Osteoblastic Cell Line Saos-2 Research Articles

Protecting Orthopaedic Implants from Infection: Antimicrobial Peptide Mel4 Is Non-Toxic to Bone Cells and Reduces Bacterial Colonisation When Bound to Plasma Ion-Implanted 3D-Printed PAEK Polymers.

Even with the best infection control protocols in place, the risk of a hospital-acquired infection of the surface of an implanted device remains significant. A bacterial biofilm can form and has the potential to escape the host immune system and develop resistance to conventional antibiotics, ultimately causing the implant to fail, seriously impacting patient well-being. Here, we demonstrate a 4 log reduction in the infection rate by the common pathogen S. aureus of 3D-printed polyaryl ether ketone (PAEK) polymeric surfaces by covalently binding the antimicrobial peptide Mel4 to the surface using plasma immersion ion implantation (PIII) treatment. The surfaces with added texture created by 3D-printed processes such as fused deposition-modelled polyether ether ketone (PEEK) and selective laser-sintered polyether ketone (PEK) can be equally well protected as conventionally manufactured materials. Unbound Mel4 in solution at relevant concentrations is non-cytotoxic to osteoblastic cell line Saos-2. Mel4 in combination with PIII aids Saos-2 cells to attach to the surface, increasing the adhesion by 88% compared to untreated materials without Mel4. A reduction in mineralisation on the Mel4-containing surfaces relative to surfaces without peptide was found, attributed to the acellular portion of mineral deposition.

MO551: Targeted Ablation of PTEN in Osteocytes Induces Severe Anemia and Increases FGF23 Production in Renal Insufficiency

Abstract BACKGROUND AND AIMS Renal disease patients present bone alterations due to an imbalance of mineral metabolism. Previous studies have demonstrated that mice with conditional ablation of the phosphatase and tensin homolog (PTEN) in osteoblasts show increases in bone volume. The aim of this study was to investigate whether PTEN ablation in osteocytes has potential benefits on bone and mineral metabolism in the context of renal insufficiency. METHOD A few 2-month-old mice with constitutive ablation of PTEN in osteocytes (Ocy-PTEN-cKO) underwent 5/6 nephrectomy (5/6Nx) and were fed a 0.9% phosphate diet. After 2 weeks, 24-h urine samples were collected and animals were sacrificed to obtain blood samples for biochemical studies and organs for histological and biochemical analyses. Bone histomorphometry and microcomputed tomography analyses were performed in femurs. Fibroblast growth factor 23 (FGF23) expression was quantified in bone lysates and intact and c-terminal FGF23 concentrations were measured in plasma samples. Animals with normal renal function and Cre-negative littermates were used as controls. In addition, the effects of PTEN silencing were assessed in vitro in osteocyte-like bone marrow mesenchymal stem cells and in the osteosarcoma cell line SAOS-2. RESULTS Ocy-PTEN-cKO mice showed increased bone volume, mainly in the cortical compartment which showed higher cortical thickness and lower porosity. Of interest, we observed lower haematocrit in the Ocy-PTEN-cKO mice that was much lower in those with 5/6Nx. Anaemia was not associated with a lower renal EPO expression. Bone mRNA and protein expression of FGF23 were increased in Ocy-PTEN-cKO mice with normal and reduced renal function. 5/6Nx resulted in higher plasma intact FGF23, which remained similar between both, Ocy-PTEN-cKO and control groups, due to increased FGF23 cleavage as indicated by the higher plasma c-terminal FGF23 levels in the Ocy-PTEN-cKO groups, resulting in increased plasma phosphate. In vitro, PTEN silencing in bone marrow mesenchymal stem cells differentiated into osteoblasts or in the osteoblastic cell line SAOS-2 did not show significant changes regarding FGF23 levels, indicating that the effects must be indirect. CONCLUSION PTEN ablation in osteocytes increases cortical bone volume and exacerbates anemia in mice with reduced renal function. Moreover, bone FGF23 production was increased but subsequent proteolysis resulted in higher plasma phosphate. These results demonstrate a major role of the osteocyte in the development of anaemia in renal disease, which could be associated with FGF23 metabolism.

Influence of Periodontal Ligament Stem Cell-Derived Conditioned Medium on Osteoblasts.

Mesenchymal stem cells (MSC) are involved in the regeneration of various missing or compromised periodontal tissues, including bone. MSC-derived conditioned medium (CM) has recently been explored as a favorable surrogate for stem cell therapy, as it is capable of producing comparable therapeutic effects. This study aimed to evaluate the influence of periodontal ligament stem cells (PDLSC)-CM on osteoblasts (OB) and its potential as a therapeutic tool for periodontal regeneration. Human PDLSC were isolated and characterized, and CM from these cells was collected. The presence of exosomes in the culture supernatant was observed by immunofluorescence and by transmission electron microscopy. CM was added to a cultured osteoblastic cell line (Saos-2 cells) and viability (MTT assay) and gene expression analysis (real-time PCR) were examined. A cell line derived from the periodontal ligament and showing all the characteristics of MSC was successfully isolated and characterized. The addition of PDLSC-CM to Saos-2 cells led to an enhancement of their proliferation and an increased expression of some osteoblastic differentiation markers, but this differentiation was not complete. Saos-2 cells were involved in the initial inflammation process by releasing IL-6 and activating COX2. The effects of PDLSC-CM on Saos-2 appear to arise from a cumulative effect of different effective components rather than a few factors present at high levels.

