Mouse Osteoblastic Cells Research Articles

Osteoprotegerin secretion and its inhibition by RANKL in osteoblastic cells visualized using bioluminescence imaging

Bone remodeling is regulated by the interaction between receptor activator of nuclear factor kappa-B ligand (RANKL) and its receptor RANK on osteoblasts and osteoclasts, respectively. Osteoprotegerin (OPG) is secreted from osteoblasts and inhibits osteoclast differentiation by acting as a decoy receptor for RANKL. Despite its importance, the mechanism underlying the secretion of OPG remains poorly understood. Here, we applied a method of video-rate bioluminescence imaging using a fusion protein with Gaussia luciferase (GLase) and visualized the secretion of OPG from living mouse osteoblastic MC3T3-E1 cells. The bioluminescence imaging revealed that the secretion of OPG fused to GLase (OPG-GLase) occurred frequently and widely across the cell surface. Notably, co-expression of RANKL significantly reduced the secretion of OPG-GLase, indicating an inhibitory role of RANKL on OPG secretion within cells. Further imaging and biochemical analyses using deletion mutants of OPG and RANKL, as well as RANKL mutants that cause autosomal recessive osteopetrosis, demonstrated the essential role of protein-protein interaction between OPG and RANKL in the inhibition of OPG secretion. Treatment with proteasome inhibitors resulted in increased levels of OPG in both culture medium and cell lysates. However, the fold-increase of OPG was similar regardless of the presence or absence of RANKL, suggesting that the regulation of OPG secretion by RANKL is independent of proteasome activity. This report visualized the secretion of OPG from living cells and provided evidence for a novel intracellular inhibitory effect of RANKL on OPG secretion.

Zinc-doped phosphate coatings for enhanced corrosion resistance, antibacterial properties, and biocompatibility of AZ91D Mg alloy

Magnesium and its alloys have been foreseen as promising bone-implant owing to good biocompatibility, high strength, mechanical properties, and biodegradability to replace the conventional bio-metals (Titanium, stainless-steel, Co-Cr alloys) in orthopedic applications. However, high corrosion rate and high degradation rates adversely effects like abrupt evolution of H2 gas and localized alkalization in a physiological environment limit its medical applications. To overcome these issues, phosphate-based surface coating is developed on Mg-alloy to resist the high biodegradation and corrosion rate utilizing magnesium substrate as source of magnesium ion through a single-step hydrothermal process. Controlling the fast corrosion rate and localized alkalization would reduce the antibacterial character of magnesium-based implants therefore, the simple phosphate-based coatings has been induced by doping it with zinc ions due to its physiological tolerance and excellent antibacterial efficacy. The doping of zinc ions may provide a sustained drug-free antibacterial characteristic for potential application in orthopedics. The synthesized phosphate-based coating was characterized using advanced techniques like Scanning Electron Microscopy, X-ray Diffraction, and wettability test, followed by comprehensive electrochemical testing to assess its corrosion behavior. The in vitro immersion test in simulated body fluid (SBF) indicates that deposition of phosphate and zinc doped phosphate coatings reduces the degradation rate consequently minimized the fast dissolution of Mg2+ ions and OH- in physiological environment. Additionally, cytotoxicity and biocompatibility were evaluated using Alamar Blue and live/dead assays on the MC3T3-E1 mouse osteoblast cell line. These assays demonstrated good cell viability, indicating the coatings possess favorable cytocompatibility and support cellular metabolic activity.

Effects of Different No-Ozone Cold Plasma Treatment Methods on Mouse Osteoblast Proliferation and Differentiation.

