Mouse Osteoblastic MC3T3-E1 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.

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.

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.

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.

Study on Bioresponsive Gelatin-Hyaluronic Acid-Genipin Hydrogel for High Cell-Density 3D Bioprinting.

The Development of bioresponsive extrudable hydrogels for 3D bioprinting is imperative to address the growing demand for scaffold design as well as efficient and reliable methods of tissue engineering and regenerative medicine. This study proposed genipin (5 mg) cross-linked gelatin (1 to 1.5 g)-hyaluronic acid (0.3 g) hydrogel bioink (20 mL) tailored for 3D bioprinting. The focus is on high cell loading and a less artificial extra-cellular matrix (ECM) effect, as well as exploring their potential applications in tissue engineering. The bioresponsiveness of these hydrogel scaffolds was successfully evaluated at 37 °C and room temperature (at pH 2.5, 7.4, and 9). The rheological and mechanical properties (more than three times) increased with the increase in gelatin content in the hydrogel; however, the hydrogel with the least amount of gelatin showed the best extrusion capability. This optimized hydrogel's high extrusion ability and post-printing shape fidelity were evident from 3D and four-axis printing of complex structures such as hollow tubes, stars, pyramids, and zigzag porous tubular (four-axis) scaffolds (printed at 90 kPa pressure, 70 mm/s speed, 22G needle, fourth axis rotation of 4 rpm). 3 million/mL MC3T3-E1 mouse osteoblast cells were used in preparing 3D bioprinted samples. The in vitro cell culture studies have been carried out in a CO2 incubator (at 37 °C, 5% CO2). In the cytocompatibility study, almost three times more cell viability was observed in 3 days compared to day 1 control, proving the non-toxicity and cell-supportiveness of these hydrogels. High cell viability and cell-to-cell interactions observed at the end of day 3 using this moderately stable hydrogel in 3D bioprinting exhibit high potential for precise cell delivery modes in tissue engineering as well as regenerative medicine.

Upregulation of TGF-β-induced HSP27 by HSP90 inhibitors in osteoblasts

BackgroundHeat shock protein (HSP) 90 functions as a molecular chaperone and is constitutively expressed and induced in response to stress in many cell types. We have previously demonstrated that transforming growth factor-β (TGF-β), the most abundant cytokine in bone cells, induces the expression of HSP27 through Smad2, p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in mouse osteoblastic MC3T3-E1 cells. This study investigated the effects of HSP90 on the TGF-β-induced HSP27 expression and the underlying mechanism in mouse osteoblastic MC3T3-E1 cells.MethodsClonal osteoblastic MC3T3-E1 cells were treated with the HSP90 inhibitors and then stimulated with TGF-β. HSP27 expression and the phosphorylation of Smad2, p44/p42 MAPK, p38 MAPK, and SAPK/JNK were evaluated by western blot analysis.ResultHSP90 inhibitors 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) and onalespib significantly enhanced the TGF-β-induced HSP27 expression. TGF-β inhibitor SB431542 reduced the enhancement by 17-DMAG or onalespib of the TGF-β-induced HSP27 expression levels. HSP90 inhibitors, geldanamycin, onalespib, and 17-DMAG did not affect the TGF-β-stimulated phosphorylation of Smad2. Geldanamycin did not affect the TGF-β-stimulated phosphorylation of p44/p42 MAPK or p38 MAPK but significantly enhanced the TGF-β-stimulated phosphorylation of SAPK/JNK. Onalespib also increased the TGF-β-stimulated phosphorylation of SAPK/JNK. Furthermore, SP600125, a specific inhibitor for SAPK/JNK, significantly suppressed onalespib or geldanamycin’s enhancing effect of the TGF-β-induced HSP27 expression levels.ConclusionOur results strongly suggest that HSP90 inhibitors upregulated the TGF-β-induced HSP27 expression and that these effects of HSP90 inhibitors were mediated through SAPK/JNK pathway in osteoblasts.

