Search Results (710)

Anterior cruciate ligament reconstruction relies on interference screw fixation, yet insufficient graft osseointegration remains a critical clinical challenge. This study aimed to develop and characterize a 3D-printed polymeric–hydroxyapatite composite interference screw with an osteoinductive surface to enhance localized osteogenic responses. Screws were designed, modeled, and fabricated using fused deposition modeling 3D printing with a polycaprolactone-poly(lactic-co-glycolic acid)-hydroxyapatite composite. Physico-chemical characterization was performed using scanning electron microscopy. Biocompatibility was assessed through mesenchymal stem cell metabolic activity assays and morphological analysis. Osteogenic gene expression was quantified by RT-qPCR following culture in osteogenic differentiation medium. In vivo osseointegration was evaluated histologically at five and nine weeks following implantation in the proximal tibial epiphysis of a rat model. 3D printing successfully produced screws with consistent geometry and surface characteristics. The composite material supported robust mesenchymal stem cell proliferation without cytotoxicity or morphological abnormalities. Histological examination revealed progressive bone formation with no adverse tissue reactions, including the absence of cyst formation, osteolysis, or excessive fibrosis. RT-qPCR revealed upregulation of osteogenic markers in those enhanced screws. These results indicate that the 3D-printed polymeric–hydroxyapatite composite screws are biocompatible and capable of stimulating localized osteogenic activity, supporting their potential as a biological foundation for future evaluation in anterior cruciate ligament reconstruction applications. Full article

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

Background: To investigate whether coating xenogeneic bone grafts with hyaluronic acid influences early osteogenic and fibrotic marker expression in vitro. Methods: Three xenograft materials were evaluated, including one hyaluronic acid-coated product and two uncoated deproteinized bovine bone mineral products, all commercially available. Human osteoblast-like cells (U2OS) and bone marrow stromal cells (HS5) were cultured with material extracts. Proliferation was assessed using XTT assay at 24 and 48 h. Cell adhesion was evaluated through fluorescence microscopy. Osteogenic markers (RUNX2, COL1A1) and fibrotic markers (COL3A1, TGF-β3) were quantified using quantitative real-time PCR. Statistical analysis employed one-way ANOVA with Benjamini–Krieger–Yekutieli (BKY) two-stage FDR correction for datasets that met the normality assumption, and the Kruskal–Wallis test with Dunn’s post hoc test for non-normally distributed data (HS5 XTT assay). Pairwise comparisons were restricted to each xenograft group versus the untreated control; an adjusted p-value < 0.05 was considered statistically significant. Results: At 48 h, the HA-coated xenograft (Xeno1) showed the highest mean metabolic activity in U2OS cells (0.538 ± 0.056) compared with the uncoated Xeno2 (0.450 ± 0.120) and Xeno3 (0.439 ± 0.073); however, after FDR correction no statistically significant differences were observed between groups. The coated material was associated with upregulation of early osteogenic markers, 2.61-fold RUNX2 upregulation (p = 0.01) compared to untreated cells. Both coated and uncoated xenografts demonstrated equivalent suppression of fibrotic markers in HS5 cells, reducing COL3A1 by 92.7% (p = 0.001) and TGF-β3 by 92.1% (p = 0.001). Conclusions: These exploratory in vitro findings suggest that HA coating may enhance early osteogenic marker expression. The observed effects on stromal markers warrant further investigation using primary cells, additional fibrotic endpoints (e.g., TGF-β1, ACTA2), and in vivo models before translational conclusions can be drawn. Full article