Background and Objectives: Enhanced osteoblast differentiation may be leveraged to prevent and treat bone-related diseases such as osteoporosis. No-ozone cold plasma (NCP) treatment is a promising and safe strategy to enhance osteoblast differentiation. Therefore, this study aimed to determine the effectiveness of direct and indirect NCP treatment methods on osteoblast differentiation. Mouse osteoblastic cells (MC3T3-E1) were treated with NCP using different methods, i.e., no NCP treatment (NT group; control), direct NCP treatment (DT group), direct NCP treatment followed by media replacement (MC group), and indirect treatment with NCP-treated media only (PAM group). Materials and Methods: The MC3T3-E1 cells were subsequently assessed for cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, and ALP and osteocalcin mRNA expression using real-time polymerase chain reaction. Results: Cell proliferation significantly increased in the NCP-treated groups (DT and PAM; MC and PAM) compared to the NT group after 24 h (p < 0.038) and 48 h (p < 0.000). ALP activity was increased in the DT and PAM groups at 1 week (p < 0.115) and in the DT, MC, and PAM groups at 2 weeks (p < 0.000) compared to the NT group. Calcium deposition was higher in the NCP-treated groups than in NT group at 2 and 3 weeks (p < 0.000). ALP mRNA expression peaked in the MC group at 2 weeks compared to the NP group (p < 0.014). Osteocalcin mRNA expression increased in the MC group at 2 weeks (p < 0.000) and was the highest in the PAM group at 3 weeks (p < 0.000). Thus, the effects of direct (DT and MC) and indirect (PAM) treatment varied, with MC direct treatment showing the most significant impact on osteoblast activity. Conclusions: The MC group exhibited enhanced osteoblast differentiation, indicating that direct NCP treatment followed by media replacement is the most effective method for promoting bone formation.

Biological Effects of Double-Layered Hydroxyapatite and Zirconium Oxide Depositions on Titanium Surfaces.

This study aimed to confirm the synergy effect of these two materials by evaluating osteoblast and antibacterial activity by applying a double-layered hydroxyapatite(HA) zirconium oxide(ZrO2) coating to titanium. The specimens used in this study were divided into four groups: a control group (polished titanium; group T) and three experimental groups: Group TH (RF magnetron sputtered HA deposited titanium), Group Z (ZrO2 ALD deposited titanium), and Group ZH (RF magnetron sputtered HA and ZrO2 ALD deposited titanium). The adhesion of Streptococcus mutans (S.mutans) to the surface was assessed using a crystal violet assay. The adhesion, proliferation, and differentiation of MC3T3-E1 cells, a mouse osteoblastic cell line, were assessed through a WST-8 assay and ALP assay. Group Z showed a decrease in the adhesion of S. mutans (p < 0.05) and an improvement in osteoblastic viability (p < 0.0083). Group TH and ZH showed a decrease in adhesion of S. mutans (p < 0.05) and an increase in osteoblastic cell proliferation and cell differentiation (p < 0.0083). Group ZH exhibited the highest antibacterial and osteoblastic differentiation. In conclusion double-layered HA and ZrO2 deposited on titanium were shown to be more effective in inhibiting the adhesion of S. mutans, which induced biofilm formation, and increasing osteoblastic differentiation involved in osseointegration by the synergistic effect of the two materials.

Activation of Nuclear Factor Erythroid 2-Related Factor 2 Transcriptionally Upregulates Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 Expression and Inhibits Ectopic Calcification in Mice.

Calcification plays a key role in biological processes, and breakdown of the regulatory mechanism results in a pathological state such as ectopic calcification. We hypothesized that ENPP1, the enzyme that produces the calcification inhibitor pyrophosphate, is transcriptionally regulated by Nrf2, and that Nrf2 activation augments ENPP1 expression to inhibit ectopic calcification. Cell culture experiments were performed using mouse osteoblastic cell line MC3T3-E1. Nrf2 was activated by 5-aminolevulinic acid and sodium ferrous citrate. Nrf2 overexpression was induced by the transient transfection of an Nrf2 expression plasmid. ENPP1 expression was monitored by real-time RT-PCR. Because the promoter region of ENPP1 contains several Nrf2-binding sites, chromatin immunoprecipitation using an anti-Nrf2 antibody followed by real-time PCR (ChIP-qPCR) was performed. The relationship between Nrf2 activation and osteoblastic differentiation was examined by alkaline phosphatase (ALP) and Alizarin red staining. We used mice with a hypomorphic mutation in ENPP1 (ttw mice) to analyze whether Nrf2 activation inhibits ectopic calcification. Nrf2 and Nrf2 overexpression augmented ENPP1 expression and inhibited osteoblastic differentiation, as indicated by ALP expression and calcium deposits. ChIP-qPCR showed that some putative Nrf2-binding sites in the ENPP1 promoter region were bound by Nrf2. Nrf2 activation inhibited ectopic calcification in mice. ENPP1 gene expression was transcriptionally regulated by Nrf2, and Nrf2 activation augmented ENPP1 expression, leading to the attenuation of osteoblastic differentiation and ectopic calcification in vitro and in vivo. Nrf2 activation has a therapeutic potential for preventing ectopic calcification.