Factors secreted by monosodium urate crystal-stimulated macrophages promote a proinflammatory state in osteoblasts: a potential indirect mechanism of bone erosion in gout

BackgroundTophi are lesions commonly present at sites of bone erosion in gout-affected joints. The tophus comprises a core of monosodium urate (MSU) crystals surrounded by soft tissue that contains macrophages and other immune cells. Previous studies found that MSU crystals directly reduce osteoblast viability and function. The aim of the current study was to determine the indirect, macrophage-mediated effects of MSU crystals on osteoblasts.MethodsConditioned medium from the RAW264.7 mouse macrophage cell line cultured with MSU crystals was added to the MC3T3-E1 mouse osteoblastic cell line. Conditioned medium from the THP-1 human monocytic cell line cultured with MSU crystals was added to primary human osteoblasts (HOBs). Matrix mineralization was assessed by von Kossa staining. Gene expression was determined by real-time PCR, and concentrations of secreted factors were determined by enzyme-linked immunosorbent assay.ResultsIn MC3T3-E1 cells cultured for 13 days in an osteogenic medium, the expression of the osteoblast marker genes Col1a1, Runx2, Sp7, Bglap, Ibsp, and Dmp1 was inhibited by a conditioned medium from MSU crystal-stimulated RAW264.7 macrophages. Mineral staining of MC3T3-E1 cultures on day 21 confirmed the inhibition of osteoblast differentiation. In HOB cultures, the effect of 20 h incubation with a conditioned medium from MSU crystal-stimulated THP-1 monocytes on osteoblast gene expression was less consistent. Expression of the genes encoding cyclooxygenase-2 and IL-6 and secretion of the proinflammatory mediators PGE2 and IL-6 were induced in MC3T3-E1 and HOBs incubated with conditioned medium from MSU crystal-stimulated macrophages/monocytes. However, inhibition of cyclooxygenase-2 activity and PGE2 secretion from HOBs indicated that this pathway does not play a major role in mediating the indirect effects of MSU crystals in HOBs.ConclusionsFactors secreted from macrophages stimulated by MSU crystals attenuate osteoblast differentiation and induce the expression and secretion of proinflammatory mediators from osteoblasts. We suggest that bone erosion in joints affected by gout results from a combination of direct and indirect effects of MSU crystals.

Exosomes from dental pulp cells attenuate bone loss in mouse experimental periodontitis.

Exosomes are small vesicles secreted from many cell types. Their biological effects largely depend on their cellular origin and the physiological state of the originating cells. Exosomes secreted by mesenchymal stem cells exert therapeutic effects against multiple diseases and may serve as potential alternatives to stem cell therapies. We previously established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this study, we aimed to investigate the effect of local administration of HHH-DPC exosomes in a mouse model of periodontitis. Exosomes purified from HHH-DPCs were subjected to particle size analysis, and expression of exosome markers was confirmed by western blotting. We also confirmed the effect of exosomes on the migration of both HHH-DPCs and mouse osteoblastic MC3T3-E1 cells. A mouse experimental periodontitis model was used to evaluate the effect of exosomes in vivo. The morphology of alveolar bone was assessed by micro-computed tomography (μCT) and histological analysis. The effect of exosomes on osteoclastogenesis was evaluated using a co-culture system. The exosomes purified from HHH-DPCs were homogeneous and had a spherical membrane structure. HHH-DPC exosomes promoted the migration of both human DPCs and mouse osteoblastic cells. The MTT assay showed a positive effect on the proliferation of human DPCs, but not on mouse osteoblastic cells. Treatment with HHH-DPC exosomes did not alter the differentiation of osteoblastic cells. Imaging with µCT revealed that the exosomes suppressed alveolar bone resorption in the mouse model of periodontitis. Although no change was apparent in the dominance of TRAP-positive osteoclast-like cells in decalcified tissue sections upon exosome treatment, HHH-DPC exosomes significantly suppressed osteoclast formation in vitro. HHH-DPC exosomes stimulated the migration of human DPCs and mouse osteoblastic cells and effectively attenuated bone loss due to periodontitis.