Poly(acrylic acid)-mediated synthesis of cerium oxide nanoparticles with variable oxidation states and their effect on regulating the intracellular ROS level.

Cerium oxide nanoparticles (CeNPs) possess multiple redox enzyme mimetic activities in scavenging reactive oxygen species (ROS) as a potential biomedicine. These enzymatic activities of CeNPs are closely related to their surface oxidation state. Here we have reported a synthetic method to modify CeNPs' surface oxidation state by changing the conformation of the poly(acrylic acid) (PAA) polymers adsorbed onto the CeNP surface. The synthesized PAA-CeNPs exhibited the same core size, morphology, crystal structure, and colloidal stability, with the only variation being their surface oxidation state (Ce3+ percentage). The modification mechanism can be attributed to the polymers chemisorbed onto the metal oxide surface forming a metal complexation structure. Such adsorption further modified CeNPs' surface oxidation state in a temperature-dependent manner. The series of PAA-CeNPs exhibited multiple redox enzyme mimetic activities (superoxide dismutase, catalase, peroxidase, and oxidase) directly related to their surface oxidation state. In vitro experiments showed no cytotoxic effect of these PAA-CeNPs on the osteoblastic cell line SAOS-2 at high loadings. Microscopic images confirmed the internalization of PAA-CeNPs in the cells. All tested PAA-CeNPs can reduce the basal and hydrogen peroxide-induced intracellular ROS level in the cells, indicating their effective intracellular ROS scavenging effect. However, we did not observe a positive correlation between the CeNP surface oxidation state and their capacities to reduce the intracellular ROS levels. We propose that CeNPs can maintain a dynamic state of Ce3+/Ce4+ during their catalytic activities, exhibiting a non-linear correlation between the CeNP surface oxidation state and their effect on regulating the intracellular ROS level.

A new way to prepare gold nanoparticles by sputtering - Sterilization, stability and other properties.

We have developed a novel simple method for effective preparing gold nanoparticles (AuNPs) intended for utilization in biomedicine. The method is based on gold sputtering into liquid poly(ethylene glycol) (PEG). The PEG was used as a basic biocompatible stabilizer of the AuNP colloid. In addition, two naturally occurring polysaccharides - Chitosan (Ch) and Methylcellulose (MC) - were separately diluted into the PEG base with the aims to enhance the yield of the sputtering without changing the sputtering parameters, and to further improve the stability and the biocompatibility of the colloid. The colloids were sterilized by steam, and their stability was measured before and after the sterilization process by dynamic light scattering and UV-Vis spectrophotometry. The results indicated a higher sputtering yield in the colloids containing the polysaccharides. The colloids were also characterized by atomic absorption spectroscopy (AAS) to reveal the composition of the prepared nanoparticles by transmission electron microscopy (TEM) to visualize the nanoparticles and to evaluate their size and clustering, and by rheometry to estimate the viscosity of the colloids. The zeta-potential of the AuNPs was also determined as an important parameter indicating the stability and the biocompatibility of the colloid. In addition, in vitro tests of antimicrobial activity and cytotoxicity were carried out to estimate the biological activity and the biocompatibility of the colloids. Antimicrobial tests were performed by a drip test on two bacterial strains - Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli. AuNP with chitosan proved to possess the highest antibacterial activity, especially towards the Gram-positive S. epidermidis. In vitro tests on eukaryotic cells, i.e. human osteoblastic cell line SAOS-2 and primary normal human dermal fibroblasts (NHDF), were performed after a 7-day cultivation to determine the effect and the toxic dose of the colloids on human cells. The studied colloid concentrations were in the range from 0.6 μg/ml to 6 μg/ml. Toxicity of the colloids started to reappear at a concentration of 4.5 μg/ml, especially with chitosan in the colloid, where the colloid with a concentration of 6 μg/ml proved to be the most toxic, especially towards the SAOS-2 cell line. However, the PEG and PEG-MC containing colloids proved to be relatively non-toxic, even at the highest concentration, but with a slowly decreasing tendency of the cell metabolic activity.

Imatinib mesylate and nilotinib decrease synthesis of bone matrix in vitro.

