Human Osteoblastic MG-63 Cells Research Articles

Icariin, a phytoestrogen, exerts rapid estrogenic actions through crosstalk of estrogen receptors in osteoblasts.

Icariin, a flavonoid glycoside derived from Epimedium brevicornum Maxim, exerts bone protective effects via estrogen receptors (ERs). This study aimed to investigate the role of ER-α66, ER-α36, and GPER in bone metabolism in osteoblasts following treatment with icariin. Human osteoblastic MG-63 cells and osteoblast-specific ER-α66 knockout mice were employed. The ERs crosstalk in the estrogenic action of icariin was evaluated in ER-α66-negative human embryonic kidney HEK293 cells. Icariin, like E2, regulated ER-α36 and GPER protein expression in osteoblasts by downregulating them and upregulating ER-α66. ER-α36 and GPER suppressed the actions of icariin and E2 in bone metabolism. However, the in vivo administration of E2 (2 mg/kg/day) or icariin (300 mg/kg/day) restored bone conditions in KO osteoblasts. ER-α36 and GPER expression increased significantly and rapidly activated and translocated in KO osteoblasts after treatment with E2 or icariin. ER-α36 overexpression in KO osteoblasts further promoted the OPG/RANKL ratio induced by E2 or icariin treatment. This study showed icariin and E2 elicit rapid estrogenic responses in bone through recruiting ER-α66, ER-α36, and GPER. Notably, in osteoblasts lacking ER-α66, ER-α36, and GPER mediate the estrogenic effects of icariin and E2, while in intact osteoblasts, ER-α36 and GPER act as negative regulators of ER-α66.

Synthesis and Biological Properties of Alanine-Grafted Hydroxyapatite Nanoparticles.

Hydroxyapatite attracts great attention as hard tissues implant material for bones and teeth. Its application in reconstructive medicine depends on its biocompatibility, which is in a function of composition and surface properties. The insertion of a protein element in the composition of implants can improve the cell adhesion and the osseointegration. Having this in mind, the proposal of this work was to develop L-alanine-grafted hydroxyapatite nanoparticles and to study their biocompatibility. Two L-alanine sources and three grafting methods were used for hydroxyapatite surface functionalization. The efficiency of grafting was determined based on X-ray powder diffraction, Fourier-transform infrared spectroscopy, thermal analyses, and field-emission scanning electron microscopy. The results indicated the formation of hydroxyapatite with 8-25 wt% of organic content, depending on the grafting method. Protein adsorption, cell adhesion, and viability studies were carried out to evaluate biological properties of grafted materials. The viability of MG-63 human osteoblastic cells following 24 h incubation with the alanine-grafted hydroxyapatite samples is well preserved, being in all cases above the viability of cells incubated with hydroxyapatite. The alanine-grafted hydroxyapatite prepared in situ and by simple mixture showed higher protein adsorption and cell adhesion, respectively, indicating their potential toward use in regenerative medicine.

Fluorosilicic acid and cotinine, separately and in combination, induce genotoxicity and telomeric reduction in human osteoblast cell line MG63

Skeletal fluorosis is a severe case in which bone deformations and bone tissue weakening occur due to excessive fluorine deposition. Recently, data on smoking have been published that smoke constituents can indirectly influence bone mass and interfere in the metabolism of fluorides in humans. Thus, the present in vitro study aimed to assess the genetic instability in human osteoblast MG63 cells exposed to fluorosilicic acid (FA) and cotinine (COT), separately and in combination, in concentrations found in human plasma. For this, cell cytotoxicity was performed by MTT assay; DNA damage was performed by alkaline comet assay (CA), modified by repair endonucleases (+FPG); micronuclei test (MN) using CBMN-Cyt assay; and telomere length (TL) by qPCR in MG63 cells. No cytotoxicity was observed for all concentrations tested in this study. Alkaline CA results showed a significant increase in DNA damage at all FA concentrations (0.03125–0.300 mg/L), in the two highest concentrations of COT (125 and 250 ng/mL), and the highest concentration of FA+COT (0.300 mg/L+250 ng/mL). Alkaline CA+FPG test was used to detect oxidized nucleobases, which occurred at the two highest concentrations of FA, COT, and FA+COT. Micronuclei test showed an increase in the frequency of MN at all concentrations of FA (0.075–0.300 mg/L) except in the lowest concentration (0.03125 mg/L), in the two highest concentrations of COT (125 and 250 ng/mL), and all concentrations of FA+COT. There was no significant difference in nuclear division index, binucleated cells, nucleoplasmic bridge, and nuclear bud. A TL reduction was observed in cells treated with the highest concentrations of FA alone (0.300 mg/L) and FA+COT (0.300 mg/L+250 ng/mL). Finally, our study showed that FA and COT (mainly alone) at concentrations found in human plasma induced oxidative damage and genetic instability in human osteoblast cells.