Mineralized polyplexes for gene delivery: Improvement of transfection efficiency as a consequence of calcium incubation and not mineralization

Gene therapy is an emerging field in which nucleic acids are used to control protein expression. The necessity of delivering nucleic acids to specific cell types and intracellular sites demands the use of highly specialized gene carriers. As a carrier modification technique, mineralization has been successfully used to modify viral and non-viral carriers, providing new properties that ultimately aim to increase the transfection efficiency. However, for the specific case of polyplexes used in gene therapy, recent literature shows that interaction with calcium, a fundamental step of mineralization, might be effective to increase transfection efficiency, leaving an ambiguity about of the role of mineralization for this type of gene carriers. To answer this question and to reveal the properties responsible for increasing transfection efficiency, we mineralized poly(aspartic acid) coated polyplexes at various CaCl2 and Na3PO4 concentrations, and evaluated the resultant carriers for physicochemical and morphological characteristics, as well as transfection and delivery efficiency with MC3T3-E1 mouse osteoblastic cells. We found that both mineralization and calcium incubation positively affected the transfection efficiency and uptake of polyplexes in MC3T3-E1 cells. However, this effect originated from the properties achieved by polyplexes after the calcium incubation step that are maintained after mineralization, including particle size increase, improved pDNA binding, and adjustment of zeta potential. Considering that mineralization can be a longer process than calcium incubation, we find that calcium incubation might be sufficient and preferred if improved transfection efficiency in vitro is the only effect desired.

Serpinb1a suppresses osteoclast formation

Serpinb1a, a serine protease inhibitor family protein, has been implicated in immunoregulation and several metabolic disorders, such as diabetes and obesity; however, its roles in bone remain unknown. Therefore, we herein investigated the physiological functions of Serpinb1a in osteoclastic and osteoblastic differentiation using mouse cell lines. Serpinb1a overexpression markedly reduced the number of tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells increased by receptor activator nuclear factor κB ligand (RANKL) in mouse preosteoclastic RAW 264.7 cells. Moreover, it significantly decreased the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP and cathepsin K in these cells. Regarding osteoblasts, Serpinb1a overexpression significantly reduced the mRNA levels of alkaline phosphatase (ALP) and osteocalcin as well as ALP activity induced by bone morphogenetic protein-2 (BMP-2) in mouse mesenchymal ST2 cells, although it did not alter osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. Concerning the pathophysiological relevance of Serpinb1a, Serpinb1a mRNA levels were decreased in the soleus and gastrocnemius muscles of mice 4 weeks after bilateral sciatic nerve resection. In conclusion, we herein revealed for the first time that Serpinb1a inhibited osteoclast formation induced by RANKL in RAW 264.7 cells and suppressed BMP-2-induced ALP activity in ST2 cells.

Synthesis and evaluation of TaC nanocrystalline coating with excellent wear resistance, corrosion resistance, and biocompatibility

To improve the mechanical properties, corrosion resistance, and biocompatibility of implanted titanium alloys, a TaC nanocrystalline coating was deposited on Ti–6Al–4V alloy using a double-cathode glow discharge method. The microstructure of the newly developed coating was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The coating exhibits a dense and uniform structure, composed of equiaxed TaC grains with an average grain size of 15.2 nm. The mechanical properties of the TaC-coated Ti–6Al–4V alloy were evaluated by a scratch tester, a nanoindentation tester, and a ball-on-disc tribometer. The average hardness of the TaC nanocrystalline coating is about 6 times higher than that of uncoated Ti–6Al–4V alloy and the specific wear rate of the coating is two orders of magnitude lower than that for Ti–6Al–4V at applied normal loads of 4.9 N under dry sliding condition. The electrochemical behavior of the TaC nanocrystalline coating after soaking in Ringer's solution for different periods was investigated by potentiodynamic polarization and electrochemical impedance spectroscopy (EIS). Furthermore, in vitro cytocompatibility of the coating was assessed using MC3T3-E1 mouse osteoblastic cells. The results showed that the TaC coating exhibits better corrosion resistance and biocompatibility as compared to uncoated Ti–6Al–4V alloy.