Tyrosine kinase inhibitors (TKIs), such as imatinib (IMA) and nilotinib (NIL), are the cornerstone of chronic myeloid leukemia (CML) treatment via the blockade of the oncogenic BCR-ABL1 fusion protein. However, skeletal side effects are commonly observed in pediatric patients receiving long-term treatment with IMA. Additionally, in vitro studies have shown that IMA and NIL alter vitamin D metabolism, which may further impair bone metabolism. To determine whether TKIs directly affect bone cell function, the present study treated the human osteoblastic cell line SaOS-2 with IMA or NIL and assessed effects on their mineralization capacity as well as mRNA expression of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG), two cytokines that regulate osteoclastogenesis. Both TKIs significantly inhibited mineralization and downregulated osteoblast marker genes, including alkaline phosphatase, osteocalcin, osterix, as well as genes associated with the pro-osteogenic Wnt signaling pathway; NIL was more potent than IMA. In addition, both TKIs increased the RANKL/OPG ratio, which is known to stimulate osteoclastogenesis. The present results suggested that the TKIs IMA and NIL directly inhibited osteoblast differentiation and directly promoted a pro-osteoclastogenic environment through the RANKL-OPG signaling axis. Thus, we propose that future work is required to determine whether the bone health of CML patients undergoing TKI-treatment should be routinely monitored.

Effects of Vitamin E-Stabilized Ultra High Molecular Weight Polyethylene on Oxidative Stress Response and Osteoimmunological Response in Human Osteoblast.

High Crosslink process was introduced in the development of joint prosthetic devices, in order to decrease the wear rate of ultrahigh molecular weight polyethylene (UHMWPE), but it also triggers the formation of free radicals and oxidative stress, which affects the physiological bone remodeling, leading to osteolysis. Vitamin E stabilization of UHMWPE was proposed to provide oxidation resistance without affecting mechanical properties and fatigue strength. The aim of this study is to evaluate the antioxidant effect of vitamin E added to UHMWPE on oxidative stress induced osteolysis, focusing in particular on the oxidative stress response in correlation with the production of osteoimmunological markers, Sclerostin and DKK-1, and the RANKL/OPG ratio compared to conventional UHMWPE wear debris. Human osteoblastic cell line SaOS2 were incubated for 96 h with wear particles derived from crosslinked and not crosslinked Vitamin E-stabilized, UHMWPE without Vitamin E, and growth medium as control. Cellular response to oxidative stress, compared to not treat cells, was evaluated in terms of proteins O-GlcNAcylation, cellular levels of OGA, and OGT proteins by immunoblotting. O-GlcNAcylation and its positive regulator OGT levels are increased in the presence of Vitamin E blended UHMWPE, in particular with not crosslinked Vit E stabilized UHMWPE. Conversely, the negative regulator OGA increased in the presence of UHMWPE not blended with Vitamin E. Vitamin E-stabilized UHMWPE induced a decrease of RANKL/OPG ratio compared to UHMWPE without Vitamin E, and the same effect was observed for Sclerostin, while DKK-1 was not significantly affected. In conclusion, Vitamin E stabilization of UHMWPE increased osteoblast response to oxidative stress, inducing a cellular mechanism aimed at cell survival. Vitamin E antioxidant effect influences the secretion of osteoimmunological factors, shifting the bone turnover balance toward bone protection stimuli. This suggests that Vitamin E-Stabilization of UHMWPE could contribute to reduction of oxidation-induced osteolysis and the consequent loosening of the prosthetic devices, therefore improving the longevity of total joint replacements.

The antifibrotic drug pirfenidone inhibits spondyloarthritis fibroblast-like synoviocytes and osteoblasts in vitro

BackgroundThe pathogenesis of spondyloarthritis (SpA) involves both inflammation and new bone formation in the spine. In line with this, the disease has been characterized as both inflammatory and fibrotic. The current treatment dampens inflammation while new bone formation can progress. Therefore, there is an unmet therapeutic need for the treatment of new bone formation in SpA. Fibrosis is mediated by myofibroblasts and new bone formation is the result of increased osteoblast mineralization and decreased osteoclast resorption. Here, we evaluate the potential effect of the newly approved anti-fibrotic agent pirfenidone (PFD) on fibrosis and new bone formation in cell culture models of SpA.MethodsFibroblast-like synoviocytes (FLSs) were isolated from SpA patients (n = 6) while the osteoblast cell line Saos-2 was purchased. The cells were cultured with PFD at 0.25 0.5, or 1.0 mg/ml. The proliferation of FLSs was analyzed with light microscopy and flow cytometry. The differentiation and activation of FLSs was assessed with flow cytometry, a membrane-based antibody array and enzyme-linked immunosorbant assays. The mineralization capacity of osteoblasts was studied with an assay measuring deposition of hydroxyapatite.ResultsPFD reduced the Ki67 expression 7.1-fold in untreated FLSs (p = 0.001) and 11.0-fold in FLSs stimulated with transforming growth factor beta (TGFβ), tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) (p = 0.022). There were no statistically significant changes in membrane expression of alpha smooth muscle actin (αSMA), intercellular adhesion molecule 1 (ICAM-1), or human leukocyte antigen DR (HLA-DR). In supernatants from FLSs stimulated with TGFβ, TNFα, and IFNγ, PFD decreased the secretion of 3 of 12 proteins more than 2-fold in the membrane-based antibody array. The changes in secretion of monocyte chemoattractant protein 1 (MCP-1) and chitinase-3-like protein 1 (CHI3L1, YKL-40) were validated with ELISA. PFD decreased the secretion of both Dickkopf-related protein 1 (DKK1) (p = 0.006) and osteoprotegerin (OPG) (p = 0.02) by SpA FLSs stimulated with TGFβ, TNFα, and IFNγ. Finally, PFD inhibited the deposition of hydroxyapatite by osteoblasts in a dose-dependent manner (p = 0.0001).ConclusionsPFD inhibited SpA FLS proliferation and function and osteoblast mineralization in vitro. This encourages studies of the in vivo effect of PFD in SpA.