Design of the double-layer biocompatible coating on AZ31 magnesium alloy for highly effective corrosion resistance

A double-layer phytic acid@cerium/polycaprolactone@cerium substituted hydroxyapatite (PA@Ce/PCL@Ce-HA) composite coating was prepared on the AZ31 Mg alloys combining the cyclic assembly and electrospinning techniques. SEM images showed that the out layer of PA@Ce/PCL@Ce-HA coating displayed a typical disorderly distributed nanofiber structure, and the average diameter of the PCL@Ce-HA fibers was 680 ± 45 nm. ATR-FTIR and XPS analysis demonstrated the existence of typical characteristic peaks of PO43− groups in the composite coating, indicating PA molecular and Ce-HA nanoparticles were successfully anchored to the alkaline pretreated Mg substrate. The results showed that this composite coating was composed of an inner PA@Ce complex and an outer PCL@Ce-HA nanofiber coating. The cross-section images verified that the thickness of the composite coating was 74.94 ± 2.14 μm. The coating adhesion tests indicated that the bonding strength between outer fibrous PCL@Ce-HA and substrates were improved, owning to the mechanical interaction and entanglement between fibrous coatings and the rough PA@Ce coating. Electrochemical tests showed the corrosion current density of PA@Ce/PCL@Ce-HA coated sample significantly dropped by two orders of magnitude compared to uncoated sample, indicating this coating significantly reduced the corrosion rate of the substrates. In addition, the lowest amount of hydrogen evolution and the minimal variation of pH value in the in-vitro immersion tests further confirmed that the PA@Ce/PCL@Ce-HA coating had a long-term stable corrosion protection for the substrates. Furthermore, the cell experiments showed that the double-layer coating provided an interface similar to the extracellular matrix (ECM), which was conducive to the attachment and proliferation of human osteoblast MG-63 cells. The hemolysis rate (HR) of PA@Ce/PCL@Ce-HA coated sample was 1.69%, which met the requirements of biomedical material applications. Thus, the outcomes of this work provided a new sight for corrosion protection of the Mg alloys and this coating has great potential in orthopedic applications.

Mechanically Stable β-TCP Structural Hybrid Scaffolds for Potential Bone Replacement

The authors report on the manufacturing of mechanically stable β-tricalcium phosphate (β-TCP) structural hybrid scaffolds via the combination of additive manufacturing (CerAM VPP) and Freeze Foaming for engineering a potential bone replacement. In the first step, load bearing support structures were designed via FE simulation and 3D printed by CerAM VPP. In the second step, structures were foamed-in with a porous and degradable calcium phosphate (CaP) ceramic that mimics porous spongiosa. For this purpose, Fraunhofer IKTS used a process known as Freeze Foaming, which allows the foaming of any powdery material and the foaming-in into near-net-shape structures. Using a joint heat treatment, both structural components fused to form a structural hybrid. This bone construct had a 25-fold increased compressive strength compared to the pure CaP Freeze Foam and excellent biocompatibility with human osteoblastic MG-63 cells when compared to a bone grafting Curasan material for benchmark.

Anti-Osteoporotic Activity of Pueraria lobata Fermented with Lactobacillus paracasei JS1 by Regulation of Osteoblast Differentiation and Protection against Bone Loss in Ovariectomized Mice

Osteoporosis is the most common bone disease associated with low bone mineral density. It is the process of bone loss and is most commonly caused by decreased estrogen production in women, particularly after menopause. Pueraria lobata, which contains various metabolites, especially isoflavone, is widely known as regulator for bone mineral contents. In this study, the effects of the P. lobata extract (PE) with or without fermentation with Lactobacillus paracasei JS1 (FPE) on osteoporosis were investigated in vitro and in vivo. The effects of PE and FPE on human osteoblastic MG63 cells, RAW 264.7 cells, and ovariectomized (OVX)-induced model mice were analyzed at various ratios. We found that FPE increased calcium deposition and inhibited bone resorption by in vitro assay. Furthermore, treatment with PE and FPE has significantly restored destroyed trabecular bone in the OVX-induced bone loss mouse model. Overall, FPE demonstrated bioactivity to prevent bone loss by decreasing bone turnover.

