María José Feito

Inhibition of bacterial adhesion on biocompatible zwitterionic SBA-15 mesoporous materials

In this manuscript in vitro bacterial adhesion assays using Escherichia coli on different SBA-15 nanostructured ceramics have been performed. For this purpose pure silica, NH(2) or COOH monofunctionalized, and NH(2)/COOH bifunctionalized SBA-15 mesoporous materials have been used. Material characterization reveals that both NH(2)/COOH and NH(2) functionalized SBA-15 materials exhibit a zwitterionic character due to the presence of -NH(3)(+)/COO(-) or -NH(3)(+)/SiO(-) moieties, respectively. In vitro adhesion assays have been carried out at the pH at which the zwitterionic nature of both of these samples is preserved, i.e. pH 5.5. The results show that the presence of both positive and negative moieties with an overall neutral charge leads to reduced E. coli adhesiveness. In vitro tests with cultured human Saos-2 osteoblasts have been carried out to evaluate the biocompatibility of the different materials at the physiological pH of 7.4. The results demonstrate that all materials exhibit good biocompatibility, with Saos-2 osteoblasts adhering, proliferating and maintaining their morphological and functional characteristics. This novel family of zwitterionic mesoporous materials opens up promising expectations in diverse biomedical applications, such as preventing some side-effects associated with bone implant infections.

Osteostatin improves the osteogenic activity of fibroblast growth factor-2 immobilized in Si-doped hydroxyapatite in osteoblastic cells.

Si-doped hydroxyapatite (Si-HA) is a suitable ceramic for the controlled release of agents to improve bone repair. We recently showed that parathyroid hormone-related protein (PTHrP) (107-111) (osteostatin) has remarkable osteogenic features in various in vitro and in vivo systems. Fibroblast growth factor (FGF)-2 modulates osteoblastic function and induces angiogenesis, and can promote osteoblast adhesion and proliferation after immobilization on Si-HA. In the present study we examined whether osteostatin might improve the biological efficacy of FGF-2-coated Si-HA in osteoblastic MC3T3-E1 cells in vitro. We found that Si-HA/FGF-2 in the presence or absence of osteostatin (100 nM) similarly increased cell growth (by about 50%). However, addition of the latter peptide to Si-HA/FGF-2 significantly enhanced gene expression of Runx2, osteocalcin, vascular endothelial growth factor (VEGF) and the VEGF receptors 1 and 2, without significantly affecting that of FGF receptors in these cells. Moreover, secreted VEGF in the MC3T3-E1 cell conditioned medium, which induced the proliferation of pig endothelial-like cells, was also enhanced by these combined factors. The synergistic action of osteostatin and Si-HA/FGF-2 on the VEGF system was abrogated by a mitogen-activated protein kinase inhibitor (U0126) and by the calcium antagonist verapamil. This action was related to an enhancement of alkaline phosphatase activity and matrix mineralization in MC3T3-E1 cells, and also in primary human osteoblastic cells. These in vitro data show that osteostatin increases the osteogenic efficacy of a Si-HA/FGF-2 biomaterial by a mechanism involving mitogen-activated protein kinases and intracellular Ca(2+). These findings provide an attractive strategy for bone tissue engineering.

Covalently bonded dendrimer-maghemite nanosystems: nonviral vectors for in vitrogene magnetofection

In this work novel nonviral nanosystems for in vitrogene magnetofection are presented. The multifunctional vectors consist of superparamagnetic iron oxide nanoparticles functionalized with low generations of poly(propyleneimine) dendrimers. The dendrimers are attached to the iron oxide nanoparticles through covalent bonds via a one-pot sol–gel synthetic route. This approach allows a direct dendritic decoration of the iron oxide NPs without any additional surface modification. Furthermore, this strategy avoids the multistep procedures of dendritic growth onto solid surfaces. The core–shell hybrid structures are water soluble as colloidal ferrofluids which are long-term stable at physiological pH. In vitro transfection experiments were assayed with Saos-2 osteoblasts, using as reporter gene a plasmid DNA that codes for the green fluorescent protein. Gene delivery experiments were carried out in the presence and in the absence of a magnetic field. The transfection efficiency strongly depends on the presence of the magnetic field and the dendrimer generation. The covalent bonding between the dendrimers and the magnetic nanoparticles surface ensures the vector integrity throughout storage and application. The nanosystems couple the DNA fragments and safely transport them under magnetic stimulus from the extracellular environment to the interior of the cell.