Impact of sphingolipids on osteoblast and osteoclast activity in Gaucher disease

Gaucher disease (GD) is an inherited disorder in which mutations in the GBA1 gene lead to deficient β-glucocerebrosidase activity and accumulation of its substrate glucosylceramide. Bone disease is present in around 84% of GD patients, ranging from bone loss including osteopenia and osteonecrosis to abnormal bone remodelling in the form of Erlenmeyer flask formation. The range of severity and variety of types of bone disease found in GD patients indicate the involvement of several mechanisms. Here we investigate the effects of exogenous sphingolipids on osteoclasts, osteoblasts, plasma cells and mesenchymal stem cells (MSC) and the interactions between these cell types. Osteoclasts were differentiated from the peripheral blood of Gaucher patients and control subjects. Osteoblasts were differentiated from mesenchymal stem cells isolated from bone marrow aspirates of Gaucher patients and control subjects. The human osteoblast cell line SaOS-2 was also investigated. Osteoclasts, osteoblasts and a human myeloma plasma cell line NCI-H929 were cultured with relevant exogenous sphingolipids to assess effects on cellular viability and function. Calcium deposition by osteoblasts differentiated from Gaucher patient MSC's was on average only 11.4% of that deposited by control subject osteoblasts. Culture with glucosylsphingosine reduced control subject MSC viability by 10.4%, SaOS-2 viability by 17.4% and plasma cell number by 40%. Culture with glucosylceramide decreased calcium deposition by control MSC-derived osteoblasts while increasing control subject osteoclast generation by 55.6%, Gaucher patient osteoclast generation by 37.6% and plasma cell numbers by up to 29.7%. Excessive osteoclast number and activity and reduced osteoblast activity may have the overall effect of an uncoupling between osteoclasts and osteoblasts in the GD bone microenvironment.

Olive Fruit Blends Modulate Lipid-Sensing Nuclear Receptor PPARγ, Cell Survival, and Oxidative Stress Response in Human Osteoblast Cells

ABSTRACTObjective: The aim of the present study was to investigate how different extravirgin olive oils (EVOOs), obtained by blending Olea europea cultivars, could influence the cell growth, the response to inflammatory stimuli, and oxidative stress in a culture of the osteosarcoma cell line Saos-2.Methods: Three different extravirgin olive oils were physicochemically characterized, determining the free acidity, the oxidation status, the polyphenols content, and the antioxidative activity. Moreover, the effects on Saos-2 cell culture were determined, studying the mRNA expression level by real-time polymerase chain reaction (PCR) assays and the antioxidative activity using fluorescent probes.Results: The cultivars used in the south of Italy, yield extravirgin oils with different amount of fatty acids and polyphenols, which counteract induction of proinflammatory cytokines and regulate free radical production in hydrogen peroxide-stimulated cells. In vitro analysis using the human osteoblast cell line Saos-2 showed that the addition of oils to cell culture simulated a hypoxic stress followed by a reoxygenation period, during which the antioxidant activity of extravirgin olive oils protected cells from oxidative damages. On the other hand, the mRNA expression levels of factors involved in inflammatory processes, cell growth recovery, and antioxidant response, as heme oxygenase-1, were differently stimulated by EVOOs. Moreover, peroxisome proliferator activated receptor γ (PPARγ) was differently modulated by EVOOs.Conclusion: These findings show that the blending of different extravirgin olive oil can impact an osteoblast cell line, in particular regarding cell growth recovery and oxidative stress.

