scaffold architecture is a key determinant of cell behavior and tissue regeneration in bone tissue engineering, yet the influence of pore size under dynamic culture conditions remains incompletely understood. This study aimed to evaluate the effects of scaffold pore size on osteogenic differentiation of porcine bone marrow-derived mesenchymal stem cells (pBMSCs) cultured in a rotational oxygen-permeable bioreactor system (ROBS). Three-dimensionally (3D) printed beta-tricalcium phosphate (β-TCP) scaffolds with pore sizes of 500 µm and 1000 µm were seeded with pBMSC and cultured for 7 and 14 days under dynamic perfusion conditions. Gene expression analysis revealed significantly higher levels of osteogenic markers (Runx2, BMP-2, ALP, Osx, Col1A1) in the 1000 µm group, particularly at the early time point, with the later-stage marker Osteocalcin (Ocl) rising faster and higher in the 1000 µm group, after a lower expression at 7 days. ALP activity assays corroborated these findings. Despite having lower mechanical strength, the 1000 µm scaffolds supported a homogeneous cell distribution and high viability across all regions. These results suggest that larger pore sizes enhance early osteogenic commitment by improving nutrient transport and fluid flow in dynamic culture. These findings also support the use of larger-pore scaffolds in bioreactor-based preconditioning strategies and underscore the clinical importance of promoting early osteogenic differentiation to reduce in vitro culture time, an essential consideration for the timely preparation of implantable grafts in bone tissue engineering. Full article

β-catenin is a key regulator of osteoblast differentiation, proliferation, and bone homeostasis. Through its interaction with transcription factors such as TCF/LEF, Runx2, and Osx, it coordinates gene expression essential for osteogenesis. The aim of this review is to demonstrate how β-catenin signaling is modulated by various physiological and pathological factors, including mechanical loading, oxidative stress, HIV-1 gp120, fluoride, implant topography, and microRNAs. These factors influence Wnt/β-catenin signaling through different mechanisms, often exerting opposing effects on osteoblast function. By integrating these modulators, we provide a comprehensive view of the dynamic regulation of β-catenin in bone biology. Understanding this complexity may provide insight into novel therapeutic strategies targeting β-catenin in bone regeneration, metabolic bone diseases, and pathologies such as HIV-associated bone loss or osteosarcoma. Full article

The aim of this in vitro study is to investigate the adhesion, proliferation, and differentiation of human adipose-derived mesenchymal stem cells (hASC) on Trabecular Titanium scaffolds manufactured with different manufacturing processes (EBM and SLM). The in vitro adhesion and proliferation of hASC on titanium scaffolds with WST assays have been carried out. The comparison of the gene expression profiles of typical bone genes (Alp, Bglap, Col1a1, and Osx) through real-time PCR assays and the evaluation of extracellular matrix composition with immunofluorescence and SEM analysis have been performed. In addition, the possible osteoinductive properties of the two scaffolds have been investigated through real-time PCR and ALP assays. Data showed that Trabecular Titanium supports human adipose-derived mesenchymal stem cell colonization and induces differentiation in bone with the deposition of the abundant extracellular mineralized matrix regardless of the manufacturing process, proving that the micro- and macro-design features are the key factors responsible for the osteoinduction behavior. These features can only be achieved through tailored 3D printing process parameters. Full article

This study aimed to investigate the effects of shear stress on osteogenic differentiation of human dental pulp stem cells (hDPSCs). The hDPSCs were subjected to shear stress for 24 h before osteogenic induction for 21 days. The mRNA expression of osteogenic markers such as RUNX2, OSX, ALP, COL1A1, OCN, and OPN was evaluated by real-time RT-PCR. Alkaline Phosphatase (ALP) activity and Alizarin Red S (ARS) staining were investigated to confirm osteogenic differentiation and mineralization of hDPSCs, respectively. The protein expression of osterix was shown by immunofluorescence staining and Western blotting. RNA sequencing was performed to investigate how shear stress affects the osteogenic differentiation of hDPSCs, which was validated through p38 inhibitor (SB203580) treatment. Real-time RT-PCR revealed that shear stress enhanced osteogenic marker-gene expression. The increased osterix protein expression was detected on Day 14 in the shear-stress loading group compared to the static group. Shear stress enhanced ALP activity and mineralization, observed on Days 14 and 21. A volcano plot exhibited up- and downregulated genes, while the p38 inhibitor markedly inhibited osteogenic differentiation of hDPSCs triggered by shear stress. In conclusion, shear stress promotes the osteogenic differentiation of hDPSCs through the p38 mitogen-activated protein kinase signaling pathway. Full article

