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In the oral cavity, dental implants—most often made of commercially pure titanium—come in contact with bacteria, and antibacterial management has been researched extensively to improve patient care. With antibiotic resistance becoming increasingly prevalent, this has resulted in copper being investigated as an antibacterial element in alloys. In this study, the objective was to investigate the copper ion concentrations at which cyto-toxicity is avoided while bacterial inhibition is ensured, by comparing Cu ion effects on selected eukaryotes and prokaryotes. To determine relevant copper ion concentrations, ion release rates from copper and a 10 wt. % Cu Ti-alloy were investigated. Survival studies were performed on MC3T3 cells and Staphylococcus epidermidis bacteria, after exposure to Cu ions concentrations ranging from 9 × 10−3 to 9 × 10−12 g/mL. Cell survival increased from <10% to >90% after 24 h of exposure, by reducing Cu concentrations from 9 × 10−5 to 9 × 10−6 g/mL. Survival of bacteria also increased in the same range of Cu concentrations. The maximum bacteria growth was found at 9 × 10−7 g/mL, probably due to stress response. In conclusion, the minimum inhibitory concentrations of Cu ions for these prokaryotes and eukaryotes were found in the range from 9 × 10−5 to 9 × 10−6 g/mL. Interestingly, the Cu ion concentration correlating to the release rate of the 10 wt. % Cu alloy (9 × 10−8 g/mL) did not kill the bacteria, although this alloy has previously been found to be antibacterial. Further studies should investigate in depth the bacteria-killing mechanism of copper.

Brain‐derived neurotrophic factor (BDNF) has been reported to participate in fracture healing, whereas the mechanism is still unclear. Since osteoblast migration is important for fracture healing, investigating effects of BDNF on osteoblasts migration may help to reveal its mechanism. Here, MC3T3‐E1 cells were used in vitro while closed femur fracture mice were applied in vivo. Cells migration was assessed with Transwell assay. The protein expression was analysed by immunoblotting. X‐ray and Micro‐CT were performed at different time after fracture. Our results showed that BDNF promoted MC3T3‐E1 cells migration, integrin β1 expression and ERK1/2 and AKT phosphorylation. K252a, a specific inhibitor for TrkB, suppressed BDNF‐induced migration, integrin β1 expression and activation of ERK1/2 and AKT. PD98059 (an ERK1/2 inhibitor) and LY294002 (an AKT inhibitor) both inhibited BDNF‐induced migration and integrin β1 expression while integrin β1 blocking antibody only suppressed cell migration. X‐ray and Micro‐CT analyses showed that the adenoviral carried integrin β1 shRNA group had slower fracture healing at 7 and 21 days, but not 35 days compared to the control group. Thus, we proposed that BDNF stimulated MC3T3‐E1 cells migration by up‐regulating integrin β1 via TrkB mediated ERK1/2 and AKT signalling, and this may help to enhance the fracture healing.

Scaffolds used for bone tissue engineering need to have a variety of features to accommodate bone cells. The scaffold should mimic natural bone, it should have appropriate mechanical strength, support cell differentiation to the osteogenic lineage, and offer adequate porosity to allow vascularization and bone in-growth. In this work, we aim at developing a new process to fabricate such materials by creating a porous composite material made of silk fibroin and cellulose as a suitable scaffold of bone tissue engineering. Silk fibroin and cellulose are both dissolved together in N,N-dimethylacetamide/LiCl and molded to a porous structure using NaCl powder. The hydrogels are prepared by a sequential regeneration process: cellulose is solidified by water vapor treatment, while the remaining silk fibroin in the hydrogel is insolubilized by methanol, which leads to a cellulose framework structure embedded in a silk fibroin matrix. Finally, the hydrogels are soaked in water to dissolve the NaCl for making a porous structure. The cellulose composition results in improving the mechanical properties for the hydrogels in comparison to the silk fibroin control material. The pore size and porosity are estimated at around 350 µm and 70%, respectively. The hydrogels support the differentiation of MC3T3 cells to osteoblasts and are expected to be a good scaffold for bone tissue engineering.

Background: The increasing demand for bone implants with improved osseointegration properties has prompted researchers to develop various coating types for metal implants. Atomic layer deposition (ALD) is a method for producing nanoscale coatings conformally on complex three-dimensional surfaces. We have prepared hydroxyapatite (HA) coating on titanium (Ti) substrate with the ALD method and analyzed the biocompatibility of this coating in terms of cell adhesion and viability. Methods: HA coatings were prepared on Ti substrates by depositing CaCO3 films by ALD and converting them to HA by wet treatment in dilute phosphate solution. MC3T3-E1 preosteoblasts were cultured on ALD-HA, glass slides and bovine bone slices. ALD-HA and glass slides were either coated or non-coated with fibronectin. After 48h culture, cells were imaged with scanning electron microscopy (SEM) and analyzed by vinculin antibody staining for focal adhesion localization. An 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test was performed to study cell viability. Results: Vinculin staining revealed similar focal adhesion-like structures on ALD-HA as on glass slides and bone, albeit on ALD-HA and bone the structures were thinner compared to glass slides. This might be due to thin and broad focal adhesions on complex three-dimensional surfaces of ALD-HA and bone. The MTT test showed comparable cell viability on ALD-HA, glass slides and bone. Conclusion: ALD-HA coating was shown to be biocompatible in regard to cell adhesion and viability. This leads to new opportunities in developing improved implant coatings for better osseointegration and implant survival.