Influence of surface termination of ultrananocrystalline diamond films coated on titanium on response of human osteoblast cells: A proteome study

Successful osseointegration, i.e. the fully functional connection of patient's bone and artificial implant depends on the response of the cells to the direct contact with the surface of the implant. The surface properties of the implant which trigger cell responses leading to its integration into the surrounding bone can be tailored by surface modifications or coating with thin layers. One potential material for such applications is ultrananocrystalline diamond (UNCD). It combines the exceptional mechanical properties of diamond with good biocompatibility and possibility of coating as thin uniform films on different substrates of biological interest. In the current work we firstly deposited UNCD films on titanium-coated substrates and applied oxygen or ammonia plasma to modify their surface properties. The as-grown and modified UNCD exhibited relatively smooth surfaces with topography dominated by rounded features. The modifications induced oxygen- or amino-terminated surfaces with increased hydrophilicity. In addition, the UNCD coatings exhibited very low coefficient of friction when diamond was used as a counterpart. As-grown and modified UNCD samples were applied to study the responses of human osteoblast MG63 cells triggered by surfaces with various terminations assessed by proteomic analysis. The results revealed that the coating of Ti with UNCD as well as the plasma modifications resulting in O- or NH2-terminated UNCD induced upregulation of proteins specific for cytoskeleton, cell membrane, and extracellular matrix (ECM) involved in the cell-ECM-surface interactions. Proteins from each of these groups, namely, vimentin, cadherin and fibronectin were further studied immunocytochemically and the results confirmed their increased abundance leading to improved cell-to-surface adhesion and cell-to-cell interactions. These findings demonstrate the potential of implant coating with UNCD and its surface modifications for better osseointegration and bone formation.

Development and Characterization of Biomedical Porous Ti–20Nb–5Ag Alloy: Microstructure, Mechanical Properties, Surface Bioactivity and Cell Viability Studies

In this study, antibacterial Ag element added to synthesis of porous Ti–20Nb–5Ag (wt%) alloy using powder metallurgy space holder route. The microstructural, mechanical property, surface bioactivity and cytotoxicity behavior of porous Ti–20Nb–5Ag alloy have been investigated. The developed porous alloy obtained the porosities ranging from 22.5 to 68%. The porous sample having a porosity of about 43% is found to be in the optimum condition, which possess a modulus of about 5.8 GPa with an excellent compressive strength about 205 MPa. XRD result shows that the formation of small amount of α-Ti, β-Ti, along with α״ martensite and Ti2Ag are key phase constituents of sintered porous Ti–20Nb–5Ag alloys. The compression strength and elastic modulus of the sintered alloys were showed that decreased with increase of porosity. Surface bioactivity result revealed that the significant formation of hydroxyapatite on the alkali-heat treated (5 M NaOH) porous Ti–20Nb–5Ag alloy which is found after the in vitro test in SBF. Further, the cell viability test was conducted on as-synthesized porous Ti–20Nb–5Ag alloy for 1, 4, and 7 days using MG-63 human osteoblast cells and result shows an excellent cell proliferation, and the cytotoxicity test confirms the non-toxic nature of the porous alloy which is very much suitable for implant application.

Enhanced Osseointegration by the Hierarchical Micro-Nano Topography on Selective Laser Melting Ti-6Al-4V Dental Implants.

