Una nueva biocerámica nanocomposite para regeneración tisular ósea

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Química Inorgánica y Bioinorgánica, leída el 23/04/2014Depto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEunpu

Tailoring hierarchical meso- macroporous 3D scaffolds: from nano to macro

Bone tissue regeneration requires the use of 3D scaffolds which mimic the architecture of the natural extracellular matrix, creating an adequate microenvironment for bone cell growth. Such 3D scaffolds need surface properties suitable for biological recognition in the early stage of cell adhesion, necessary to ensure complete cell colonization, retained cell functionality, and subsequently bone regeneration. Herein, hierarchical 3D scaffolds based on new hydroxyapatite/mesoporous glass nanocomposite bioceramic (MGHA) exhibiting different scales of porosity have been synthesized. These 3D scaffolds possess: (i) highly ordered mesopores with diameters of 10 nm; (ii) macropores with diameters in the 30-80 mu m range with interconnections of 1-10 mu m; and (iii) large macropores of ca. 500 mu m. To improve their surface properties, 3D scaffolds were modified through direct functionalization with amine propyl groups, which notably improve preosteoblast adhesion, proliferation (2.3 fold), differentiation (4.8 fold) and further cell colonization of these scaffolds. The observed enhancement can be related to these amine groups which favour early adhesion, e. g., based on nonspecific protein adsorption as was demonstrated by ellipsometry. These results suggest that the combination of hierarchical structure design and amine surface modification of hydroxyapatite/mesoporous nanocomposite scaffolds yields a double increase in cell proliferation, as well as a quadruple increase in cell differentiation, demonstrating the potential of these nanocomposite materials for bone tissue regeneration purposes

MC3T3-E1 pre-osteoblast response and differentiation after graphene oxide nanosheet uptake

Nano-graphene oxide (GO) and its functionalized derivatives have aroused a great interest for drug delivery, tissue engineering and photothermal cancer therapy, but their biocompatibility has not yet been fully assessed. The aim of the present study was to evaluate the proliferation and differentiation of MC3T3-E1 pre-osteoblasts after the uptake of GO nanosheets (c.a. 400nm), functionalized with poly(ethylene glycol-amine) (PEG) and labelled with fluorescein isothiocyanate (FITC). Significant proliferation decrease and apoptosis increase were observed 3days after incorporation of FITC-PEG-GO by MC3T3-E1 cells. However, alterations on healthy pre-osteoblast differentiation into cells exhibiting osteoblast phenotype were not observed, as they showed normal alkaline phosphatase levels and matrix mineralization 12days after nanosheet uptake. The results suggest that 40μg/mL concentrations of these nanosheets would not affect the differentiation of healthy pre-osteoblasts, thus these PEG-GO nanosheets have potential to be used for biomedical applications after their internalization, as the induction of local hyperthermia on bone cancer.Spanish Ministerio de Economía y CompetitividadDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Biocompatibility and levofloxacin delivery of mesoporous materials

A comparative study of mesoporous matrices designed for both drug-loading methods, impregnation (IP) and surfactant-assisted drug loading (also denoted as one-pot, OP), has been carried out evaluating their physicochemical characteristics, cell response, drug delivery profiles, and antibacterial activity. Surfactant-free (calcined) and surfactant-templated (non-calcined) mesoporous silica have been used as IP and OP starting matrices, respectively. Both non-calcined and calcined matrices do not exert any cytotoxic effect on osteoblasts. However, non-calcined matrices induce on fibroblasts a significant proliferation delay with morphological alterations and dose-dependent increases in fibroblast size, internal complexity, and intracellular calcium content but without cell lysis and apoptosis. Residual ethanol and the surface silanol groups in these non-calcined matrices are involved in the observed fibroblast changes. Finally, both IP and OP matrices have been loaded with levofloxacin to compare them as drug delivery systems. Both IP and OP matrices exhibit similar in vitro levofloxacin release profiles, showing an initial fast delivery followed by a sustained release during long time periods. These profiles and the antimicrobial activity results suggest the use of these IP and OP matrices as local drug delivery systems in the osteomyelitis and other bone infection treatments.Ministerio de Ciencia e Innovación(España)Comunidad de MadridDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

New nanocomposite system with nanocrystalline apatite embedded into mesoporous bioactive glass

Crystalline nanoparticles are very attractive building blocks for the preparation of nanostructured materials. These particles can be dispersed in different noncrystalline mesostructured matrixes in order to obtain nano-composite systems which combine the properties of both components broadening their functionality. In the present study, a novel nanocomposite bioceramic formed by nanocrystalline apatite particles uniformly embedded into a mesostructured SiO2−CaO−P2O5 glass wall has been synthesized through the evaporation-induced self-assembly (EISA) method, commonly used for mesoporous bioactive glass synthesis, but accelerating the sol−gel apatite crystallization rate by strong acidification. Moreover, the use of F127 surfactant as a structure directing agent in this synthesis has allowed the homogeneous nanocrystalline apatite particles incorporation inside of the amorphous mesoporous glass. In vitro bioactive assays have shown a fast apatite-like phase formation similar to that exhibited by mesoporous bioactive glasses. Furthermore, the response of L929 fibroblasts and Saos-2 osteoblasts to this new nanocomposite has indicated a significant improvement in its biocompatibility compared with conventional mesoporous bioactive glasses.Ministerio de Ciencia y Tecnología(España)Comunidad de MadridDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Biopolymer-coated hydroxyapatite foams: A new antidote for heavy metal intoxication