Mechanical Properties, Drug Release, Biocompatibility, and Antibacterial Activities of Modified Emulsified Gelatin Microsphere Loaded with Gentamicin Composite Calcium Phosphate Bone Cement In Vitro.

Bone defects are commonly addressed with bone graft substitutes; however, surgical procedures, particularly for open and complex fractures, may pose a risk of infection. As such, a course of antibiotics combined with a drug carrier is often administered to mitigate potential exacerbations. This study involved the preparation and modification of emulsified (Em) crosslinking-gelatin (gel) microspheres (m-Em) to reduce their toxicity. The antibiotic gentamicin was impregnated into gel microspheres (m-EmG), which were incorporated into calcium phosphate bone cement (CPC). The study investigated the effects of m-EmG@CPC on antibacterial activity, mechanical properties, biocompatibility, and proliferation and mineralization of mouse progenitor osteoblasts (D1 cells). The average size of the gel microspheres ranged from 22.5 to 16.1 μm, with no significant difference between the groups (p > 0.05). Most of the oil content within the microspheres was transferred through modification, resulting in reduced cytotoxicity. Furthermore, antibiotic-impregnated m-EmG did not compromise the intrinsic properties of the microspheres and exhibited remarkably antibacterial effects. After combining with CPC (m-EmG@CPC), the microspheres did not significantly hinder the CPC reaction and produced the main product, hydroxyapatite (HA). However, the compressive strength of the largest microsphere content of 0.5 wt.% m-EmG in CPC decreased significantly from 59.8 MPa of CPC alone to 38.7 MPa of 0.5m-EmG@CPC (p < 0.05). The 0.5m-EmG@CPC composite was effective against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in drug release and antibacterial tests. Compared with m-EmG alone, the 0.5m-EmG@CPC composite showed no toxicity to mouse fibroblast cells (L929). Additionally, the proliferation and mineralization of mouse osteoblastic osteoprogenitor cells (D1 cells) did not have a negative impact on the 0.5m-EmG@CPC composite over time in culture compared with CPC alone. Results suggest that the newly developed antibacterial 0.5m-EmG@CPC composite bone cement did not negatively affect the performance of osteoprogenitor cells and could be a new option for bone graft replacement in surgeries.

Suppression of bone resorption in ovariectomized mice using estrogen-immobilized polyphosphodiesters

Estrogens play a crucial role in bone remodeling. In this study, we synthesized two types of bone-targeted polyphosphodiesters (PPDEs): poly(diethyl sodium phosphate) (PEP•Na) and β-estradiol 17-valerate terminated PEP•Na (E2V-PEP•Na), and determined the inhibitory effect of the terminal estrogen on ovariectomy-induced bone resorption. We first assessed the impact of these polymers on the differentiation of osteoblasts and osteoclasts in vitro. Both PPDEs effectively promoted the differentiation of mouse osteoblastic MC3T3-E1 cells, with E2V-PEP•Na significantly enhancing the mineralization of these cells. Conversely, the density of osteoclasts formed from bone marrow mononuclear cells (BMNCs) significantly decreased when exposed to E2V-PEP•Na. Additionally, the formation of resorption pits on calcium phosphate-coated plates was reduced after treating BMNCs with both PPDEs. We then investigated the role of these polymers in treating osteoporosis using ovariectomized (OVX) mice. PEP•Na or E2V-PEP•Na was separately injected into the tail vein of the mice three times at 2-week intervals, starting 1 day after OVX treatment. Two weeks after the final injection, we observed that E2V-PEP•Na was distributed in bone tissue in a dose-dependent manner. The injection of E2V-PEP•Na significantly reduced bone resorption, and all bone parameters of OVX mice treated with E2V-PEP•Na were superior to those of sham mice. In contrast, PEP•Na did not affect bone mineral metabolism. In conclusion, our study is the first to demonstrate that E2V-PEP•Na can inhibit bone resorption in vivo, offering a promising new polymeric drug for preventing osteoporosis.

Organic Matrices of Calcium Carbonate Biominerals Improve Osteoblastic Mineralization.

Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown that nacreous biominerals induce osteoblastic extracellular mineralization. Among them, Pinctada margaritifera is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other Pinctada species (Pinctada radiata, Pinctada maxima, and Pinctada fucata) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the Pinctada-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the Pinctada genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the Pinctada genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO3 biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.

Construction of Magnesium Phosphate Chemical Conversion Coatings with Different Microstructures on Titanium to Enhance Osteogenesis and Angiogenesis.