Currently, selective laser melting (SLM) has been thriving in implant dentistry for on-demand fabricating dental implants. Based on the coarse microtopography of SLM titanium surfaces, constructing nanostructure to form the hierarchical micro-nano topography is effective in enhancing osseointegration. Given that current nanomodification techniques of SLM implants, such as anodization and hydrothermal treatment, are facing the inadequacy in costly specific apparatus and reagents, there has been no recognized nanomodified SLM dental implants. The present study aimed to construct hierarchical micro-nano topography on self-made SLM dental implants by a simple and safe inorganic chemical oxidation, and to evaluate its contribution on osteoblastic cells bioactivity and osseointegration. The surface chemical and physical parameters were characterized by FE-SEM, EDS, profilometer, AFM, and contact angle meter. The alteration on bioactivity of MG-63 human osteoblastic cells were detected by qRT-PCR. Then the osseointegration was assessed by implanting implants on the femur condyle of New Zealand Rabbits. The hierarchical micro-nano topography was constituted by the microrough surface of SLM implants and nanoneedles (diameter: 20∼50 nm, height: 150∼250 nm), after nanomodifying SLM implants in 30% hydrogen peroxide and 30% hydrochloride acid (volume ratio 1:2.5) at room temperature for 36 h. Low chemical impurities content and high hydrophilicity were observed in the nanomodified group. Cell experiments on the nanomodified group showed higher expression of mitophagy related gene (PINK1, PARKIN, LC3B, and LAMP1) at 5 days and higher expression of osteogenesis related gene (Runx2 and OCN) at 14 days. In the early stage of bone formation, the nanomodified SLM implants demonstrated higher bone-to-implant contact. Intriguingly, the initial bone-to-implant contact of nanomodified SLM implants consisted of more mineralized bone with less immature osteoid. After the cessation of bone formation, the bone-to-implant contact of nanomodified SLM implants was equal to untreated SLM implants and marketable TixOs implants. The overall findings indicated that the inorganic chemical oxidized hierarchical micro-nano topography could enhance the bioactivity of osteoblastic cells, and consequently promote the peri-implant bone formation and mineralization of SLM dental implants. This study sheds some light on improvements in additive manufactured dental implants.

Regulation of human β-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human β-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

Polyhedral Oligomeric Silsesquioxane /Platelets Rich Plasma/Gelrite-Based Hydrogel Scaffold for Bone Tissue Engineering.

Polyhedral oligomeric silsesquioxane (POSS) is a monomer with silicon structure and an internal nanometric cage. The purpose of this study was to provide an injectable hydrogel that could be easily located in open or closed bone fractures and injuries, and also to reduce the possible risks of infections caused by bone graft either as an allograft or an autograft. Various formulations of temperature sensitive hydrogels containing hydroxyapatite, Gelrite, POSS and platelets rich plasma (PRP), such as the co-gelling agent and cell growth enhancer, were prepared. The hydrogels were characterized for their injectability, gelation time, phase transition temperature and viscosity. Other physical properties of the optimized formulation including compressive stress, compressive strain and Young's modulus as mechanical properties, as well as storage and loss modulus, swelling ratio, biodegradation behavior and cell toxicity as rheometrical parameters were studied on human osteoblast MG-63 cells. Alizarin red tests were conducted to study the qualitative and quantitative osteogenic capability of the designed scaffold, and the cell adhesion to the scaffold was visualized by scanning electron microscopy. The results demonstrated that the hydrogel scaffold mechanical force and injectability were 3.34±0.44 Mpa and 12.57 N, respectively. Moreover, the scaffold showed higher calcium granules production in alizarin red staining compared to the control group. The proliferation of the cells in G4.5H1P0.03PRP10 formulation was significantly higher than in other formulations (p<0.05). The optimized Gelrite/Hydroxyapatite/POSS/PRP hydrogel scaffold has useful impacts on osteoblasts activity, and may be beneficial for local drug delivery in complications including a break or bone loss.

Efficacy and Cytotoxicity of Binary Mixtures as Root Canal Filling Solvents.

Objectives: This study reports the efficacy of two solvent mixtures on the dissolution of gutta-percha and AH Plus sealer, together with the cytotoxicity. Methods: Methyl ethyl ketone (MEK), orange oil, tetrachloroethylene, MEK/tetrachloroethylene (1:1), MEK/orange oil (1:1), and chloroform (control) were tested. Twelve groups (n = 15) of standardized stainless-steel molds filled with softened gutta-percha cones and twelve (n = 15) filled with AH Plus were immersed in the corresponding mixture or individual solvent, in an ultrasonic bath, for either 2 or 5 min. The effect of the solvents was assessed qualitatively by a topographical analysis (scanning electron microscopy) and chemical analysis (Fourier transform infrared spectroscopy), and quantitatively by a weight loss and viscoelastic property (dynamic mechanical analysis) evaluation. The cytotoxicity was assessed on MG63 human osteoblastic cells. Results: The mixtures did not show the formation of new compounds. Both presented significantly higher efficacies compared to their individual solvents, suggesting a synergistic effect. Their dissolution efficacy was similar to that of chloroform, showing high cytocompatibility. Conclusions: The proposed strategy, incorporating ultrasound agitation and profiting from the synergy of adequate solvents, might enhance root canal cleanliness allowing a single-step procedure to dissolve gutta-percha and the sealer remnants, while assuring cytocompatibility with the periapical tissues.