Arbutin ameliorates glucocorticoid-induced osteoporosis through activating autophagy in osteoblasts.

Chronic long-term glucocorticoid use causes osteoporosis partly by interrupting osteoblast homeostasis and exacerbating bone loss. Arbutin, a natural hydroquinone glycoside, has been reported to have biological activities related to the differentiation of osteoblasts and osteoclasts. However, the role and underlying mechanism of arbutin in glucocorticoid-induced osteoporosis are elusive. In this study, we demonstrated that arbutin administration ameliorated osteoporotic disorders in glucocorticoid dexamethasone (Dex)-induced mouse model, including attenuating the loss of bone mass and trabecular microstructure, promoting bone formation, suppressing bone resorption, and activating autophagy in bone tissues. Furthermore, Dex-stimulated mouse osteoblastic MC3T3-E1 cells were treated with arbutin. Arbutin treatment rescued Dex-induced repression of osteoblast differentiation and mineralization, the downregulation of osteogenic gene expression, reduced autophagic marker expression, and decreased autophagic puncta formation. The application of autophagy inhibitor 3-MA decreased autophagy, differentiation, and mineralization of MC3T3-E1 cells triggered by arbutin. Taken together, our findings suggest that arbutin treatment fends off glucocorticoid-induced osteoporosis, partly through promoting differentiation and mineralization of osteoblasts by autophagy activation.

Contribution of Glucosylceramide Synthase to the Proliferation of Mouse Osteoblasts.

Glycosphingolipids are known to be involved in bone metabolism. However, their roles and regulatory mechanisms in osteoblast proliferation are largely unknown. In this study, we examined the effects of inhibitors of glucosylceramide synthase (GCS), which is responsible for the generation of all glycosphingolipids, on osteoblast proliferation. We analyzed the expression of glycosphingolipids and cell growth in MC3T3-E1 mouse osteoblast cells treated with the GCS inhibitors miglustat, D-PDMP and D-PPMP. We also conducted microarray analysis and RNA interference to identify genes involved in cell growth regulated by GCS. Glycosphingolipids GD1a and Gb4 expressed in MC3T3-E1 cells, were suppressed by GCS inhibitors. Furthermore, the proliferation of MC3T3-E1 cells was suppressed by the inhibitors. Using microarray analysis, we predicted nine genes (Fndc1, Acta2, Igfbp5, Cox6a2, Cth, Mymk, Angptl6, Mab21l2, and Igsf10) suppressed by all three inhibitors. Furthermore, partial silencing of Angptl6 by RNA interference reduced MC3T3-E1 cell growth. These results show that GCS regulates proliferation through Angptl6 in osteoblasts.

Cytotoxicity of Ti–6Al–4V–5Cu Alloy to MC3T3-E1 Cells

The aim of this work was to study the cytotoxicity of Ti–6Al–4V–5Cu (TC4–Cu) alloy for dental applications and evaluate the cell viability of different fabricated TC4–Cu alloys in contact with MC3T3-E1 cells in vitro. Ti–6Al–4V (TC4) alloy was used as a negative control to evaluate the cytotoxicity level of TC4–Cu alloy so as to provide basic support for the dental clinical application. Control group TC4 and experimental group TC4–Cu with different fabrications were incubated in the cell culture medium. The absorbance value of mouse osteoblast MC3T3-E1 cells was detected by CCK-8 assay after 1, 3 and 7 days, respectively. The relative proliferation rate of cells was calculated, and then the toxicity level was valued for each group. Cell morphology on the surface was also studied by observing the cytoskeleton through F-actin filament staining. The experimental results showed that the absorbance values for the experimental and the negative control groups were not same at different time points. Compared with different fabricated TC4–Cu alloys, the annealing-TC4–Cu alloy showed much better biocompatibility. The mouse osteoblast MC3T3-E1 cells cultured with annealing-TC4–Cu, rolling-TC4–Cu and solution + aging-TC4–Cu have no toxic effects, and these alloys could promote the proliferation of mouse osteoblasts.

