Javier Linares

Endocytic Mechanisms of Graphene Oxide Nanosheets in Osteoblasts, Hepatocytes and Macrophages

Nano-graphene oxide (GO) has attracted great interest in nanomedicine due to its own intrinsic properties and its possible biomedical applications such as drug delivery, tissue engineering and hyperthermia cancer therapy. However, the toxicity of GO nanosheets is not yet well-known and it is necessary to understand its entry mechanisms into mammalian cells in order to avoid cell damage and human toxicity. In the present study, the cellular uptake of pegylated GO nanosheets of ca. 100 nm labeled with fluorescein isothiocyanate (FITC-PEG-GOs) has been evaluated in the presence of eight inhibitors (colchicine, wortmannin, amiloride, cytochalasin B, cytochalasin D, genistein, phenylarsine oxide and chlorpromazine) that specifically affect different endocytosis mechanisms. Three cell types were chosen for this study: human Saos-2 osteoblasts, human HepG2 hepatocytes and murine RAW-264.7 macrophages. The results show that different mechanisms take part in FITC-PEG-GOs uptake, depending on the characteristics of each cell type. However, macropinocytosis seems to be a general internalization process in the three cell lines analyzed. Besides macropinocytosis, FITC-PEG-GOs can enter through pathways dependent on microtubules in Saos-2 osteoblasts, and through clathrin-dependent mechanisms in HepG2 hepatocytes and RAW-264.7 macrophages. HepG2 cells can also phagocytize FITC-PEG-GOs. These findings help to understand the interactions at the interface of GO nanosheets and mammalian cells and must be considered in further studies focused on their use for biomedical applications.

In vitro evaluation of graphene oxide nanosheets on immune function

HYPOTHESIS Graphene oxide (GO) has attracted the scientific community attention due to its novel properties and wide range of potential applications including hyperthermia cancer therapy. However, little is known about the GO effects on the immune function which involves both innate and adaptive defence mechanisms through the activation of different cell populations and secretion of several cytokines. The effect of different GO nanosheets designed for hyperthermia cancer therapy on macrophage and lymphocyte function should be determined before using GO for this application. EXPERIMENTS The effects of GO nanosheets with 1 (1-GOs) and 6 arms (6-GOs) of polyethylene glycol on RAW-264.7 macrophages and primary splenocytes (as approximation to the in vivo situation) were evaluated through the proinflammatory cytokine secretion and the modulation of cell proliferation in the presence of specific stimuli for either T-lymphocytes (concanavalin A, anti-CD3 antibody) or B-lymphocytes/macrophages (lipopolysaccharide). FINDINGS 6-GOs significantly increased the secretion of TNF-α by RAW-264.7 macrophages without alteration of IL-6 and IL-1β levels. The treatment of primary splenocytes with 1-GOs and 6-GOs in the presence of concanavalin A, anti-CD3 antibody and lipopolysaccharide, produced significant dose-dependent decreases of cell proliferation and IL-6 levels, revealing weak inflammatory properties of GOs which are favourable for hyperthermia cancer therapy.

Triggering cell death by nanographene oxide mediated hyperthermia

Graphene oxide (GO) has been proposed as an hyperthermia agent for anticancer therapies due to its near-infrared (NIR) optical absorption ability which, with its small two-dimensional size, could have a unique performance when compared to that of any other nanoparticle. Nevertheless, attention should be given to the hyperthermia route and the kind of GO-cell interactions induced in the process. The hyperthermia laser irradiation parameters, such as exposure time and laser power, were investigated to control the temperature rise and consequent damage in the GOs containing cell culture medium. The type of cell damage produced was evaluated as a function of these parameters. The results showed that cell culture temperature (after irradiating cells with internalized GO) increases preferentially with laser power rather than with exposure time. Moreover, when laser power is increased, necrosis is the preferential cell death leading to an increase of cytokine release to the medium.

Nanocrystalline silicon substituted hydroxyapatite effects on osteoclast differentiation and resorptive activity

In the present study, the effects of nanocrystalline hydroxyapatite (nano-HA) and nanocrystalline Si-substituted hydroxyapatite (nano-SiHA) on osteoclast differentiation and resorptive activity have been evaluated in vitro using osteoclast-like cells. The action of these materials on proinflammatory and reparative macrophage populations was also studied. Nano-SiHA disks delayed the osteoclast differentiation and decreased the resorptive activity of these cells on their surface, as compared to nano-HA samples, without affecting cell viability. Powdered nano-SiHA also induced an increase of the reparative macrophage population. These results along with the beneficial effects on osteoblasts previously observed with powdered nano-SiHA suggest the potential of this biomaterial for modulating the fundamental processes of bone formation and turnover, preventing bone resorption and enhancing bone formation at implantation sites in treatment of osteoporotic bone and in bone repair and regeneration.

Response of osteoblasts and preosteoblasts to calcium deficient and Si substituted hydroxyapatites treated at different temperatures.

Hydroxyapatite (HA) is a calcium phosphate bioceramic widely used for bone grafting and augmentation purposes. The biological response of HA can be improved through chemical and microstructural modifications, as well as by manufacturing it as macroporous implants. In the present study, calcium deficient hydroxyapatite (CDHA) and Si substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to obtain different microstructural properties, the scaffolds have been treated at 700°C and 1250°C. The scaffolds have been characterized and tested as supports for both osteoblast growth and pre-osteoblast differentiation, as fundamental requisite for their potential use in bone tissue engineering. Morphology, viability, adhesion, proliferation, cell cycle, apoptosis, intracellular content of reactive oxygen species and interleukin-6 production were evaluated after contact of osteoblasts-like cells with CDHA and SiHA materials. An adequate interaction of osteoblasts-like cells and preosteoblasts-like cells with all these scaffolds was observed. However, the higher bone cell proliferation and differentiation on CDHA and SiHA scaffolds treated at 1250°C and the lower adsorption of albumin and fibrinogen on these materials in comparison to those treated at 700°C, suggest a better tissue response to CDHA and SiHA materials treated at high temperature.

