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

Nanocrystallinity effects on osteoblast and osteoclast response to silicon substituted hydroxyapatite

Hypothesis: Silicon substituted hydroxyapatites (SiHA) are highly crystalline bioceramics treated at high temperatures (about 1200ºC) which have been approved for clinical use with spinal, orthopedic, periodontal, oral and craniomaxillofacial applications. The preparation of SiHA with lower temperature methods (about 700ºC) provides nanocrystalline SiHA (nano-SiHA) with enhanced bioreactivity due to higher surface area and smaller crystal size. The aim of this study has been to know the nanocrystallinity effects on the response of both osteoblasts and osteoclasts (the two main cell types involved in bone remodelling) to silicon substituted hydroxyapatite. Experiments: Saos-2 osteoblasts and osteoclast-like cells (differentiated from RAW-264.7 macrophages)have been cultured on the surface of nano-SiHA and SiHA disks and different cell parameters have been evaluated: cell adhesion, proliferation, viability, intracellular content of reactive oxygen species, cell cycle phases, apoptosis, cell morphology, osteoclast-like cell differentiation and resorptive activity. Findings: This comparative in vitro study evidences that nanocrystallinity of SiHA affects the cell/biomaterial interface inducing bone cell apoptosis by loss of cell anchorage (anoikis), delaying osteoclast-like cell differentiation and decreasing the resorptive activity of this cell type. These results suggest the potential use of nano-SiHA biomaterial for preventing bone resorption in treatment of osteoporotic bone

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

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 TNF alpha) 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 TNF alpha 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. (C) 2015 Elsevier Inc. All rights reserved

Effects of immobilized VEGF on endothelial progenitor cells cultured on silicon substituted and nanocrystalline hydroxyapatites

Vascular endothelial growth factor (VEGF) plays an essential role in angiogenesis and vascular homeostasis. Endothelial progenitor cells (EPCs) are primitive bone marrow cells participating in neovascularization and revascularization processes, which also promote bone regeneration. Synthetic hydroxyapatite (HA) has been widely used in bone repair and implant coatings. In HA-based materials, small levels of ionic substitution by silicon (Si) have significant effects on osteoclastic and osteoblastic responses. Moreover, nanocrystalline hydroxyapatites (nano-HA) display enhanced bioreactivity and beneficial effects in bone formation. In this work, the angiogenic potential of VEGF-121 adsorbed on crystalline and nanocrystalline HAs with different Si proportion is evaluated with endothelial-like cells derived from EPCs cultured on nano-HA, nano-SiHA0.25, nano-SiHA0.4, HA, SiHA0.25 and SiHA0.4 disks. The Si amount incorporated for x ¼ 0.25 is enough to yield changes in the textural parameters and surface charge without decomposing the HA phase. Si substitution for x ¼ 0.4 does not result in pure Si-substituted apatites. Si probably remains at the grain boundaries as amorphous silica in nano-SiHA0.4 and SiHA0.4 is decomposed in a-TCP and HA after 1150 �C treatment. Immobilized VEGF on nano-HA, nano-SiHA0.25, nano-SiHA0.4, HA, SiHA0.25 and SiHA0.4 maintains its function exerting a local regulation of the cell response. The crystallite size and topography of nanocrystalline HAs could produce insufficient and weak contacts with endothelial-like cells triggering anoikis. Concerning Si proportion, the best results are obtained with SiHA0.25/VEGF and nano- SiHA0.25/VEGF disks. All these results suggest the potential utility of SiHA0.25/VEGF and nano-SiHA0.25/VEGF for bone repair and tissue engineering by promoting angiogenesis

In vitro evaluation of graphene oxide nanosheets on immune function

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

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

Response of osteoblasts and preosteoblasts to calcium deficient and Sisubstituted hydroxyapatites treated at different temperatures

tHydroxyapatite (HA) is a calcium phosphate bioceramic widely used for bone grafting and augmenta-tion purposes. The biological response of HA can be improved through chemical and microstructuralmodifications, as well as by manufacturing it as macroporous implants. In the present study, calciumdeficient hydroxyapatite (CDHA) and Si substituted hydroxyapatite (SiHA) macroporous scaffolds havebeen prepared by robocasting. In order to obtain different microstructural properties, the scaffolds havebeen treated at 700◦C and 1250◦C. The scaffolds have been characterized and tested as supports forboth osteoblast growth and pre-osteoblast differentiation, as fundamental requisite for their potentialuse in bone tissue engineering. Morphology, viability, adhesion, proliferation, cell cycle, apoptosis, intra-cellular content of reactive oxygen species and interleukin-6 production were evaluated after contactof osteoblasts-like cells with CDHA and SiHA materials. An adequate interaction of osteoblasts-like cellsand preosteoblasts-like cells with all these scaffolds was observed. However, the higher bone cell pro-liferation and differentiation on CDHA and SiHA scaffolds treated at 1250◦C and the lower adsorptionof albumin and fibrinogen on these materials in comparison to those treated at 700◦C, suggest a bettertissue response to CDHA and SiHA materials treated at high temperature

Hidroxiapatitas y nanopartículas de óxido de grafeno: respuesta celular y aplicación biomédica