Investigation of The Cellular Response to Bone Fractures: Evidence for Flexoelectricity

The recent discovery of bone flexoelectricity (strain-gradient-induced electrical polarization) suggests that flexoelectricity could have physiological effects in bones, and specifically near bone fractures, where flexoelectricity is theoretically highest. Here, we report a cytological study of the interaction between crack stress and bone cells. We have cultured MC3T3-E1 mouse osteoblastic cells in biomimetic microcracked hydroxyapatite substrates, differentiated into osteocytes and applied a strain gradient to the samples. The results show a strong apoptotic cellular response, whereby mechanical stimulation causes those cells near the crack to die, as indicated by live-dead and caspase staining. In addition, analysis two weeks post-stimulation shows increased cell attachment and mineralization around microcracks and a higher expression of osteocalcin –an osteogenic protein known to be promoted by physical exercise. The results are consistent with flexoelectricity playing at least two different roles in bone remodelling: apoptotic trigger of the repair protocol, and electro-stimulant of the bone-building activity of osteoblasts.

Haem oxygenase-1 induction prevents glucocorticoid-induced osteoblast apoptosis through activation of extracellular signal–regulated kinase1/2 signalling pathway

BackgroundHigh-dose glucocorticoid (GC) therapy always causes osteoporosis partly by inducing osteoblast apoptosis. However, the underlying mechanisms of GC-induced apoptosis remain elusive. Haem oxygenase-1 (HO-1) is a cytoprotective protein that rescues cells from H2O2 or high glucose–induced apoptosis. In bone metabolism, HO-1 also participates in osteoclast and osteoblast differentiation. ObjectiveThe present study aimed to investigate the protective role of HO-1 against GC-induced osteoblast apoptosis and to elucidate the underlying mechanism. MethodsMouse osteoblastic MC3T3-E1 cells were treated with dexamethasone (Dex) for 24 h in the presence or absence of cobalt (III) protoporphyrin IX chloride (CoPP, an inducer of HO-1). In some experiments, U0126 was added to the culture 1 h before CoPP treatment. The induction of apoptosis was determined by flow cytometry. Cell viability was evaluated using a cell counting kit-8 (CCK-8) assay. The expression levels of Bax and bcl-2 were measured by real-time polymerase chain reaction and Western blot. HO-1, extracellular signal–regulated kinase (ERK)-1/2 and pERK1/2 protein levels were measured by Western blot analysis. ResultsDex promoted apoptosis and inhibited cell viability in MC3T3-E1 cells. In addition, Dex significantly increased Bax expression and reduced Bcl-2 expression. The expression of HO-1 was also reduced after Dex treatment. HO-1 induction by CoPP significantly attenuated Dex-induced apoptosis as evidenced by Annexin V/PI staining. The mRNA expression level of antiapoptotic gene Bcl-2 was also increased after CoPP treatment. Moreover, CoPP treatment increased the phosphorylation of ERK1/2. U0126, an inhibitor of ERK activation, significantly abrogated the protective effects of CoPP. ConclusionOur results demonstrate that HO-1 induction by CoPP can attenuate Dex-induced apoptosis of mouse osteoblastic MC3T3-E1 cells. The antiapoptotic effect of HO-1 induction may be correlated with the activation of ERK1/2 signalling pathway.The translational potential of this article: HO-1 induction by CoPP can prevent GC-induced osteoblast apoptosis. Our findings will highlight the therapeutic potential of HO-1 induction in GC-induced osteoporosis.

Joint Association Analysis Identified 18 New Loci for Bone Mineral Density.