Titanium (Ti) and its alloys are widely used as hard tissue substitutes in dentistry and orthopedics, but their low bioactivity leads to undesirable osseointegration defects in the early osteogenic phase. Surface modification is an important approach to overcome these problems. In the present study, novel magnesium phosphate (MgP) coatings with controllable structures were fabricated on the surface of Ti using the phosphate chemical conversion (PCC) method. The effects of the microstructure on the physicochemical and biological properties of the coatings on Ti were researched. The results indicated that accelerators in PCC solution were important factors affecting the microstructure and properties of the MgP coatings. In addition, the coated Ti exhibited excellent hydrophilicity, high bonding strength, and good corrosion resistance. Moreover, the biological results showed that the MgP coatings could improve the spread, proliferation, and osteogenic differentiation of mouse osteoblast cells (MC3T3-E1) and vascular differentiation of human umbilical vein endothelial cells (HUVECs), indicating that the coated Ti samples had a great effect on promoting osteogenesis and angiogenesis. Overall, this study provided a new research idea for the surface modification of conventional Ti to enhance osteogenesis and angiogenesis in different bone types for potential biomedical applications.

Role of Piezo1 in modulating the RANKL/OPG ratio in mouse osteoblast cells exposed to Porphyromonas gingivalis lipopolysaccharide and mechanical stress.

Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3-E1 osteoblast-like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.-LPS) and mechanical stress. To investigate the effect of P.g.-LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3-E1 cells with P.g.-LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. The RANKL/OPG ratio significantly increased in MC3T3-E1 cells cultured in a medium containing P.g.-LPS and undergoing mechanical stress compared to cells treated with P.g.-LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.-LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.-LPS and mechanical stress. MC3T3-E1 cells treated with P.g.-LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast-like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. These findings suggest that P.g.-LPS and Piezo1-mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts.

Influence of core-shell polydopamine-barium titanate doping on the osseointegration and cytotoxicity of the polypyrrole coatings applied by ECD on Nitinol orthopedic implants

In the current study, a thin polydopamine (PDA) layer of around 5 nm was created on the barium titanate (BT) nanoparticles through self-polymerization of dopamine. The produced modified PDA/BT core-shell nanoparticles, termed PBT, were co-electrodeposited with polypyrrole (PPy) onto Nitinol (NiTi) alloy substrates in concentrations ranging from 1 to 10% by weight to produce PPy/PBT composite coatings. The incorporation of these particles changed PPy's cauliflower morphology into a rougher but more compact nodular morphology. According to the atomic force microscopy results, the average surface roughness of PPy coating increased from 44.2 nm up to 80.2 nm by the co-deposition of PBT. Aside from variations in morphology and roughness values, all of the PPy and PPy/PBT coatings demonstrated hydrophilicity. Nonetheless, while the majority of the composite coatings had higher water contact angle (WCA) than the unreinforced PPy, the PPy/10PBT composite coating had the lowest WCA of all samples, measuring 56.9°. The piezoelectric responses of the coatings were also examined, and the best specimen was the PPy/5PBT composite coating with an enhanced output voltage of approximately 400 mV. Finally, MC3T3 mouse osteoblast cells were used for in vitro cell culture evaluations. While healthy cells were evenly distributed on the surface of all coatings after 3 days, indicating biocompatibility, cell density on composite coatings was higher than on unreinforced PPy coating. According to the MTT experiment, the vitality of MC3T3 osteoblast cells on the surface of PPy/5PBT composite coating was more than 30% greater than that on the surface of PPy coating.

Tmem119 is involved in bone anabolic effects of PTH through enhanced osteoblastic bone formation in mice

The intermittent administration of parathyroid hormone (PTH) exerts potent bone anabolic effects, which increase bone mineral density (BMD) and reduce fracture risk in osteoporotic patients. However, the underlying mechanisms remain unclear. Tmem119 has been proposed as a factor that is closely linked to the osteoblast phenotype, and we previously reported that PTH enhanced the expression of Tmem119 in mouse osteoblastic cells. However, roles of Tmem119 in the bone anabolic effects of PTH in vivo remain unknown. We herein investigated the roles of Tmem119 in bone anabolic effects of PTH using Tmem119-deficient mice. Tmem119 deficiency significantly reduced PTH-induced increases in trabecular bone volume and cortical BMD of femurs. Effects of Tmem119 deficiency on bone mass seemed predominant in female mice. Histomorphometric analyses with calcein labeling showed that Tmem119 deficiency significantly attenuated PTH-induced increases in the rates of bone formation and mineralization as well as numbers of osteoblasts. Moreover, Tmem119 deficiency significantly blunted PTH-induced decreases in phosphorylation of β-catenin and increases in alkaline phosphatase activity in osteoblasts. In conclusion, the present results indicate that Tmem119 is involved in bone anabolic effects of PTH through osteoblastic bone formation partly related to canonical Wnt-β-catenin signaling in mice.