Growth mechanisms of Ca- and P-rich MAO films in Ti-15Zr-xMo alloys for osseointegrative implants

In this study, a micro-arc oxidation treatment was applied to Ti-15Zr-xMo (x = 0, 5, 10 and 15 wt%) alloys to produce porous oxide layers enriched with bioactive ions (calcium and phosphorus) for use as osseointegrative implants. Biocompatibility studies, namely metabolic activity, mineralization and differentiation studies were conducted with human osteoblastic cell line SAOS-2. A typical porous coating was obtained in all samples, with similar morphologies and thicknesses, which were found to be dependent on the maximum applied voltage. Calcium and phosphorus ions were incorporated into the films, as indicated by EDX analysis. Chemical analyses indicated that the films were composed preferentially of Ti and Zr oxides. XRD patterns revealed mostly substrate Ti phases. However, cross-sectional TEM imaging and automated phase and orientation mapping showed distinct amorphous and nanocrystalline regions within the films, with a higher fraction of Ca atoms incorporated in the outer layer. After immersion in Hanks' Balanced Salt Solution (HBSS) for seven days, small amounts of calcium phosphate precipitates were observed at the surface of all samples which were confirmed by ICP-AES measurements, indicating that the MAO treatment possibly introduced a considerable bioactive response in the samples. Biological results indicate that Ti-15Zr-15Mo MAO-treated surfaces are biocompatible and induce a higher osteoblasts viability and mineralization. The combination of porous structure and bioactive composition of the oxide layers can be suitable for use as advanced biomedical implants with osseointegration ability.

MiRNA-34a inhibits human osteosarcoma proliferation by regulating the expression of ether-à-go-go 1 gene: a study in vitro

Objective To detect the influence of miRNA-34a (miR-34a) on the proliferation of osteosarcoma and the mechanisms responsible for miR-34a regulation. Methods The osteoblastic cell line MG-63 and Saos-2, human osteoblastic cell line hFOB 1.19, 10 osteosarcoma tissues and 10 normal bone tissues were selected. The expression of miRNA-34a in osteosarcoma cells and tissues was detected by quantitative real-time polymerase chain reaction (qPCR). Next, a eukaryotic expression vector named pcDNA/miR-34a was constructed. Then, osteosarcoma cells were transfected with this eukaryotic expression vector and the effects of miR-34a overexpression on the proliferation and growth of osteosarcoma were measured using CCK-8, colony formation and xenograft model of nude mice. Finally, Western blot analysis was used to detect the expression of ether-a-go-go 1 (Eag1) gene in osteosarcoma cells after transfected with pcDNA/miR-34a or a miR-34a inhibitor miR-34a-2'-O-Methyl antisense oligoribonucleotide (miR-34a-2'-O-Me). Results Compared with normal bone tissues and osteoblastic cell line, miR-34a was down-regulated in osteosarcoma cell lines and tissues. Compared with the blank group and the control group, the cell survival rates of miR-34a group of the two cell lines were significantly lower [MG-63 72 h: blank group (40.05±4.82)%, control group (36.88±4.66)%, miRNA-34a group (26.24±6.22)%; MG-63 96 h: blank group (83.55±5.95)%, control group (80.13±4.48)%, miRNA-34a group (30.21±7.26)%; Saos-2 72 h: blank group (46.45±8.15)%, control group (43.33±6.89)%, miRNA-34a group (26.81±3.17)%; Saos-2 96 h: blank group (84.79±4.10)%, control group (80.14±3.11)%, miRNA-34a group (31.77±5.17)%]. The similar results were obtained from colony formation assay (MG-63: blank group 83.40±3.29, control group 80.00±3.06, miR-34a group 24.40±2.71; Saos-2: blank group 85.00±3.32, control group 80.60±3.29, miR-34a group 30.40±4.94). The tumor volumes of osteosarcoma xenograft in the miR-34a group was significantly smaller than that in the blank group and control group after 21 days treatment (all P < 0.001). Overexpression of miR-34a could decrease Eag1 expression in osteosarcoma cell lines while inhibition of miR-34a induced the of expression Eag1(P < 0.001). Conclusion MiR-34a plays a tumor suppressor role in osteosarcoma and could suppress the proliferation and growth of osteosarcoma through the regulation of Eag1. Moreover, it may be a novel target for osteosarcoma therapy. Key words: Osteosarcoma; MicroRNAs; Cell proliferation; Mice, nude; Gene, ether-a-go-go 1

Modulatory Effects of a Nutraceutical Supplement on Saos-2 Cells Reveal Its Phlebotonic Activity

ABSTRACTObjective: Herbal extract compositions are largely used to manage vein diseases. We prepared a new composition of herbs, named FLEBO OK™, that, when administered as a nutraceutical to patients affected by peripheral vascular diseases, was able to improve their health conditions. We analyzed the effects of this nutraceutical composition on in vitro cultured cells with the aim to obtain information about its mechanisms of action.Methods: A culture of human osteoblast cell line Saos-2 was stimulated with tumor necrosis factor (TNF)-α or interleukin (IL)-1β to induce the expression of some chemokines and matrix metalloproteases (MMPs). This cell culture was then exposed to the prepared composition and the amount of expression of the genes coding for the monocyte chemotactic protein (MCP)-1, IL-8, IL-1β, MMP-2, MMP-3, MMP-9 proteins was measured by real-time polymerase chain reaction (RT-PCR). The experiments were repeated exposing the cells to the same amount of the well-known micronized purified flavonoid fraction. Moreover, we describe the effects of the administration of nutraceutical composition to 20 patients affected by peripheral vascular diseases and 20 healthy individuals.Results: The RT-PCR analyses showed that the new composition induces the expression of MMP-3 and MMP-9 and downregulates MMP-2 in cell cultures stimulated with IL-1β, whereas it induces the expression of IL-8 and represses the expression of IL-1β and MCP-1 in cell cultures stimulated with TNF-α. The induction of the expression of MMP-3 and the downregulation of MCP-1 might result in an antiplatelet activity that was not observed for the micronized purified flavonoid fraction. Interviewed patients reported an improvement in their conditions after 1 month of FLEBO OK treatment.Conclusion: These findings could provide a hypothesis for the high efficiency of the identified nutraceutical composition to management of peripheral vascular diseases.