Objective: Secretory factors, termed the secretome, in the conditioned medium (CM) from dental mesenchymal stem cells (MSCs) have shown anti-inflammatory, anti-apoptotic, and tissue regenerative potential. This cell-free product could be further developed by preconditioning cells with various biochemical agents, which lead to a change in secretome and CM profiles. Among the favorable candidates for CM production, metformin as an anti-diabetic medication is currently considered a potential agent for dental hard tissue and periodontal regeneration. Here, we aimed to assess the composition of CM from periodontal ligament stem cells (PDLSCs) grown in metformin-preconditioned media (Met-CM) compared to normal PDLSC-CM and assess the ability of Met-CM to recover the function of inflamed PDLSCs. Methods: Met-CM and normal CM were collected from PDLSCs grown with or without 50 µM metformin, respectively, under healthy culture conditions. Mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were performed to comparatively evaluate the proteomic profiles in PDLSC-CM versus Met-CM. We then treated the PDLSC cultures with lipopolysaccharide (LPS) from Porphyromonas gingivalis to induce inflammation and evaluated the osteogenic/cementogenic differentiation in the presence of Met-CM or normal PDLSC-CM by assessing alkaline phosphatase activity, intracellular calcium levels, and mRNA expression of osteogenic and cementogenic factors, including RUNX2, OCN, OSX, and CEMP-1. Subsequently, we performed RNA sequencing to identify transcriptomic changes in the treated cells. Results: We identified 202 differentially expressed proteins, 175 of which were significant, in Met-CM versus normal PDLSC-CM. Among the analyzed groups, the top three protein classes were protein-binding activity modulator, cytoskeletal protein, and extracellular matrix (ECM) protein. Treatment of PDLSCs with LPS significantly attenuated ALP activity, [Ca²⁺]_i, and the mRNA expression levels of RUNX2, OCN, OSX, and CEMP-1, whereas treatment with Met-CM alone markedly enhanced PDLSC differentiation activity compared with the control. Moreover, osteogenic/cementogenic differentiation of the LPS-treated PDLSCs was recovered through incubation in Met-CM. Transcriptomic analysis identified 511 and 3591 differentially expressed genes in the control versus Met-CM and LPS versus LPS + Met-CM groups, respectively. The enrichment of biological processes includes positive regulation of DNA-templated transcription and skeletal system morphogenesis in the control versus Met-CM comparison, as well as positive regulation of transcription from the RNA polymerase II promoter and negative regulation of the apoptotic process in the LPS versus LPS + Met-CM comparison. Molecular function analysis demonstrated the enrichment of protein-binding terms among the DEGs from each comparison. Conclusions: Metformin preconditioning enhanced the recovery effect of PDLSC-CM on LPS-induced inflamed PDLSCs. These findings suggest that metformin preconditioning could represent a practical formula for PDLSC-secretome, which may contribute to the development of future cell-free periodontal regenerative strategies. Full article