Gelatin–alginate composite hydrogels are some of the most prevalent bioinks used for extrusion-based three-dimensional (3D) bioprinting because of their combined bioactivity and ability to ionically crosslink. Ionically crosslinked gelatin–alginate constructs containing human bone marrow–derived mesenchymal stem cells (hBM-MSCs) were characterised over time under standardised in vitro conditions to assess physicochemical properties and resultant cell behaviour. Water uptake and degradation were quantified over time in phosphate-buffered saline (PBS) and collagenase type II media for up to 21 days. Cell viability and metabolic activity were quantified, and osteogenic gene expression (RUNX2, COL1A1, OCN) was assessed. Raman spectroscopy and compressive mechanical characterisation were performed. Collagen and glycosaminoglycan-related peaks were observed from extracellular matrix (ECM)-associated components, with an increased presence of protein-associated signatures later in culture. Hydrogels displayed nonlinear elastic behaviour with increased stress after longer incubation times, suggesting no degradation of mechanical integrity over the duration of the study. Hydrogels experienced rapid hydration followed by decreased swelling over time, with a maximum swelling ratio at 24 h. Degradation rates significantly increased over longer incubation times (p < 0.001) and in collagenase media compared to PBS (p < 0.001). Observed differences were likely due to both ion-exchange-mediated network disassembly and the dissolution of gelatin components. Cell metabolic activity decreased under osteogenic culture conditions, while changes in osteogenic marker expression were sequential, suggesting a transition from proliferation to early osteogenic commitment in this 3D system. This work provides both physicochemical and biological characterisation of a commonly utilised gelatin–alginate bioink system, to provide future optimisations within the field of extrusion-based bone tissue engineering, a reproducible baseline for future optimisation of bioink systems in extrusion-based bone tissue engineering. Full article

Microphysiological systems (MPSs) have emerged as promising platforms for drug discovery and in vitro pharmacological testing. MPSs aid to reproduce physiologically relevant microenvironments, in which controlled perfusion can play important role. In this study, an on-chip AC electrothermal (ACET) pump was developed for pulsatile perfusion in microfluidic cell culture systems. The proposed pump generates fluid motion through the interaction between an applied electric field and temperature-dependent gradients in the electrical properties of the fluid. Pulsatile perfusion was produced by periodic application of an AC voltage to the electrode array, and the pulsation cycle could be controlled electrically. The maximum flow velocity increased with the applied AC voltage, demonstrating tunable flow generation by the ACET pump. To evaluate the applicability of the developed system to cell culture, human mesenchymal stem cells (hMSCs) were cultured under pulsatile perfusion conditions for five days. The results showed that osteogenic differentiation under pulsatile perfusion was higher than that under static culture conditions. These findings demonstrate the potential of the proposed on-chip ACET pump as a simple and effective platform for generating physiologically relevant pulsatile perfusion in microphysiological systems. Full article

Osteoporosis is a major global health challenge, particularly among aging populations, underscoring the need for safe and effective nutritional interventions. Probiotics and their metabolites have emerged as promising candidates for modulating bone health via the gut-bone axis. In this study, we investigated the effects of a cell-free culture supernatant (CFS) from the food-grade bacterium Limosilactobacillus fermentum GBE18 on the proliferation, differentiation, and mineralization of MC3T3-E1 pre-osteoblasts. GBE18 CFS exhibited no cytotoxicity at concentrations ranging from 1% to 4% (v/v). Notably, 2% (v/v) CFS significantly enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization (p < 0.05). Transcriptomic profiling revealed that differentially expressed genes were enriched in osteoblast-related processes and two key signaling pathways: Wnt/β-catenin and PI3K/Akt. Subsequent qRT-PCR and Western blot analyses confirmed the upregulation of critical regulators (Rspo2, Pdpk1, Malat1) and demonstrated coordinated activation of Akt phosphorylation, β-catenin stabilization, and Runx2 protein expression. Our findings indicate that GBE18 CFS promotes osteogenic differentiation through coordinated modulation of the PI3K/Akt and Wnt/β-catenin pathways. Consequently, this study provides mechanistic evidence supporting the potential application of L. fermentum GBE18-derived metabolites as functional food ingredients or dietary interventions for bone health and osteoporosis management. Full article