Attenuation of Oxidative Stress-Induced Osteoblast Apoptosis by Curcumin is Associated with Preservation of Mitochondrial Functions and Increased Akt-GSK3β Signaling

Background: Osteoblast apoptosis induced by oxidative stress plays a crucial role in the development and progression of osteoporosis. Curcumin, a natural antioxidant isolated from Curcuma longa, has highly protective effects against osteoporosis. However, the effects of curcumin on oxidative stress-induced osteoblast apoptosis remain unclear. This study aimed to explore the effect of curcumin on hydrogen peroxide (H₂O₂) induced osteoblast apoptosis and the underlying mechanisms. Methods: An osteoblastic cell line (Saos-2) was exposed to various concentrations of H₂O₂ with or without curcumin treatment. Cell viability was evaluated by MTT assays. The apoptosis rate was analyzed by flow cytometry and TUNEL assays. Mitochondrial ROS and membrane potential were determined using a fluorescence microscope. Mitochondrial respiratory enzyme activity was measured using a spectrophotometer. Protein levels were detected by western blotting. Results: Curcumin was cytoprotective because it greatly improved the viability of Saos-2 cells exposed to H₂O₂ and attenuated H₂O₂-induced apoptosis. Curcumin treatment also preserved the mitochondrial redox potential, decreased the mitochondrial oxidative status, and improved the mitochondrial membrane potential and functions. Furthermore, curcumin treatment markedly increased levels of phosphorylated protein kinase B (Akt) and phosphorylated glycogen synthase kinase-3β (GSK3β). Conclusion: Curcumin administration ameliorates oxidative stress-induced apoptosis in osteoblasts by preserving mitochondrial functions and activation of Akt-GSK3β signaling. These data provide experimental evidence supporting the clinical use of curcumin for prevention or treatment of osteoporosis.

Carbon Microelectrodes for Real Time Osteoporosis Drug Screenings

Osteoporosis is a disabling skeletal disorder affecting 200 million people worldwide1. Presently, the main challenge is to develop new more effective drugs with reduced long-term side effects. Monitoring the interactions of diseased bone tissues with drugs is fundamental for osteoporosis drug development and screening. In vitro cytotoxicity assays represent the most commonly used approach for screening and validation of drug candidates in the early stage of drug discovery2. Most of these assays rely on two dimensional (2D) cell culture systems which do not allow for complex three dimensional (3D) spatial interactions (i.e. cell-cell and cell-extracellular matrix (ECM) interactions) typical in living bone and crucial for accurate results in toxicological testing3. Moreover, conventional 2D cytotoxicity screenings do not provide temporal information of the cellular response to drugs. Thus, there is a need for new and complementary 3D in vitro systems that can better mimic the in vivo microenvironment of bone architecture and enable in situ monitoring of bone cell response to osteoporosis drugs in real time. Conductive 3D substrates and scaffolds (e.g. based on metal, carbon, conductive polymers, etc.) offer the possibility for in-situ electrical sensing while providing a more in-vivo like microenvironment for real-time monitoring of cell population processes4. In the fabrication of 3D electrodes, limitations for high throughput, reproducibility, large-scale production and costs still remain a critical issue. Carbonaceous materials5, such as graphene, graphene foam, CNTs, diamond-like carbon, carbon composites and pyrolysed carbon are emerging for development of 2D and 3D electrodes. The carbon MEMS (C-MEMS) technique is a very simple and cost-effective method for electrode fabrication6, where a patterned polymer template is heated to temperatures above 900 °C in inert atmosphere to produce pyrolysed carbon electrodes. This process enables easy and reproducible fabrication of carbon electrodes with possibility for tailoring ad-hoc designs and sensitivities for specific applications7. Moreover, pyrolysed carbon electrodes exhibit a wide electrochemical potential window, chemical inertness towards a range of solvents and electrolytes, good biocompatibility, and the possibility to tune the electrical and mechanical properties of the electrodes. In order to monitor bone cell response to drugs in real time, 2D interdigitated pyrolysed carbon electrodes (IDE) have been designed and fabricated with the C-MEMS technique, using SU-8 as photoresist to define the polymer template on a Si-based substrate (Figure 1A). The IDEs are composed of two combs, one used as working (WE) and one used as counter electrode (CE). Three different designs have been evaluated by varying finger width and spacing, i.e. width/spacing of 5/8.4, 12/15, 30/30 µm. Electrochemical characterization has been performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Figure 1B shows cyclic voltammograms related to the 30/30 µm design performed in a solution of 1 mM ruthenium (II/III) hexamine chloride in PBS at a scan rate of 100 mV/s. For the same chips impedance spectra were recorded in PBS in the frequency range of 0.1 – 100000 Hz by applying 1 mV sinusoidal potential (Figure 1C). Analogue behaviors have been observed for the other geometrical designs. In order to validate the electrodes for impedance-based cell assays, adhesion and proliferation of a human osteoblastic model cell line (Saos-2) was assessed in real time. First, the effect of carbon-coated surfaces (i.e. laminin, collagen, fibronectin) on Saos-2 cells was evaluated and compared with the traditionally used polystyrene (data not shown). Fibronectin coating was shown to better promote cell adhesion and proliferation (Figure 1D inset). Saos-2 cells were seeded in a concentration of 140000 cells/cm2 on a fibronectin coated (20 µg/mL) carbon chip maintained at 37° C in a humidified atmosphere having 5% CO2 and impedance spectra were acquired every hour for 48 hours. Figure 1D shows the normalized impedance (Cell Index) profile over time, providing indication on the temporal evolution of cell adhesion and proliferation until reaching confluence. These results indicate that carbon electrodes are suitable for EIS-based cellular assays using osteoblastic cells and pave the way for the development of 3D in vitro cytotoxicity models of bone tissue for osteoporosis drug screenings.