Bacterium-triggered carious lesions implicate dental hard tissue destruction and the simultaneous initiation of regenerative events comprising dental stem cell activation. Streptococcus mutans (S. mutans) is a prominent pathogen of the oral cavity and the principal cause of caries. S. mutans generates complex products involved in interbacterial interactions, including Mutanobactin-D (Mub-D), which belongs to a group of non-ribosomal cyclic lipopeptides. In the present study, we aimed to analyse the potential role of the synthetic Mub-D peptide in cell populations involved in tissue regenerative processes. To this end, we assessed the in vitro effects of Mub-D in human dental pulp stem cells (hDPSCs) and human bone marrow stem cells (hBMSCs). Our data demonstrated a concentration-dependent effect of Mub-D on their viability and a significant increase in their proliferation and osteogenic/odontogenic differentiation. These events were associated with specific changes in gene expression, where CCDN-1, RUNX-2, OSX, OCN, DMP-1, DSPP, and BMP-2 genes were upregulated. The ability of Mub-D to modulate the osteogenic/odontogenic differentiation of both hDPSCs and hBMSCs and considerably enhance mineralisation in a controlled and concentration-dependent manner opens new perspectives for stem cell-based regenerative approaches in the clinics. Full article

Low-intensity pulsed ultrasound (LIPUS) is a form of ultrasound that utilizes low-intensity pulsed waves. Its effect on bones that heal by intramembranous ossification has not been sufficiently investigated. In this study, we examined LIPUS and the autologous bone, to determine their effect on the healing of the critical-size bone defect (CSBD) of the rat calvaria. The bone samples underwent histological, histomorphometric and immunohistochemical analyses. Both LIPUS and autologous bone promoted osteogenesis, leading to almost complete closure of the bone defect. On day 30, the bone volume was the highest in the autologous bone group (20.35%), followed by the LIPUS group (19.12%), and the lowest value was in the control group (5.11%). The autologous bone group exhibited the highest intensities of COX-2 (167.7 ± 1.1) and Osx (177.1 ± 0.9) expression on day 30. In the LIPUS group, the highest intensity of COX-2 expression was found on day 7 (169.7 ±1.6) and day 15 (92.7 ± 2.2), while the highest Osx expression was on day 7 (131.9 ± 0.9). In conclusion, this study suggests that LIPUS could represent a viable alternative to autologous bone grafts in repairing bone defects that are ossified by intramembranous ossification. Full article

An important part of regenerative endodontic procedures involving immature permanent teeth is the regeneration of the pulp–dentin complex with continuous root development. Hydraulic calcium silicate cements (HCSCs) are introduced for the pulpal treatment of immature permanent teeth. The stem-cell-derived secretome recently has been applied for the treatment of various damaged tissues. Here, we evaluated the biocompatibility and osteogenic differentiation of HCSCs combined with secretome on human dental pulp stem cells. In the Cell Counting Kit-8 test and wound healing assays, significantly higher cell viability was observed with secretome application. In alkaline phosphatase analysis, the activity was significantly higher with secretome application in all groups, except for RetroMTA on day 2 and Endocem MTA Premixed on day 4. In an Alizarin Red S staining analysis, all groups with secretome application had significantly higher staining values. Quantitative real-time polymerase chain reaction results showed that the day 7 expression of OSX significantly increased with secretome application in all groups. SMAD1 and DSPP expression also increased significantly with secretome addition in all groups except for Biodentine. In conclusion, HCSCs showed favorable biocompatibility and osteogenic ability and are predicted to demonstrate greater synergy with the addition of secretome during regenerative endodontic procedures involving immature permanent teeth. Full article