In a previous study, we demonstrated that a novel surface treatment applied to laser-melted Ti6Al4V substrates supports osteoblast-like cell adhesion, proliferation, and the activation of early osteogenic pathways. Building on these preliminary findings, the present work aimed to further investigate the ability of the same surface to promote extracellular matrix (ECM) deposition, organization, and osteogenic maturation, which are critical events for the establishment of a stable bone–implant interface in subperiosteal dental implants. Human osteoblast-like MG-63 cells were cultured on Ti6Al4V discs subjected to different surface treatments, including a proprietary surface modification (ATcs) specifically designed for subperiosteal applications. ECM formation and maturation were evaluated through scanning electron microscopy coupled with energy-dispersive spectroscopy, immunofluorescence, and semiquantitative analyses of osteogenic markers type I collagen (COL1A1), secreted protein acidic and rich in cysteine (SPARC), and dentin matrix protein 1 (DMP1) through Western blotting. The results showed that, while all tested surfaces supported cell adhesion, the ATcs surface promoted a distinct osteogenic profile characterized by enhanced DMP1 expression, organized collagen deposition, and the formation of calcium–phosphate–rich mineralized structures. Compared to surfaces that primarily stimulated cell proliferation or early matrix production, ATcs appeared to favour progression toward late-stage osteogenic maturation and matrix mineralization. Taken together, these findings extend our previous observations and indicate that this novel surface treatment not only supports osteoblast viability and early differentiation but also promotes extracellular matrix maturation, a key prerequisite for effective osseointegration. Although further in vivo studies are required, the present data provide additional biological rationale for the use of ATcs-treated Ti6Al4V surfaces in next-generation custom-made subperiosteal implant designs. Full article

Bone formation requires a substantial energy supply to sustain extracellular matrix production and mineralization, yet the temporal contribution of lipid metabolism during osteoblast maturation remains incompletely characterized. This study investigated the molecular and transcriptional remodeling of lipid metabolism. Intracellular lipid distribution was analyzed by confocal microscopy using Nile Red staining. Transcriptional modulation of lipid synthesis, storage, lipolysis, genes associated with mitochondrial fatty acid oxidation, and osteogenic markers were assessed by quantitative real-time PCR, and the biochemical composition was evaluated by Raman spectroscopy. Early stages of spheroid development showed higher expression of genes involved in lipid synthesis and storage (FASN, DGAT2, and PLIN2) together with intracellular lipid accumulation, whereas later stages displayed increased expression of lipolytic and β-oxidation markers (PNPLA2/ATGL, CPT1A, and HADHA), accompanied by the redistribution of lipid droplets. The Raman analysis revealed a time-dependent variation of lipid-associated CH₂/CH₃ bands and modulation of protein-related Amide I–III signals, consistent with biochemical remodeling during maturation. Overall, the data indicate a coordinated transcriptional shift from lipid accumulation-associated pathways toward lipid mobilization during osteogenic progression in a 3D culture. This model provides a controlled experimental platform for investigating metabolic regulation during bone formation and for studying metabolic alterations associated with skeletal disorders. Full article

The prevalence of osteoporosis, a skeletal disorder characterized by reduced bone mass, microarchitectural deterioration, and increased fracture risk, poses a substantial global healthcare burden. Although animal models and two-dimensional cell cultures have been used to advance bone research, they do not completely replicate the multicellular interactions, extracellular matrix organization, and biomechanical environment of human bone, limiting their translational relevance. This review provides a critical synthesis of recent advances in bone organoid technology, emphasizing biological complexity, technical innovation, and relevance to osteoporosis modeling. Beyond summarizing progress, we distinguish validated capabilities from aspirational claims and identify the methodological gaps that must be addressed before bone organoids can reliably support drug screening, regenerative medicine, and precision approaches. Advances in stem cell biology, tissue engineering, and three-dimensional culture systems have enabled the use of self-organizing, multicellular organoids that reproduce key physiological and pathological features of bone. These systems model estrogen-deficiency-induced bone loss, glucocorticoid-associated osteoporosis, aging-related degeneration, and genetic susceptibility. By integrating osteogenic and endothelial components within biomimetic matrices, bone organoids can support mechanistic studies and pharmacological testing. However, their incomplete vascularization, limited mechanical fidelity, instability, and lack of standardized benchmarks restrict their translational readiness. Overcoming these barriers requires technological refinement, quantitative metrics, and regulatory alignment. Full article