Effects of nanocrystalline hydroxyapatites on macrophage polarization

Silicon substituted and nanocrystalline hydroxyapatites have attracted the attention of many researchers due to their up-regulation in osteoblast cell metabolism and enhanced bioreactivity, respectively. On the other hand, the biomaterial success or failure depends ultimately on the immune response triggered after its implantation. Macrophages are the main components of the innate immune system with an important role in healing and tissue remodelling due to their remarkable functional plasticity, existing in a whole spectrum of functional populations with varying phenotypic features. The effects of nanocrystalline hydroxyapatite (nano-HA) and nanocrystalline silicon substituted hydroxyapatite (nano-SiHA) on the macrophage populations defined as pro-inflammatory (M1) and reparative (M2) phenotypes have been evaluated in the present study using RAW 264.7 cells and mouse peritoneal macrophages as in vitro models. M1 and M2 macrophage phenotypes were characterized by flow cytometry and confocal microscopy by the expression of CD80 and CD163, known as M1 and M2 markers, respectively. The polarization of primary macrophages towards the M1 or M2 phenotype was induced with the pro-inflammatory stimulus LPS or the anti-inflammatory stimulus IL-10, respectively, evaluating the biomaterial effects under these conditions. Our results show that both nano-HA and nano-SiHA favour the macrophage polarization towards an M2 reparative phenotype, decreasing M1 population and ensuring an appropriate response in the implantation site of these biomaterials designed for bone repair and bone tissue engineering.

Influence of the covalent immobilization of graphene oxide in poly(vinyl alcohol) on human osteoblast response

The differences in the response of human Saos-2 osteoblasts to nanocomposites of poly(vinyl alcohol) (PVA) and 1.5wt.% graphene oxide (GO) prepared by covalent linking (PVA/GO-c) and simple blending (PVA/GO-m) have been evaluated through different biocompatibility parameters. The effects produced on osteoblasts by these two nanocomposites were analysed in parallel and compared with the direct action of GO and with the effect of PVA films without GO. The intracellular content of reactive oxygen species (ROS) and the levels of interleukin-6 (IL-6) were measured to evaluate oxidative stress induction and protective response, respectively. The results demonstrate that the combination of GO with PVA reduces both the proliferation delay and the internal cell complexity alterations induced by GO on human osteoblasts. Moreover, the covalent attachment of GO to the PVA chains increases both cell viability and IL-6 levels, reducing both apoptosis and intracellular ROS content when compared to simple blending of both materials. The use of this strategy to modulate the biointerface reduces the toxic effects of graphene while preserving the reinforcement characteristics for application in tissue engineering scaffolds, and has enormous interest for polymer/graphene biomaterials development.

Effects of bleaching on osteoclast activity and their modulation by osteostatin and fibroblast growth factor 2.

HYPOTHESIS Dental bleaching with H2O2 is a common daily practice in dentistry to correct discoloration of anterior teeth. The aim of this study has been to determine whether this treatment of human teeth affects growth, differentiation and activity of osteoclast-like cells, as well as the putative modulatory action of osteostatin and fibroblast growth factor 2 (FGF-2). EXPERIMENTS Previously to the in vitro assays, structural, physical-chemical and morphological features of teeth after bleaching were studied. Osteoclast-like cells were cultured on human dentin disks, pre-treated or not with 38% H2O2 bleaching gel, in the presence or absence of osteostatin (100 nM) or FGF-2 (1 ng/ml). Cell proliferation and viability, intracellular content of reactive oxygen species (ROS), pro-inflammatory cytokine (IL-6 and TNFα) secretion and resorption activity were evaluated. FINDINGS Bleaching treatment failed to affect either the structural or the chemical features of both enamel and dentin, except for slight morphological changes, increased porosity in the most superficial parts (enamel), and a moderate increase in the wettability degree. In this scenario, bleaching produced an increased osteoclast-like cell proliferation but decreased cell viability and cytokine secretion, while it augmented resorption activity on dentin. The presence of either osteostatin or FGF-2 reduced the osteoclast-like cell proliferation induced by bleaching. FGF-2 enhanced ROS content, whereas osteostatin decreased ROS but increased TNFα secretion. The bleaching effect on resorption activity was increased by osteostatin, but this effect was less evident with FGF-2. CONCLUSIONS These findings further confirm the deleterious effects of tooth bleaching by affecting osteoclast growth and function as well as different modulatory actions of osteostatin and FGF-2.

High Glucose Alters the Secretome of Mechanically Stimulated Osteocyte-like Cells Affecting Osteoclast Precursor Recruitment and Differentiation.

Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte‐driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro‐survival effect conferred to osteocytic MLO‐Y4 cells and osteoblastic MC3T3‐E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre‐exposure prevented both cell survival and ERK and β‐catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO‐Y4 and MC3T3‐E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO‐Y4 cell‐conditioned medium (CM), but not by MC3T3‐E1 cell‐CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP‐1α, MIP‐1β MCP‐1, and GM‐CSF in MLO‐Y4 cell‐CM. RAW264.7 proliferation was inhibited by MLO‐Y4 CM under static or HG conditions, but it increased by FF‐CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG‐CM in static condition allowed formation of osteoclast‐like cells, which were unable to resorb hydroxyapatite. In contrast, FF‐CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL‐6 secretion or their inhibition induced by FF in MLO‐Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte‐osteoclast communication.