The marine factor 3,5-dihydroxy-4-methoxybenzyl alcohol prevents TNF-α-mediated impairment of mineralization in mouse osteoblastic MC3T3-E1 cells: Impact of macrophage activation

The phenolic antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), found in the Pacific oyster Crassostrea gigas, is a superior peroxyl radical scavenger compared to other materials, including Trolox. DHMBA may play an important role in the prevention of health disorders. This study elucidates whether DHMBA prevents the impairment of mineralization of mouse osteoblastic MC3T3-E1 cells under inflammatory conditions by using mouse macrophage RAW264.7 cells in vitro. Culturing with DHMBA (1–100 μM) did not affect the proliferation and death of MC3T3-E1 cells. DHMBA stimulated osteoblastic mineralization. DHMBA blocked the decrease in mineralization of MC3T3-E1 cells caused by culture with the inflammatory cytokine TNF-α. DHMBA inhibited the production of TNF-α by stimulation with lipopolysaccharide (LPS) in RAW264.7 cells. The growth of MC3T3-E1 cells was suppressed by coculture with macrophages under LPS stimulation through the crosstalk of both cells. Interestingly, the growth of MC3T3-E1 cells was suppressed by culturing with the conditioned medium obtained by culturing macrophages with LPS. The effect of the conditioned medium was blocked by the presence of DHMBA or Bay 11–7082, an inhibitor of the TNF-α pathway. The blocking effect of DHMBA was not further enhanced in the presence of Bay 11–7082. Mechanistically, DHMBA was found to decrease the levels of NF-κB p65 and the activity of NF-κB reporter expression in MC3T3-E1 cells. DHMBA was shown to prevent the impairment of osteoblastic mineralization via TNF-α signaling involved in macrophage activation in the bone marrow microenvironment. This study may provide a novel strategy for the therapy of osteoblastic impairment.

ZnO and Cu/ZnO-modified Magnesium orthopedic implant with improved osteoblast cellular activity: An in-vitro study

Mg-based biomaterials, due to their desirable mechanical integrity, natural bioabsorbability, and osteopromotive characteristics, are promising candidates for both orthopedic applications, such as the surgical fixation of injured musculoskeletal tissues, and cardiovascular applications like coronary artery stents. In the present study, nano- and micro-sized ZnO and Cu/ZnO bioactive particles were used to fabricate Mg-based biocomposites using friction stir processing (FSP). FSP resulted in significant microstructural modification in terms of grain refinement, uniform redistribution of secondary phase particles and homogenous dispersion of bioactive particles. Long-term biodegradation results through H2 evolution method illustrated that biodegradation rate of the bioabsorbable WE43 alloy was significantly reduced by addition of bioactive particles. In addition, fabricated biocomposites with addition of nano-sized particles resulted in lower biodegradation rate in comparison to those with micro-sized particles. Furthermore, uniform dispersion of bioactive particles resulted in superior in-vitro biocompatibility in contact with MC3T3-E1 mouse osteoblast cells. Mg–ZnO–N and Mg–Cu/ZnO–N biocomposites showed improved cell adhesion with elongated and spread morphology of MC3T3-E1 osteoblast cells. Live/dead staining and MTT assay indicated that number of healthy cells and cell viability after 24 h of incubation increased by uniform distribution of bioactive particles. Moreover, fabricated biocomposites with homogenous distribution of Zn2+ and Cu2+ ions showed higher ALP activity. Our findings propose that Mg–ZnO and Mg–Cu/ZnO biocomposites with homogenous dispersion of bioactive particles can be promising candidates for bone tissue regeneration applications.