Large-scale expansion of human mesenchymal stem cells (hMSCs) remains a major challenge due to the intrinsic trade-off between cell proliferation and the maintenance of multipotency in conventional culture systems. Stiff substrates, such as tissue culture polystyrene or rigid hydrogels, promote rapid proliferation but induce progressive loss of stemness, whereas very soft matrices preserve multipotency at the expense of cell growth. To overcome this limitation, we developed mechanically soft, phase-separated gelatin–phenol/hyaluronic acid–phenol (Gtn-Ph/HA-Ph) hydrogels with precisely controlled microstructures via enzyme-mediated crosslinking. These hydrogels consist of HA-rich, dot-like domains embedded within a continuous Gtn-rich network, allowing for independent tuning of stiffness and domain architecture. On single-component Gtn-Ph hydrogels, hMSC proliferation increased with substrate stiffness, whereas soft hydrogels with a storage modulus (G′) of approximately 0.6 kPa markedly suppressed proliferation while preserving stemness marker expression, confirming the stiffness-dependent trade-off. In contrast, phase-separated Gtn-Ph/HA-Ph hydrogels supported robust hMSC proliferation even under soft mechanical conditions while maintaining high expression of stemness-associated markers. During long-term culture, hMSCs achieved a 68- to 195-fold increase in cumulative cell yield on soft Gtn-Ph/HA-Ph hydrogels (G′ = 0.5 kPa) compared with tissue culture polystyrene. Expression of α-smooth muscle actin (α-SMA) mRNA, encoded by the ACTA2 gene and associated with cellular senescence and fibrotic activation, was completely suppressed, while hMSCs retained robust adipogenic, osteogenic, and chondrogenic differentiation capacities. These results demonstrate that phase-separated Gtn-Ph/HA-Ph hydrogels effectively resolve the proliferation–multipotency dilemma in hMSC expansion and provide a promising platform for scalable manufacturing of therapeutic stem cells. 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

Articular cartilage, a highly specialised and avascular tissue, exhibits limited regenerative potential following trauma or degenerative conditions such as osteoarthritis (OA). Conventional surgical interventions, including microfracture and autologous chondrocyte implantation (ACI), have shown limited long-term efficacy due to donor site morbidity and restricted cell proliferation. In this context, mesenchymal stromal cells (MSCs) have emerged as a promising alternative owing to their multipotency, self-renewal capacity, and low immunogenicity. While bone marrow (BM) remains the traditional source of MSCs, recent studies have reported that peripheral blood-derived mesenchymal stromal cells (PB-MSCs) may possess chondrogenic, osteogenic, and adipogenic potential comparable to that of BM-derived MSCs. PB-MSCs can be harvested through minimally invasive methods, thereby avoiding the complications associated with BM aspiration. Experimental evidence indicates that PB-MSCs exhibit strong cell viability, proliferative potential, and the ability to synthesise cartilage-specific extracellular matrix proteins, such as type II collagen and sulphated glycosaminoglycans, within three-dimensional scaffolds. Immunophenotypically, PB-MSCs express mesenchymal markers including CD29, CD44, CD90, and CD105 while lacking hematopoietic markers CD34 and CD45. Flow cytometry analyses reveal that CD105⁺ populations increase following cryopreservation, highlighting their clinical utility. In contrast to these experimentally defined PB-MSCs, the term peripheral blood stem cells (PBSCs) is used in clinical studies to describe heterogeneous, non-cultured peripheral blood-derived cell preparations, typically enriched in hematopoietic stem and progenitor cells following granulocyte colony-stimulating factor (G-CSF) mobilisation, without full mesenchymal characterisation. In vitro studies confirm successful tri-lineage differentiation, whereas in vivo investigations have demonstrated effective cartilage regeneration using PB-based clinical approaches, including postoperative intra-articular administration of hyaluronic acid (HA) combined with PBSCs, as well as implantation of PBSCs covered with a collagen membrane. Furthermore, advancements in biomaterial engineering, such as poly(ethylene glycol)–cysteine–arginine–glycine–aspartic acid (PEG-CRGD) hydrogels, have enhanced PB-MSC adhesion, proliferation, and chondrogenic differentiation while promoting immunomodulation through M2 macrophage polarisation. Despite these promising outcomes, the available evidence remains limited and heterogeneous, with substantial variability in cell definitions, experimental models, and clinical study designs, which currently constrains definitive conclusions regarding clinical efficacy. Future research should focus on optimising isolation protocols, understanding molecular pathways governing PB-MSC chondrogenesis, and standardising clinical applications. Overall, PB-MSCs represent a viable, less invasive, and translationally relevant cell source for cartilage regeneration and regenerative orthopaedic therapies Full article