Bone mineral density (BMD) at various skeletal sites have shared genetic determinants. In the present study, aiming to identify shared loci associated with BMD, we conducted a joint association study of a genomewide association study (GWAS) and a meta-analysis of BMD at different skeletal sites: (i) a single GWAS of heel BMD in 142,487 individuals from the UK Biobank, and (ii) a meta-analysis of 30 GWASs of total body (TB) BMD in 66,628 individuals from the Genetic Factors for Osteoporosis (GEFOS) Consortium. The genetic correlation coefficient of the two traits was estimated to be 0.57. We performed joint association analysis with a recently developed statistical method multi-trait analysis of GWAS (MTAG) to account for trait heterogeneity and sample overlap. The joint association analysis combining samples of up to 209,115 individuals identified 18 novel loci associated with BMD at the genomewide significance level (α = 5.0 × 10-8 ), explaining an additional 0.43% and 0.60% of heel-BMD and TB-BMD heritability, respectively. The vast majority of the identified lead SNPs or their proxies exerted local expression quantitative trait loci (cis-eQTL) activity. Credible risk variants, defined as those SNPs located within 500 kilobases (kb) of the lead SNP and with p values within two orders of magnitude of the lead SNP, were enriched in transcription factor binding sites (p = 3.58 × 10-4 ) and coding regions (p = 5.71 × 10-4 ). Fifty-six candidate genes were prioritized at these novel loci using multiple sources of information, including several genes being previously reported to play a role in bone biology but not reported in previous GWASs (PPARG, FBN2, DEF6, TNFRSF19, and NFE2L1). One newly identified gene, SCMH1, was shown to upregulate the expression of several bone biomarkers, including alkaline phosphatase (ALP), collagen type 1 (COL-I), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor 2 (RUNX2), in mouse osteoblastic MC3T3-E1 cells, highlighting its regulatory role in bone formation. Our results may provide useful candidate genes for future functional investigations. © 2019 American Society for Bone and Mineral Research.

Effects of aspirin-loaded graphene oxide coating of a titanium surface on proliferation and osteogenic differentiation of MC3T3-E1 cells

Graphene oxide (GO) has attracted considerable attention for biomedical applications such as drug delivery because of its two-dimensional structure, which provides a large surface area on both sides of the nanosheet. Here, a new method for titanium (Ti) surface modification involving a GO coating and aspirin (A) loading (A/Ti-GO) was developed, and the bioactive effects on mouse osteoblastic MC3T3-E1 cells were preliminarily studied. The X-ray photoelectron spectrometry indicated new C-O-N, C-Si-O-C, and C-N=C bond formation upon GO coating. Remarkably, the torsion test results showed stable bonding between the GO coating and Ti under a torsional shear force found in clinical settings, in that, there was no tearing or falling off of GO coating from the sample surface. More importantly, through π-π stacking interactions, the release of aspirin loaded on the surface of Ti-GO could sustain for 3 days. Furthermore, the A/Ti-GO surface displayed a significantly higher proliferation rate and differentiation of MC3T3-E1 cells into osteoblasts, which was confirmed by a water-soluble tetrazolium salt-8 (WST-8) assay and alkaline phosphatase activity test. Consequently, Ti surface modification involving GO coating and aspirin loading might be a useful contribution to improve the success rate of Ti implants in patients, especially in bone conditions.

A new insight into cystatin C contained in milk basic protein to bone metabolism: Effects on osteoclasts and osteoblastic MC3T3-E1 cells invitro.

A milk protein fraction possessing alkaline isoelectric points (milk basic protein [MBP]) improves bone metabolism invivo, and it inhibits bone resorption by osteoclasts and promotes mouse osteoblastic MC3T3-E1 cells proliferation invitro. Cystatin C (CysC) is a component of MBP and shows bone resorption inhibitory activity. Therefore, it is likely that MBP with higher CysC content improves bone metabolism more effectively. In this study, we prepared MBP with low and high contents of CysC and compared its effects on bone metabolism with standard MBP invitro. Our results showed that the CysC content in MBP was positively related to not only bone resorption inhibitory activity but also MC3T3-E1 cells proliferative activity. Furthermore, purified CysC stimulated MC3T3-E1 cells proliferation. These results indicate that CysC contributes to promotion of MC3T3-E1 cells proliferation, and MBP with higher CysC content shows enhanced bone resorption inhibitory activity and MC3T3-E1 cells proliferative activity. CysC is considered an important factor in the effect on bone metabolism of MBP.