The rapid development and application of nanotechnology to biological interfaces has impacted the bone implant field, allowing researchers to finely modulate the interface between biomaterials and recipient tissues. In the present study, oxidative anodization was exploited to generate two alumina surfaces with different pore diameters. The former displayed surface pores in the mean range of 16–30 nm, while in the latter pores varied from to 65 to 89 nm. The samples were characterized by Field Emission Scanning Electron Microscopy (FESEM) and Energy Dispersive X-ray spectroscopy (EDX) analysis prior to being tested with pre-osteoblastic MC3T3-E1 cells. In vitro cell response was studied in terms of early cell adhesion, viability, and morphology, including focal adhesion quantification. Both the alumina samples promoted higher cell adhesion and viability than the control condition represented by the standard culture dish plastic. Osteogenic differentiation was assessed through alkaline phosphatase activity and extracellular calcium deposition, and it was found that of the two nano-surfaces, one was more efficient than the other. By comparing for the first time two nano-porous alumina surfaces with different pore diameters, our data supported the role of nano-topography in inducing cell response. Modulating a simple aspect of surface texture may become an attractive route for guiding bone healing and regeneration around implantable metals.

Understanding cell migration is essential not only for fundamental biology but also for the development of targeted disease therapies. Traditional in vitro cell migration assays typically rely on optical microscopy to capture cell movements and subsequent image-based tracking to quantify cell migration characteristics, which often involve substantial experimental workload and analytical complexity. Therefore, there is a need for an automated and streamlined approach to monitor and analyze cell movements. In this work, a microfabricated impedance sensor integrating electrode pairs and selectively protein-coated channels was developed for real-time monitoring of single-cell migration. The optimized electrode dimensions with 10 μm width and 10 μm gap enabled sensitive detection of impedance magnitude increase induced by individual cells. The impedance magnitude changes were correlated with the cell coverage area on electrodes, allowing continuous tracking of single-mouse osteoblast MC3T3 cell movement across the electrode pair. Distinct impedance responses of signal duration and magnitude were observed under different surface coatings, revealing the influence of microenvironmental chemistry on cell motility and adhesion. Furthermore, comparative impedance profiling of MC3T3 and nasopharyngeal epithelial NP460 cells demonstrated that MC3T3 cells produced larger changes in impedance real part and phase due to larger spreading area and larger number of focal adhesions, whereas NP460 cells showed shorter impedance signal change durations, consistent with faster cell migration. These electrical signatures collectively captured intrinsic differences in cell morphology, adhesion, and motility. The developed impedance sensor provides a label-free approach for single-cell migration characterization and can be potentially applied to cell identification.

Elevated serum levels of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) are found in patients with phosphate wasting diseases and chronic kidney disease-mineral and bone disorder (CKD-MBD). These diseases are associated with rickets and renal osteodystrophy, respectively. FGF23 is secreted from osteoblastic cells and signals through FGFRs, membrane coreceptor alpha-Klotho (Klotho), and, possibly, a circulating form of Klotho. Despite the absence of detectable Klotho on osteoblastic cells, studies have suggested that forced FGF23 expression in osteoblasts inhibited mineralization. Thus, we examined the effects of exogenously applied FGF23 on osteoblastic MC3T3.E1 cell proliferation and differentiation, with and without soluble Klotho. MC3T3.E1 cells were cultured in osteoblast differentiation medium, supplemented with FGF23 (0.1–1,000 ng/mL), Klotho (50 ng/mL), the combination FGF23 + Klotho, and FGF2 (100 ng/mL) as a control. Neither FGF23 nor Klotho exposure affected proliferation of day 4 growth phase cells or mineralization of day 14 cultures. In contrast, FGF23 + Klotho resulted in inhibition of mineralization and osteoblast activity markers at day 14, and a slight, reproducible induction of proliferation. Inhibition of FGFR1, but not FGFR2 or FGFR3, completely restored FGF23 + Klotho-induced inhibition of alkaline phosphatase (ALP) activity at day 7. ALP activity was partially restored by the MAPK inhibitor U0126 but not inhibitors p38 and P13K. Thus, soluble Klotho enables FGF23 signaling in MC3T3.E1 cells, likely through FGFR 1(IIIc). Elevated FGF23 actions, in part, appear to parallel FGF2 with lower potency. In addition to affecting bone via indirect phosphate wasting pathways, supraphysiological FGF23 and soluble Klotho may directly impact bone in diseases with elevated FGF23 levels.