Decreased Serotonin Levels and Serotonin-Mediated Osteoblastic Inhibitory Signaling in Patients With Ankylosing Spondylitis.

Evidence suggests that serotonin is an inhibitor of bone formation. We aimed to assess: 1) serum serotonin levels in patients with ankylosing spondylitis (AS), a prototype bone-forming disease, compared with patients with rheumatoid arthritis (RA) and healthy subjects; 2) the effect(s) of TNFα blockers on serum serotonin levels in patients with AS and RA; and 3) the effect(s) of serum of AS patients on serotonin signaling. Serum serotonin levels were measured in 47 patients with AS, 28 patients with RA, and 40 healthy subjects by radioimmunoassay; t test was used to assess differences between groups. The effect of serum on serotonin signaling was assessed using the human osteoblastic cell line Saos2, evaluating levels of phospho-CREB by Western immunoblots. Serotonin serum levels were significantly lower in patients with AS compared with healthy subjects (mean ± SEM ng/mL 122.9 ± 11.6 versus 177.4 ± 24.58, p = 0.038) and patients with RA (mean ± SEM ng/mL 244.8 ± 37.5, p = 0.0004). Patients with AS receiving TNFα blockers had significantly lower serotonin levels compared with patients with AS not on such treatment (mean ± SEM ng/mL 95.8 ± 14.9 versus 149.2 ± 16.0, p = 0.019). Serotonin serum levels were inversely correlated with pCREB induction in osteoblast-like Saos-2 cells. Serotonin levels are low in patients with AS and decrease even further during anti-TNFα treatment. Differences in serotonin levels are shown to have a functional impact on osteoblast-like Saos-2 cells. Therefore, serotonin may be involved in new bone formation in AS.

A Possible Role Of The Tetraspanin CD9 In Bone Marrow Retention and Engraftment Of Human CD34+ Hematopoietic Stem/Progenitor Cells