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.

Immobilization and bioactivity evaluation of FGF-1 and FGF-2 on powdered silicon-doped hydroxyapatite and their scaffolds for bone tissue engineering

Fibroblast growth factors (FGFs) are polypeptides that control the proliferation and differentiation of various cell types including osteoblasts. FGFs are also strong inducers of angiogenesis, necessary to obtain oxygen and nutrients during tissue repair. With the aim to incorporate these desirable FGF biological properties into bioceramics for bone repair, silicon substituted hydroxyapatites (Si-HA) were used as materials to immobilize bioactive FGF-1 and FGF-2. Thus, the binding of these growth factors to powdered Si-HA and Si-HA scaffolds was carried out efficiently in the present study and both FGFs maintained its biological activity on osteoblasts after its immobilization. The improvement of cell adhesion and proliferation onto Si-HA scaffolds suggests the potential utility of these FGF/scaffolds for bone tissue engineering.

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.

Subacute Tissue Response to 3D Graphene Oxide Scaffolds Implanted in the Injured Rat Spinal Cord

The increasing prevalence and high sanitary costs of lesions affecting the central nervous system (CNS) at the spinal cord are encouraging experts in different fields to explore new avenues for neural repair. In this context, graphene and its derivatives are attracting significant attention, although their toxicity and performance in the CNS in vivo remains unclear. Here, the subacute tissue response to 3D flexible and porous scaffolds composed of partially reduced graphene oxide is investigated when implanted in the injured rat spinal cord. The interest of these structures as potentially useful platforms for CNS regeneration mainly relies on their mechanical compliance with neural tissues, adequate biocompatibility with neural cells in vitro and versatility to carry topographical and biological guidance cues. Early tissue responses are thoroughly investigated locally (spinal cord at C6 level) and in the major organs (i.e., kidney, liver, lung, and spleen). The absence of local and systemic toxic responses, along with the positive signs found at the lesion site (e.g., filler effect, soft interface for no additional scaring, preservation of cell populations at the perilesional area, presence of M2 macrophages), encourages further investigation of these materials as promising components of more efficient material-based platforms for CNS repair.

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.

Signaling Pathways of Immobilized FGF-2 on Silicon-Substituted Hydroxyapatite

Therapeutic strategies for bone regeneration involve the selection of suitable biomaterials, growth factors, and cell types to mimic the cellular microenvironment where molecular and mechanical signals control the reconstruction of bone tissue. The immobilization of basic fibroblast growth factor (FGF-2) on powdered silicon-substituted hydroxyapatite (Si-HA) allows to prepare a biofunctional biomaterial able to interact with bone cells in a very specific way. The biological activity of FGF-2/Si-HA, evaluated in Saos-2 osteoblasts and MC3T3-E1 preosteoblasts through the PLCγ and MAPK/ERK signal transduction pathways, shows that FGF-2 immobilized on Si-HA provides the right signals to cells stimulating crucial intracellular mechanisms of osteoblast proliferation and differentiation.

In vitro evaluation of glass–glass ceramic thermoseed-induced hyperthermia on human osteosarcoma cell line †

The use of biomaterials as implantable thermoseeds under the action of an external magnetic field is a very interesting methodology to focus the heat into the target tumors as osteosarcoma. In this study, biocompatible and bioactive G15GC85 thermoseeds, tailored through the combination of sol-gel glasses (G) with a magnetic glass ceramic (GC), were used to induce hyperthermia on cultured human osteosarcoma cells after exposition to alternating magnetic field (MF, 100 kHz/200 Oe). G15GC85 magnetic glass-glass ceramic thermoseeds induced in vitro effective hyperthermia with drastic reduction in proliferation of human osteosarcoma Saos-2 cells and high increase of apoptotic cells after two 40 min consecutive sessions of MF. Deep cell morphology alterations were observed after this hyperthermic treatment, and the proteomic analysis revealed modification of gamma actin molecular properties related to cytoskeleton alterations. These results indicate that G15GC85 thermoseeds allow to induce in vitro effective hyperthermia on human osteosarcoma cells.