Novel 3D-macroporous biopolymer-coated hydroxyapatite foams are potential devices for the treatment of heavy-metal intoxication by ingestion. These foams are designed to exhibit a fast and efficient metal ion immobilization into the HA structure in acidic media. The capture process of metal ions is stable, not releasing any metal ion when the foams are soaked in clean basic media afterwards. These two steps mimic a digestion process.Ministerio de Ciencia e Innovación (España)Comunidad de MadridMarie Curie EuropeanDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Differential effects of graphene oxide nanosheets on Candida albicans phagocytosis by murine peritoneal macrophages

Macrophages, as effector cells involved in the innate and adaptive immunity, play a key role in the response to nanomaterials as graphene oxide (GO) and in their cellular uptake. The interactions at the interface of GO nanosheets, macrophages and microbial pathogens need to be assessed to determine the possible impairment of the immune system induced by biomedical treatments with this nanomate-rial. Here, we have evaluated by flow cytometry and confocal microscopy the ability of murine peritoneal macrophages to phagocytose the fungal pathogen Candida albicans, alive or heat-killed, after treatment with poly(ethylene glycol-amine)-derivatized GO nanosheets (PEG-GO). After GO treatment, differences in fungal phagocytosis were observed between macrophages that had taken up GO nanosheets (GO+ pop-ulation) and those that had not (GO population). GO treatment increased the ingested alive yeasts in GO macrophages, whereas phagocytosis diminished in the GO+ population. Ingestion of heat-killed yeasts was slightly higher in both GO and GO+ populations when comparing with control macrophages. For the first time, we show that GO uptake by macrophages modulates its phagocytic capability, affecting differentially the subsequent ingestion of either alive or heat-killed yeasts. Enhanced ingestion of heat-killed yeast by GO-treated macrophages suggests a beneficial role of this nanomaterial for the clearance of dead microorganisms during infection.Ministerio de Economía y Competitividad(España)Fundação para a Ciência e a Tecnologia(Portugal)Depto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Biological performance of hydroxyapatite-biopolymer foams: in vitro cell response

Uncoated and biopolymer-coated nanocrystalline hydroxyapatite (HA) macroporous foams are presented as promising candidates as scaffolds for bone tissue regeneration. To this end, foam degradability, the cytotoxic effects on osteoblast-like cells of foam degradation by-products and biocompatibility with osteoblast-like cells were assayed on the three-dimensional (3-D) foam surface. The results show that the 3-D interconnected architectural design of these HA foams allows excellent osteoblast internalization, proliferation and differentiation, exhibiting adequate colonization over the entire scaffold surface with an appropriate degradation rate without any cytotoxic effects.Ministerio de Ciencia y Tecnología(España)Comunidad de MadridDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Diamond-graphite nanoplatelet surfaces as conductive substrates for the electrical stimulation of cell functions

The nanocarbon allotropes constitute valid alternatives when designing control and actuation devices for electrically assisted tissue regeneration purposes, gathering among them important characteristics such as chemical inertness, biocompatibility, extreme mechanical properties, and, importantly, low and tailorable electrical resistivity. In this work, coatings of thin (100 nm) vertically aligned nanoplatelets composed of diamond (5 nm) and graphite were produced via a microwave plasma chemical vapor deposition (MPCVD) technique and used as substrates for electrical stimulation of MC3T3-E1 preosteoblasts. Increasing the amount of N2 up to 14.5 vol % during growth lowers the coatings’ electrical resistivity by over 1 order of magnitude, triggers the nanoplatelet vertical growth, and leads to the higher crystalline quality of the nanographite phase. When preosteoblasts were cultured on these substrates and subjected to two consecutive daily cycles of 3 μA direct current stimulation, enhanced cell proliferation and metabolism were observed accompanied by high cell viability. Furthermore, in the absence of DC stimulation, alkaline phosphatase (ALP) activity is increased significantly, denoting an up-regulating effect of preosteoblastic maturation intrinsically exerted by the nanoplatelet substratesDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu

Magnetic colloidal nanoformulations to remotely trigger mechanotransduction for osteogenic differentiation

Nowadays, diseases associated with an ageing population, such as osteoporosis, require the development of new biomedical approaches to bone regeneration. In this regard, mechanotransduction has emerged as a discipline within the field of bone tissue engineering. Herein, we have tested the efficacy of superparamagnetic iron oxide nanoparticles (SPIONs), obtained by the thermal decomposition method, with an average size of 13 nm, when exposed to the application of an external magnetic field for mechanotransduction in human bone marrowderived mesenchymal stem cells (hBM-MSCs). The SPIONs were functionalized with an Arg-Gly-Asp (RGD) peptide as ligand to target integrin receptors on cell membrane and used in colloidal state. Then, a comprehensive and comparative bioanalytical characterization of non-targeted versus targeted SPIONs was performed in terms of biocompatibility, cell uptake pathways and mechanotransduction effect, demonstrating the osteogenicEuropean Union’s Horizon 2020Ministerio de Ciencia e InnovaciónDepto. de Química en Ciencias FarmacéuticasFac. de FarmaciaTRUEpu