Objective The aim of the present study was to evaluate the potential protective mechanism of icariin against oxidative damage caused by hydrogen peroxide in MC3T3‐E1 cells. Methods MC3T3‐E1 cells were treated with different concentrations of icariin to explore the optimal dose of icariin. MC3T3‐E1 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2O2; 0, 0.1, 0.2, 0.5, 1, and 2 mM) for 24 h to induce oxidative damage and cell viability was assessed by Cell Counting Kit‐8 (CCK‐8) assay. Then, cells were divided into five groups: control, H2O2 (0.2 mM), icariin (0.1 μM) and H2O2 (0.2 mM), + icariin (0.1 μM). Cell viability was detected by CCK‐8 assay. In addition, the content of glutathione and superoxide dismutase and the activity level of malondialdehyde in these treatment groups were determined. Alkaline phosphatase (ALP) and alizarin red S (ARS) staining were also performed to measure the early and late osteogenesis, respectively. Protein expression of β‐catenin and cyclin D1 was measured by western blot assay. Then, we used an antagonist of Wnt/β‐catenin signaling pathway (DKK‐1) and western blot analysis to further explore potential mechanism. Results After 24 h of exposure to 0.2 mM H2O2, the viability of MC3T3‐E1 cells was significantly decreased compared to that of the control cells. We first found that icariin can promote cell proliferation of MC3T3‐E1 cells in a dose‐dependent manner, with the dosage 0.1 μM showing the best pro‐proliferative effect. Furthermore, icariin could promote the protein expression of OSX and RUNX2. The results showed that icariin can reverse the inhibitory osteogenic effects of MC3T3‐E1 caused by H2O2. In addition, icariin could increase the Wnt‐signaling related proteins. The results showed that MC3T3‐E1 cells in the H2O2 (0.2 mM) + icariin (0.1 μM) + Wnt‐signaling antagonist (DKK‐1) group had weaker ALP and ARS staining compared with that observed in the control and H2O2 (0.2 mM) + icariin (0.1 μM) groups. The ALP activity and calcium content were decreased in the 0.2 mM H2O2 + 0.1 μM icariin + DKK‐1 group compared to that observed in the 0.2 mM H2O2 + 0.1 μM icariin group. Conclusion The results showed that icariin can increase the viability of MC3T3‐E1 cells, reverse the oxidative stress induced by H2O2 and protect MC3T3‐E1 cells against H2O2‐induced inhibition of osteogenic differentiation, which may occur through the Wnt/β‐catenin signaling pathway. Schematic diagram illustrating the mechanism of action of icariin against oxidative damage.

Objective This study aims to investigate how hyperoside (HYP) alleviates oxidative stress‐induced osteoporosis, its molecular mechanisms, and its impact on osteoblast differentiation, oxidative damage, and the estrogen‐PI3K/VEGF signaling pathway. Methods The osteoblast differentiation model was induced using dexamethasone, and osteoblast‐related markers like ALP, NO, GSH, MDA, and SOD were measured post‐HYP intervention. A zebrafish model was used to assess HYP's impact on ROS and bone formation. Network pharmacology identified key oxidative stress and osteoporosis targets, with HYP's binding affinity confirmed via molecular docking and simulation. RT‐qPCR verified the expression of key pathway targets. Results HYP can counteract dexamethasone‐induced inhibition of osteoblast differentiation, boost ALP, NO, GSH, and SOD levels, and lower MDA levels in osteoblasts. It also reduces ROS accumulation and enhances bone formation in zebrafish. Network pharmacology identified a common oxidative stress and osteoporosis target, with molecular docking confirming HYP's stable binding. RT‐qPCR showed HYP significantly upregulates SRC, PI3K, AKT1, and e‐NOS, activating the estrogen‐PI3K/VEGF pathway. Conclusion HYP plays an anti‐oxidative stress effect through targeted regulation of estrogen‐PI3K/VEGF signal axis, and then promotes osteoblast differentiation and bone formation, which provides a new potential candidate drug and experimental basis for the treatment of osteoporosis. This study explores the potential of hyperoside to activate the estrogen‐PI3K/VEGF axis, thereby suppressing oxidative stress and enhancing osteogenic differentiation to mitigate osteoporosis. The initial proposal of the estrogen‐PI3K/VEGF signaling pathway axis as a potential therapeutic approach for osteoporosis. The pharmacological effects of oxidative stress and osteoporosis have been confirmed through dual experiments conducted on MC3T3‐E1 cells and zebrafish.