Enterococcus faecalis induces necroptosis in human osteoblastic MG63 cells through the RIPK3 / MLKL signalling pathway.

To explore the activation of necroptosis triggered by Enterococcus faecalis in human osteoblastic MG63 cells and provide new insights into the pathogenesis of refractory apical periodontitis. The viability of MG63 cells exposed to live E.faecalis was investigated using the cell counting kit-8 assay. The relative expressions of specific markers for necroptosis, namely p-RIPK3 and p-MLKL, were determined by western blotting. Cells pretreated with necrosulfonamide and GSK'872, which are specific inhibitors for MLKL and RIPK3, respectively, were then subjected to lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry analysis and Hoechst 33342/PI double fluorescence staining. Lentiviral-delivered short hairpin RNA (shRNA) targeting MLKL was employed to further confirm the activation of necroptosis in MG63 cells infected with E.faecalis. Transmission electron microscopy was additionally used to observe the morphological characteristics. Statistical analysis was conducted using Student's t-tests or one-way ANOVA followed by the Student-Newman-Keuls test. The infection with E.faecalis significantly inhibited the viability of MG63 cells in a multiplicity of infection- and infection time-dependent manners (P<0.05). In line with this, the expression levels of necroptosis-related markers, p-RIPK3 and p-MLKL, were significantly increased postinfection (P<0.05). Significant reductions in death rate were detected in the case of E.faecalis-infected MG63 cells following pretreatment with the inhibitors of RIPK3 and MLKL (P<0.01). Furthermore, silencing of MLKL by shRNA significantly decreased LDH release (P<0.01) and resulted in less mitochondrial swelling and vacuole-like changes, as well as reduced endoplasmic reticulum expansion. Enterococcus faecalis infection-induced necroptosis of MG63 cells via the RIPK3/MLKL signalling pathway, which may exert a negative influence on the healing process of refractory apical periodontitis. This study may offer novel insights into the pathogenesis and potential therapeutic targets of refractory apical periodontitis.

Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells.

BackgroundAvicularin is a plant-derived flavonoid used in traditional Chinese medicine to treat conditions that include ankle fracture. Bradykinin stimulated MG-63 human osteoblastic osteosarcoma cells has previously been studied in an in vitro model. This study aimed to investigate the effects of avicularin on bradykinin-treated MG-63 human osteoblastic osteosarcoma cells in vitro.Material/MethodsMG-63 cells were treated with increasing concentrations of avicularin for 48 hours, followed by 1 μM of bradykinin for 24 h. The MTT assay was used to measure cell viability. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure the expression of inflammatory mediators, interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) mRNA and protein, respectively. The expression of oxidative stress factors, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase were measured. Western blot and qRT-PCR were performed to determine p38, p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) protein levels and mRNA expression, respectively.ResultsAvicularin had no cytotoxic effect on MG-63 cells. Avicularin significantly upregulated the expression levels IL-1β, IL-6, and TNF-α in the bradykinin treated group in a dose-dependent manner. Avicularin reduced the level of MDA and the activity of SOD and catalase in the bradykinin treated MG-63 cells, reduced p-p38, p-p65, iNOS, and COX-2 expression, and decreased the p-p38/p38 ratio and the p-p65/p65 ratio in bradykinin treated MG-63 cells in a dose-dependent manner.ConclusionsAvicularin reduced the expression of inflammatory cytokines and the levels of markers of oxidative stress in MG-63 human osteoblastic osteosarcoma cells in vitro.

Tocotrienol-rich fraction from annatto ameliorates expression of lysyl oxidase in human osteoblastic MG-63 cells.