The stromal cell-derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing, engraftment and retention of hematopoietic stem/progenitor cells. We previously demonstrated that expression of CD9 is a downstream signal of the SDF-1/CXCR4 axis, and that CD9 regulates short-term (20 hours) homing of cord blood (CB) CD34+ cells in the NOD/SCID mouse xenotransplantation model (Leung et al, Blood, 2011). Here, we provided further evidence that pretreatment of CB CD34+ cells with a CD9-neutralizing antibody significantly reduced their long-term (6 weeks) engraftment, as indicated by the presence of human CD45+ cells, in the recipient bone marrow and spleen by 70.9% (P = .0089) and 87.8% (P = .0179), respectively (n = 6). However, CD9 blockade did not bias specific lineage commitment, including the CD14+ monocytic, CD33+ myeloid, CD19+ B-lymphoid and CD34+ stem/progenitor cells (n = 4). We also observed an increase of the CD34+CD9+ subsets in the bone marrow (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4). These data indicate that CD9 possesses important functions in regulating stem cell engraftment and its expression level on CD34+ cells is up-regulated in the target hematopoietic organs. Analysis of paired bone marrow (BM) and peripheral blood (PB) samples from healthy donors revealed a higher CD9 expression in BM-resident CD34+ cells (57.3% ± 8.1% CD9+ cells in BM vs. 29.3% ± 5.8% in PB; n = 5, P = 0.0478). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (33.8% ± 3.0% CD9+ cells, n = 24), when compared with those in BM (56.4% ± 4.9% CD9+ cells, n = 8, P = 0.0025). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.55-fold increase, n = 4, P = 0.0103), concomitant with a 75.2% reduction in the CD34+CXCR4+ subsets (P = 0.0118). Treatment of NOD/SCID chimeric mice with G-CSF increased the frequency of circulating CD45+ cells (3.4-fold) and CD34+ cells (3.3-fold), and substantially decreased the CD34+CD9+ subsets in the BM from 75.8% to 30.8%. Importantly, the decline in CD9 levels during G-CSF mobilization was also observed in the CD34+CD38-/low primitive stem cell subpopulation. Interestingly, in vitro treatment of BM CD34+ cells with G-CSF did not affect CD9 expression (n = 3), suggesting that a signaling intermediate is required for G-CSF-mediated CD9 down-regulation in vivo. Transwell migration assay revealed a significant enrichment of CD9- cells that were migrated towards a SDF-1 gradient (n = 4 for BM CD34+ cells, P = 0.0074; n = 7 for CB CD34+ cells, P = 0.0258), implicating that CD9 might negatively regulate stem cell motility. In contrast, pretreatment with the CD9-neutralizing antibody inhibited adhesion of CD34+ cells to the osteoblastic cell line Saos-2 by 33.5% (n = 2). Our results collectively suggest a previously unrecognized role of CD9 in stem cell retention by dual regulation of cell motility and adhesion, and reveal a dynamic regulation of CD9 expression in the BM microenvironment, which might represent an important event in controlling stem cell homing and mobilization. Disclosures:No relevant conflicts of interest to declare.

Rivaroxaban, a direct inhibitor of the coagulation factor Xa interferes with hormonal-induced physiological modulations in human female osteoblastic cell line SaSO2

The use of anticoagulants has been associated with systemic osteoporosis and increased risk for poor fracture healing but is inevitable following major orthopedic surgery of lower limbs. Rivaroxaban A (R) is an anticoagulant recently introduced in the clinical setting, which is a specific factor Xa inhibitor. We reported previously that R significantly inhibited cell growth, energy metabolism and alkaline phosphatase activity in human osteoblastic cell line SaOS2, with no effect on mineralization, indicating transient inhibition of bone formation. We now investigated the effects of R on SaOS2 response to osteoblast-modulating hormones. At sub-confluence cells were treated with: estradiol-17β (E2), the phytoestrogens daidzein (D) and biochainin A (BA), the carboxy-pytoestrogenic derivative carboxy-D (cD), the estrogen receptor α (ERα) agonist PPT, the estrogen receptor β (ERβ) agonist DPN, parathyroid hormone (PTH) and several vitamin D metabolites and analogs with/without R for 24h. All hormones tested stimulated significantly DNA synthesis (DNA), creatine kinase (CK) and alkaline phosphatase (ALP) specific activities, but all these stimulations were totally inhibited when given together with R. R had no effect on mRNA expression of ERα, ERβ and 25 Hydroxy-vitamin D3-1α hydroxylase (1OHase), but inhibited hormonal modulations of mRNA expressions. In conclusion R inhibited significantly hormonal stimulation of different parameters indicating inhibition of not only the early stages of bone formation, but also the stimulatory effects of bone modulating hormones with a yet unclear mechanism. The relevance of these findings to human bone physiology is yet to be investigated.

The anti-osteoporotic effect of Moringa oliefera on osteoblastic cells: SaOS 2

M.oliefera oliefera (MO) is a middle-sized tree, cultivated throughout India. It is a multipurpose tree, used as a vegetable, spice, a source of cooking and cosmetic oil and as a medicinal plant. It is reported to contain alkaloids, flavonoids, anthocyanins. It possesses anti-inflammatory, antioxidant, antimicrobial, antihyperlipidaemic, antifertility and anticancer activities. An in-vivo study in our laboratory has proved the osteoprotective potential of the plant. In the present study an attempt is made to explore the osteoblastogenic potential of the different parts of the plant on the osteoblast cell line SaOS2. Methanolic extracts of MO components showed a positive effect, and of all the three components, flower and fruit were found to have significant osteoblast stimulating property. Flower extract was found to be increasing the number of osteoblastic cells; while the fruit extract was having more elaborative effect as it increased ALP activity, induced bone formation, increased hydroxyproline content and bone mineral formation. In conclusion, Methanolic Extact of MO on SaOS2 cell line affirmed the beneficial effects and thus indicates its potential in preventing osteoporosis. Keywords- MO, MO Flower, MO Fruit, MO Leaves, Moringa oliefera, Osteoblasts, SaOS2

Controlled adhesion and proliferation of a human osteoblastic cell line by tuning the nanoporosity of titania and silica coatings.

The engineering of surfaces to control cell adhesion represents an active area of biomaterials research. Herein, we demonstrate that it is possible to tune the adhesion and proliferation of a human osteoblastic cell line (Saos-2) by tailoring the nanopore size of an oxide film coating.