To counteract the effect of zoledronate and decrease the risk of osteonecrosis of the jaw (BRONJ) development in patients undergoing guided bone regeneration surgery, the use of geranylgeraniol (GGOH) has been proposed. Collagen membranes may act as biomimetical drug carriers. The objective of this study was to determine the capacity of collagen-based membranes doped with GGOH to revert the negative impact of zoledronate on the growth and differentiation of human osteoblasts. MG-63 cells were cultured on collagen membranes. Two groups were established: (1) undoped membranes and (2) membranes doped with geranylgeraniol. Osteoblasts were cultured with or without zoledronate (50 μM). Cell proliferation was evaluated at 48 h using the MTT colorimetric method. Differentiation was tested by staining mineralization nodules with alizarin red and by gene expression analysis of bone morphogenetic proteins 2 and 7, alkaline phosphatase (ALP), bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7), type I collagen (Col-I), osterix (OSX), osteocalcin (OSC), osteoprotegerin (OPG), receptor for RANK (RANKL), runt-related transcription factor 2 (Runx-2), TGF-β1 and TGF-β receptors (TGF-βR1, TGF-βR2, and TGF-βR3), and vascular endothelial growth factor (VEGF) with real-time PCR. One-way ANOVA or Kruskal–Wallis and post hoc Bonferroni tests were applied (p < 0.05). Scanning electron microscopy (SEM) observations were also performed. Treatment of osteoblasts with 50 μM zoledronate produced a significant decrease in cell proliferation, mineralization capacity, and gene expression of several differentiation markers if compared to the control (p < 0.001). When osteoblasts were treated with zoledronate and cultured on GGOH-doped membranes, these variables were, in general, similar to the control group (p > 0.05). GGOH applied on collagen membranes is able to reverse the negative impact of zoledronate on the proliferation, differentiation, and gene expression of different osteoblasts’ markers. Full article

Ossiculoplasty is a surgical operation performed to restore auditory transmission through the reconstruction of the ossicular chain using prosthetics. Tissue bioengineering has assumed a pivotal role in implementing alternatives to conventional ossicular middle ear replacement prostheses, to overcome extrusion while preserving acoustic properties. This in vitro study aims to explore, for the first time in current literature, the feasibility of a biohybrid middle ear prosthesis, composed of titanium surrounded by a bone extracellular matrix as bio-coating. We have hereby studied the adhesion and proliferation of human adipose-derived mesenchymal stem cells (hASC) on titanium scaffolds in vitro. Moreover, we identified the osteogenic differentiation of hASC using an immunofluorescence assay to analyze osteoblasts’ gene expression profiles (Alp, Runx2, Col1a1, Osx, and Bglap), and we counted the presence of collagen as a marker of hASC’s ability to secrete an extracellular matrix. We utilized scanning electron microscopy to evaluate the presence of an extracellular matrix on the scaffolds. Our preliminary data demonstrated the titanium’s ability to support human adipose-derived mesenchymal stem cell colonization, proliferation, and osteoblastic differentiation, in order to obtain a biohybrid device. Our experience seems encouraging; thus, we advocate for further in vivo research to corroborate our results regarding bone transplantation. Full article

Bone tissue engineering using different scaffolds is a new therapeutic approach in regenerative medicine. This study explored the osteogenic potential of human dental pulp stem cells (hDPSCs) grown on a hydrolytically modified poly(L-lactide-co-caprolactone) (PLCL) electrospun scaffold and a non-woven hyaluronic acid (HYAFF-11™) mesh. The adhesion, immunophenotype, and osteogenic differentiation of hDPSCs seeded on PLCL and HYAFF-11™ scaffolds were analyzed. The results showed that PLCL and HYAFF-11™ scaffolds significantly supported hDPSCs adhesion; however, hDPSCs’ adhesion rate was significantly higher on PLCL than on HYAFF-11™. SEM analysis confirmed good adhesion of hDPSCs on both scaffolds before and after osteogenesis. Alizarin red S staining showed mineral deposits on both scaffolds after hDPSCs osteogenesis. The mRNA levels of runt-related transcription factor 2 (Runx2), collagen type I (Coll-I), osterix (Osx), osteocalcin (Ocn), osteopontin (Opn), bone sialoprotein (Bsp), and dentin sialophosphoprotein (Dspp) gene expression and their proteins were higher in hDPSCs after osteogenic differentiation on both scaffolds compared to undifferentiated hDPSCs on PLCL and HYAFF-11™. These results showed that PLCL scaffolds provide a better environment that supports hDPSCs attachment and osteogenic differentiation than HYAFF-11™. The high mRNA of early osteogenic gene expression and mineral deposits observed after hDPSCs osteogenesis on a PLCL mat indicated its better impact on hDPSCs’ osteogenic potential than that of HYAFF-11™, and hDPSC/PLCL constructs might be considered in the future as an innovative approach to bone defect repair. Full article