Lysyl oxidase (LOX) is required for the formation of bone collagen cross-links. Inactivation of the LOX gene in osteoblasts by DNA methylation and JAK signaling has been reported to cause loss of cross-links and an increased risk of fractures. Tocotrienols (T3s) have proven benefits on bone strength, but their potential effects on LOX remain largely unknown. Thus, the present study investigates the in vitro effects of T3s on LOX expression in human osteoblastic MG-63 cells. Results indicated that Tocotrienol-Rich Fraction (TRF), the δ-T3 rich oil extracted from Annatto was the most effective and significantly increased LOX expression. TRF treatment decreased de-novo methyltransferases (DNMTs), DNMT3A and DNMT3B levels. In addition, TRF significantly inhibited JAK2 activation and decreased expression of Fli1, a transcription factor of DNMTs. We conclude that TRF induced an increase in LOX expression via inhibition of de-novo methylation and reduction of Fli1 expression by the inactivation of JAK2.Abbreviations: CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; Fli1: friend leukemia virus integration 1; JAK: janus kinase; LOX: lysyl oxidase; PCR: polymerase chain reaction; STAT: signal transducers and activators of transcription; T3s: tocotrienols; TPs: tocopherols; TRF: Tocotrienol-Rich Fraction.

Terminal chemical functions of polyamidoamine dendrimer surfaces and its impact on bone cell growth

Besides their use for drug and gene delivery, dendrimer molecules are also favorable for the design of new surface coatings for orthopedic and dental implants due to the wide variety of functional terminal groups and their multivalent character. The purpose of this work was to observe how covalently immobilized polyamidoamine (PAMAM) dendrimer molecules with different terminal chemical groups influenced serum protein adsorption and osteoblast behavior. To this end, fifth-generation PAMAM dendrimers were immobilized on silicon surfaces with an anhydride-containing silane coupling agent which results in positively charged terminal NH2-groups. Coatings with a net negative charge were generated by introduction of terminal CO2H- or CH3-groups. Surface characterization was performed by static and dynamic contact angle and zeta potential. The in vitro studies with human MG-63 osteoblastic cells focused on cell adhesion, morphology, cell cycle, apoptosis and actin formation within 24 h. This work demonstrated that cell growth was dependent on surface chemistry and correlated strongly with the surface free energy and charge of the material. The positively charged NH2 surface induced tight cell attachment with well-organized actin stress fibers and a well spread morphology. In contrast, CO2H- and CH3-functional groups provoked a decrease in cell adhesion and spreading and indicated higher apoptotic potential, although both were hydrophilic. The knowledge about the cell-material dialogue is of relevance for the development of bioactive implants in regenerative medicine.

Halloysite Nanotube-Reinforced Ion-Incorporated Hydroxyapatite-Chitosan Composite Coating on Ti-6Al-4 V Alloy for Implant Application

To develop the corrosion resistance and improve the biological performance of a titanium implant (Ti6Al4V alloy), a series of mineral (M = Zn and Mg)-substituted hydroxyapatite (MHA), chitosan-MHA (CS-MHA), halloysite nanotube-MHA (HNT-MHA), and HNT-CS-MHA composite coatings were fabricated on the anodized titanium alloy by electrodeposition. The surface morphology and cross section of various coated composites were investigated by high-resolution scanning electron microscopy (HR-SEM). Furthermore, the functional groups and phase structure of the composite coatings were investigated by Fourier transform infrared spectroscopy (FTIR) and X-ray diffractometry (XRD). Corrosion behaviors of the composite coatings were also investigated by polarization and impedance spectroscopy (EIS). Moreover, the cell-material interaction of the composite coating was observedin vitrowith human osteoblast MG63 cells for cell proliferation at 1, 4, and 7 days of incubation. Consequently, HNT-CS-MHA-Ti may have potential applications in the field of orthopedic and dental implants.

Enterococcus faecalis induces apoptosis and pyroptosis of human osteoblastic MG63 cells via the NLRP3 inflammasome.