New hard tissue formation helps create a more stable seal in endodontic treatment. To achieve this, a novel class of endodontic sealers containing the pro-osteogenic element, strontium (within a BG), embedded in a polydimethylsiloxane matrix (Sr-PDMS) was produced. The properties of this sealer were compared with a commercially available bioactive endodontic sealer, Guttaflow Bioseal (GFBS). Glass was prepared via the melt quench method and incorporated into the GFBS matrix. Its physical properties were tested against the International Organisation for Standardisation (ISO) 6876. For biocompatibility assessment, dose–response proliferation of OCCM-30 cells was quantified by measuring DNA levels in varying concentrations of exogenous calcium and strontium, in culture media conditioned with the novel BG powder, and in sealer discs of the GFBS and novel Sr-PDMS. Two-way ANOVA followed by one-way ANOVA and the Bonferroni post hoc test were applied to the cell viability data. Both the GFBS and novel Sr-PDMS sealants demonstrated physical properties that met ISO 6876, but Sr-PDMS displayed greater radiopacity (p < 0.05), lower solubility, and increased setting time. Both sealants released ions into the immersion solution, with the additional release of Sr from the novel sealer. GFBS displayed evidence of apatite formation. As expected, high concentrations of BG-conditioned media were cytotoxic, but the levels released by the BG in the Sr-PDMS were not cytotoxic with 1:000 dilution and resulted in significantly increased (p < 0.01) cell proliferation compared to the control group. Full article

Background: Well-known risk factors for soft tissue heterotopic ossification (HO) include aging and mechanical stress, which may be linked to oxidative stress and downstream nucleotide metabolites. Thus, we investigated the involvement of extracellular ATP (ex-ATP) and its metabolites in the oxidative stress-induced mineralization of TT-D6 cells and primary mouse tendon cells. Methods: An osteogenic culture with the intermittent addition of hydrogen peroxide was monitored for two weeks using metabolomic and gene expression analyses. Results: Calcium deposition was significantly enhanced by 0.3 mM hydrogen peroxide in the osteogenic media after 2 weeks, with minimal calcification in its absence. Similar results were observed in a medium transfer experiment using 3-day-old hydrogen peroxide-treated conditioned medium, which led to an increased expression of osterix and alkaline phosphatase. Metabolomic analysis revealed a gradual increase in ex-ATP and its metabolites, including ADP, AMP, and adenosine, in the medium. The metabolite increase was enhanced by hydrogen peroxide after 12 h. Moreover, exogenous adenosine (100 μM) increased mineralization in osteogenic media. Additionally, 1 μM dipyridamole, an inhibitor of equilibrative nucleoside transporter 1 (Ent1), also increased it in response to low-dose (0.1 mM) hydrogen peroxide. Conclusions: The enhanced osteogenic calcification of the tendon cell culture by hydrogen peroxide was associated with an increase in extracellular nucleotide metabolites, especially adenosine, with some evidence of causality. Full article

The success of titanium dental implants rely on osseointegration, influenced by surface properties and early immune responses. While sandblasted and acid-etched (SLA) titanium surfaces have shown clinical success, macrophage-mediated immune responses at these interfaces remain poorly understood. Anatase nanostructures have been shown to influence macrophage polarization on smooth titanium, but their effects on micro-rough SLA surfaces are not fully explored. This study investigates the immunomodulatory effects of micro-nanostructured anatase coatings on SLA titanium using human monocyte-derived macrophages (MDMs). M0-MDMs, were cultured and polarized to M1 and M2- macrophages on Ti-machined, Ti-SLA, Ti-SLA-anatase, and coverslip control surfaces for 48 h. Macrophage behavior was assessed using CCK-8 assay, confocal microscopy, SEM, ELISA, and qRT-PCR. All surfaces demonstrated excellent cytocompatibility, with similar macrophage viability across all investigated groups. M1 macrophages showed upregulation of CCR7 and TNF-α, while M2 macrophages expressed CD209 and CCL13 across all surfaces. Importantly, Ti-SLA-anatase did not significantly alter M1 or M2 markers, cytokine secretion, or gene expression, and did not exacerbate inflammatory responses. Micro-nanostructured anatase coatings on SLA titanium are immunologically well-tolerated and do not increase inflammation. These findings, combined with previously reported enhanced osteogenic properties, suggest the clinical potential of anatase-coated SLA surfaces. Full article