Globo-series Gb4 activates ERK and promotes the proliferation of osteoblasts

ObjectivesGlobo-series Gb4 (globoside) is involved in the immune system and disease pathogenesis. We recently reported that systemic Gb4 deficiency in mice led to decreased bone formation due to a reduction in osteoblast number. However, it remains unclear whether Gb4 expressed in osteoblasts promotes their proliferation. Therefore, we investigated the role of Gb4 in osteoblast proliferation in vitro. MethodsWe examined osteoblast proliferation in Gb3 synthase knockout mice lacking Gb4. We investigated the effects of Gb4 synthase knockdown in the mouse osteoblast cell line MC3T3-E1 on its proliferation. Furthermore, we administered Gb4 to MC3T3-E1 cells in which Gb4 was suppressed by a glucosylceramide synthase (GCS) inhibitor and evaluated its effects on their proliferation. To elucidate the mechanisms by which Gb4 promotes osteoblast proliferation, the phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) levels were measured in MC3T3-E1 cells. ResultsOsteoblast proliferation was lower in Gb3 synthase knockout mice lacking Gb4 than in wild-type mice. Proliferation was inhibited by Gb4 synthase knockdown in MC3T3-E1 cells. Furthermore, the administration of Gb4 to MC3T3-E1 cells, in which a GCS inhibitor suppressed Gb4, promoted their proliferation. Moreover, it increased the phosphorylated ERK1/2 levels in MC3T3-E1 cells. ConclusionsOur results suggest that Gb4 expressed in osteoblasts promotes their proliferation through ERK1/2 activation.

Fabrication of water-dispersible and cell-stimulating calcium phosphate nanoparticles immobilizing basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a therapeutic protein that can enhance angiogenesis, wound healing, and tissue regeneration; however, it is extremely unstable even under a normal physiological environment. Biocompatible calcium phosphate (CaP) nanoparticles (NPs) co-immobilizing bFGF, heparin, and ferucarbotran would be useful as a multifunctional delivery carrier of bFGF. In this study, such NPs were successfully fabricated by a coprecipitation process, using a labile supersaturated CaP solution containing bFGF, heparin, and ferucarbotran. The NPs showed relatively high negative zeta potential (-12mV) because of the negatively charged heparin, which enabled their stable dispersion in water. The hydrodynamic diameter of the NPs was around 200nm. Immunoreactive bFGF was released from the NPs in an acellular medium dose-dependently. The NPs promoted proliferation of baby hamster kidney fibroblasts (BHK-21 cells) and mouse osteoblastic MC3T3-E1 cells at a certain dose range, although they inhibited proliferation of rat pheochromocytoma (PC-12) cells. These results demonstrated that the effect of the NPs on cell proliferation was dependent on the cell type and dose, the details of which should be investigated in a future study.

Guar-Based Injectable Hydrogel for Drug Delivery and In Vitro Bone Cell Growth.

Injectable hydrogels offer numerous advantages in various areas, which include tissue engineering and drug delivery because of their unique properties such as tunability, excellent carrier properties, and biocompatibility. These hydrogels can be administered with minimal invasiveness. In this study, we synthesized an injectable hydrogel by rehydrating lyophilized mixtures of guar adamantane (Guar-ADI) and poly-β-cyclodextrin (p-βCD) in a solution of phosphate-buffered saline (PBS) maintained at pH 7.4. The hydrogel was formed via host-guest interaction between modified guar (Guar-ADI), obtained by reacting guar gum with 1-adamantyl isocyanate (ADI) and p-βCD. Comprehensive characterization of all synthesized materials, including the hydrogel, was performed using nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and rheology. The in vitro drug release study demonstrated the hydrogel's efficacy in controlled drug delivery, exemplified by the release of bovine serum albumin (BSA) and anastrozole, both of which followed first-order kinetics. Furthermore, the hydrogel displayed excellent biocompatibility and served as an ideal scaffold for promoting the growth of mouse osteoblastic MC3T3 cells as evidenced by the in vitro biocompatibility study.