To investigate the ability of E.faecalis to cause apoptosis and pyroptosis of human osteoblastic MG63 cells and whether the inflammasome is involved in this process. MG63 cells were infected with E.faecalis at a multiplicity of infection (MOI) of 10, 100, 500 and 1000 for 6 and 12h. The cell proliferation was determined with the Cell Counting Kit-8. Apoptosis and pyroptosis after E.faecalis infection was evaluated by flow cytometry and a lactate dehydrogenase (LDH) cytotoxicity assay kit. Then, MG63 cells transfected with specific small interfering RNAs (siRNAs) targeting the NLR family pyrin domain-containing 3 (NLRP3) gene were infected with E.faecalis and subjected to the LDH release and apoptotic cell assays. One-way anova test and Student-Newman-Keuls test were used to evaluate the differences amongst groups in each experiment. An independent sample t-test was performed to analyse the differences between the 12- and 6-h time-points. P<0.05 was regarded as significant. E.faecalis induced apoptosis and inflammatory cell death (pyroptosis) of MG63 cells in a MOI dose-dependent manner. The NLRP3 inflammasome and caspase-1 were activated in E.faecalis-infected MG63 cells. However, the percentages of induced apoptotic and pyroptic cell death decreased significantly when the NLRP3 inflammasome was downregulated using siRNAs (P<0.05). E.faecalis promoted apoptosis and pyroptosis of MG63 cells via the NLRP3 inflammasome. This indicates that E.faecalis infection may result in the delayed reconstruction of periapical lesions, and NLRP3 is the potential target of new intracanal therapeutic agents.

Hypoxia regulates angeogenic-osteogenic coupling process via up-regulating IL-6 and IL-8 in human osteoblastic cells through hypoxia-inducible factor-1α pathway

Inappropriate angiogenesis and osteogenesis are considered as the crucial factors of osteoporotic fracture. Hypoxia is a primary driving force for regulating the angiogenic-osteogenic coupling process. Our recent results indicated that hypoxia could improve angiogenesis as well as differentiation and activity of osteoblastic cells via up-regulating VEGF through HIF-1α pathway. Here we demonstrated that in human osteoblastic MG-63, U2-OS and Saos-2 cells, besides VEGF, the other two pro-angiogenic factors IL-6 and IL-8 were also up-regulated by hypoxia and CoCl2 (a mimic of hypoxia). Mechanism studies indicated overexpression of HIF-1α (generated from transfection with a plasmid encoding sense HIF-1α) markedly increased the levels of IL-6 and IL-8 in osteoblastic cells. Furthermore, a luciferase reporter assay was performed using the reporter vector containing the IL-6 or IL-8 promoter sequence to illustrate observably increased activity of hypoxia-induced IL-6 and IL-8 promoter caused by overexpression of HIF-1α. Additionally, chromatin immune-precipitation analysis showed hypoxia increased the DNA binding ability of HIF-1α to IL-6 or IL-8 promoter. Analysis in vitro by MTT test and Boyden chamber assay showed exogenous IL-6 and IL-8 (a relatively short period of treatment with recombinant IL-6 or IL-8 equivalent to the autocrine levels) could significantly promote the proliferation of human osteoblastic, endothelial and monocytic cells, as well as the migration of human endothelial cells. Taken together, these results indicate that IL-6 and IL-8 in osteoblastic cells may also contribute to the angiogenic-osteogenic coupling process via HIF-1α pathway. Besides VEGF, IL-6- or IL-8-targeted adjunctive therapy maybe a new strategy to improve the treatment of osteoporosis.

Iron excess upregulates SPNS2 mRNA levels but reduces sphingosine-1-phosphate export in human osteoblastic MG-63 cells.

Systemic iron excess favors the development of osteoporosis, especially during genetic hemochromatosis. The cellular mechanisms involved are still unclear despite numerous data supporting a direct effect of iron on bone biology. Therefore, the aim of this study was to characterize mechanisms involved in the iron-related osteoblast impairment. We studied, by using the MG-63 cell lines, the effect of iron excess on SPNS2 gene expression which was previously identified by us as potentially iron-regulated. Cell-type specificity was investigated with hepatoma HepG2 and enterocyte-like Caco-2 cell lines as well as in iron-overloaded mouse liver. The SPNS2-associated function was also investigated in MG-63 cells by fluxomic strategy which led us to determinate the S1P efflux in iron excess condition. We showed in MG-63 cells that iron exposure strongly increased the mRNA level of the SPNS2 gene. This was not observed in HepG2, in Caco-2 cells, and in mouse livers. Fluxomic study performed concomitantly on MG-63 cells revealed an unexpected decrease in the cellular capacity to export S1P. Iron excess did not modulate SPHK1, SPHK2, SGPL1, or SGPP1 gene expression, but decreased COL1A1 and S1PR1 mRNA levels, suggesting a functional implication of low extracellular S1P concentration on the S1P/S1PR signalizing axis. Our results indicate that iron impacts the S1P/S1PR signalizing axis in the MG-63 cell line and suggest that iron can affect the bone-associated S1P pathway and favor the occurrence of osteoporosis during chronic iron overload.