Proliferation and differential regulation of osteoblasts cultured on surface-phosphorylated cellulose nanofiber scaffolds

Phosphorus-containing polymers have received much attention for their excellent ability to regulate bone cell differentiation and calcification. Given the increasing concern about environmental issues, it is promising to utilize “green” biomaterials to construct novel cell culture scaffolds for bone tissue engineering. Herein, surface-phosphorylated cellulose nanofibers (P-CNFs) were fabricated as a novel green candidate for osteoblast culture. Compared with native CNF, P-CNFs possessed shorter fiber morphology with tunable phosphate group content (0–1.42 mmol/g). The zeta-potential values of CNFs were enhanced after phosphorylation, resulting in the formation of uniform and stable scaffolds. The cell culture behavior of mouse osteoblast (MC3T3-E1) cells showed a clear phosphate content-dependent cell proliferation. The osteoblast cells adhered well and proliferated efficiently on P-CNF0.78 and P-CNF1.05, with phosphate contents of 0.78 and 1.05 mmol/g, respectively, whereas the cells grown on native CNF substrate formed aggregates due to poor cell attachment and exhibited limited cell proliferation. In addition, the P-CNF substrates with optimal phosphate content provided a favorable cellular microenvironment and significantly promoted osteogenic differentiation and calcification, even in the absence of a differentiation inducer. The bio-based P-CNFs are expected to mimic the bone components and provide a means to regulate osteoblast proliferation and differentiation in bone tissue engineering.

Metformin activates AMPK and mTOR to Inhibit RANKL-stimulated osteoclast formation.

Metformin is a medication used to treat type 2 diabetes by inhibiting hepatic glucose production through adenosine monophosphate-activated protein kinase (AMPK) activation. Autophagy is closely related to the homeostasis and stress mechanisms of the body. In recent years, much research has arisen on therapeutic methods utilizing autophagy mechanisms to treat diagnoses such as metabolic diseases, tumors, and Alzheimer's disease. This study thus aimed to investigate the effects of metformin treatment on the differentiation of osteoclasts and changes in AMPK mechanisms, which play an important role in regulating energy homeostasis, and mTOR, a highly conserved kinase that regulates autophagy. Experimentation, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, tartrate-resistant acid phosphate (TRAP) staining, pit formation assay, immunofluorescence, western blotting, and real-time polymerase chain reaction (PCR) was performed to investigate the effects of metformin on osteoclast differentiation. Additionally, to investigate its association with AMPK and pathways, the AMPK inhibitor compound C and mammalian targets of rapamycin (mTOR) activator leucine were used to examine the expression of osteoclast- or autophagy-related proteins, genes, and TRAP-positive cells. Metformin showed no cytotoxic effects on mouse osteoblastic cell lines (MC3T3-E1) and murine macrophage cell lines (RAW264.7) but did inhibit osteoclast differentiation. Furthermore, metformin was found to inhibit osteoclast differentiation through AMPK activation and mTOR inhibition. In turn, AMPK inhibition using compound C promoted osteoclast differentiation, and mTOR activation using leucine inhibited autophagy and osteoclast differentiation. Metformin activates the AMPK pathway while functioning as an activator of mTOR, thereby leading to the inhibition of autophagy and osteoclast differentiation.

Vitamin D may assist the UPR against sodium fluoride-induced damage by reducing RIPK1, ATG5, BECN1, oxidative stress and increasing caspase-3 in the osteoblast MC3T3-E1 cell line

BackgroundOut of all measure systemic exposure to fluorides can cause defect of skeletal and dental fluorosis. Endoplasmic reticulum (ER) stress is caused by fluorine-induced oxidative stress and importance of vitamin D in its prevention is not known enough in bone cells. This study was carried out to investigate fluorine-induced oxidative stress, ER stress, and death pathways and the effect of vitamin D on them. MethodsMC3T3-E1 mouse osteoblast cell line was used as the material of the study. The NaF and vitamin D concentrations were determined by the MTT assay. NaF treatments and vitamin D supplementation (pre-add, co-add, and post-add) was administered in the cell line at 24th and 48th hours. The expression of the genes in oxidative stress, ER stress, and death pathways was determined using RT-qPCR and Western blotting techniques. ResultsVitamin D significantly reduced mRNA expression levels of SOD2, CYGB, ATF6, PERK, IRE1, ATG5 and BECN1 whereas caused an increase in levels GPX1, SOD1, NOS2 and Caspase-3 in MC3T3-E1 mouse osteoblast cell line of NaF-induced. In addition, GPX1, SOD1, ATF6, PERK, IRE1, BECN1, Caspase-3 and RIPK1 protein levels were examined by Western blot analysis, and it was determined that vitamin D decreased IRE1 and PERK protein levels, but increased GPX1, SOD1, ATF6 and Caspase-3 protein levels. ConclusionThe findings of the study suggest that vitamin D has protective potential against NaF-induced cytotoxicity reasonably through the attenuation of oxidative stress, ER stress, ATG5, IRE1 and by increasesing caspase-3 in vitro conditions.