En la presente Tesis Doctoral Hidroxiapatitas y nanopartículas de óxido de grafeno: Respuesta celular y aplicación biomédica se analizan a nivel celular los efectos de dos tipos de materiales con fines biomédicos muy diferentes: a hidroxiapatitas para regeneración ósea y tratamiento de osteoporosis y b nanopartículas de óxido de grafeno para tratamiento antitumoral por hipertermia. CAPÍTULO I El aumento de la esperanza de vida conlleva una mayor incidencia de patologías asociadas a la edad como la osteoporosis, debida a un desequilibrio en el remodelado óseo. Por esta razón, existe gran demanda de biomateriales para la prevención y tratamiento local de fracturas osteoporóticas. La hidroxiapatita sustituida con silicio SiHA despierta gran interés por su mayor bioactividad y por el papel de este elemento en la calcificación ósea. Las hidroxiapatitas nanocristalinas nanoHA , debido a su similitud con el componente mineral del hueso y su osteoconductividad, ocupan una posición clave respecto a otras biocerámicas. En la presente Tesis Doctoral se ha evaluado el efecto del factor de crecimiento fibroblástico básico unido a SiHA como inductor de los procesos de osteogénesis y angiogénesis. Asimismo, se han investigado los efectos de nanoHA y nanoSiHA sobre macrófagos y linfocitos T y sobre la diferenciación y actividad de osteoclastos, analizando los posibles beneficios de estos materiales para tratar la osteoporosis. Los resultados obtenidos se recogen en el Capítulo I y dieron lugar a las siguientes publicaciones: Matesanz MC et al. Macromol Biosci 12: 446, 2012. Lozano D et al. Acta Biomater 8: 2770, 2012. Matesanz MC et al. J Colloid Interface Sci 416: 59, 2014. Matesanz MC et al. J Mater Chem B 2: 2910, 2014. Todos estos estudios indican el elevado potencial de nanoSiHA como biomaterial biocompatible para la combinación con factores de crecimiento y la modulación del remodelado óseo lo que le convierte en candidato ideal para tratamiento de osteoporosis y regeneración ósea. CAPÍTULO II El osteosarcoma es una patología ósea de gran importancia al afectar mayoritariamente a adolescentes. La inducción de hipertemia local mediante nanopartículas está en estudio actualmente como alternativa a la quimioterapia y la radioterapia. Las nanopartículas de óxido de grafeno nanoGO presentan un gran potencial para su aplicación biomédica debido a su capacidad para inducir hipertermia cuando se exponen a radiación infrarroja cercana NIR , no invasiva, inocua, y que penetra la piel. Una vez internado el nanoGO en las células tumorales, se podría inducir el incremento de temperatura local sobre el material y provocar la muerte celular. En la presente Tesis Doctoral se ha llevado a cabo el estudio de la cinética de incorporación de nanoGO en diferentes tipos celulares, la evaluación de su biocompatibilidad y localización intracelular, así como los efectos de distintos tratamientos de fototermia con nanoGO sobre osteoblastos humanos Saos2 derivados de osteosarcoma. Los resultados obtenidos se recogen en el Capítulo II y han dado lugar a las siguientes publicaciones: Vila M et al. Nanotechnology 23: 465103, 2012. Matesanz MC et al. Biomaterials 34: 1562, 2013. Vila M et al. Nanotechnology 25: 035101, 2014; Global Medical Discovery, Key Nanotechnology Articles, June 27,2014. Estos estudios han permitido conocer los efectos de nanoGO sobre diferentes tipos celulares tras su incorporación, su localización intracelular, así como la importancia de la elección de las condiciones adecuadas de tiempo y potencia de exposición a luz NIR para controlar el tipo de muerte celular, reduciendo los efectos secundarios para su posible utilización en el tratamiento local del osteosarcoma. Los resultados obtenidos permiten conocer las respuestas celulares específicas a nanoSiHA y nanoGO, esenciales para la posible aplicación biomédica de estos materiales en patologías óseas como osteoporosis y osteosarcoma, respectivamente

Response of macrophages and neural cells in contact with reduced graphene oxide microfibers

Graphene-based materials are revealing a great promise for biomedical applications and demonstrating attractiveness for neural repair. In the context of neural tissue damage, the dialogue between neural and immune cells appears critical for driving regeneration, thus making the understanding of their relations pivotal. Herein, the acute response of RAW-264.7 macrophages on nanostructured reduced graphene oxide (rGO) microfibers has been evaluated through the analysis of cell parameters including proliferation, viability, intracellular content of reactive oxygen species, cell cycle, apoptosis, and cell size and complexity. The influence of the direct contact of rGO microfibers on their polarization towards M1 and M2 phenotypes has been studied by analyses of both M1 (CD80) and M2 (CD163) markers and the secretion of the inflammatory cytokines TNF-α and IL-6. Finally, the capability of these rGO microfibers to regulate neural stem cell differentiation has been also evaluated. Findings reveal that rGO microfibers inhibit the proliferation of RAW-264.7 macrophages without affecting their viability and cell cycle profiles. The presence of M1 and M2 macrophages on these microfibers was confirmed after 24 and 48 h, respectively, accompanied by a decrease in TNF-α and an increase in IL-6 cytokine secretion. These rGO microfibers were also able to support the formation of a highly interconnected neural culture composed of both neurons (map2+ cells) and glial cells (vimentin+ cells). These findings encourage further investigation of these microfibers as attractive biomaterials to interact with immune and neural cells, attempting to support wound healing and tissue repair after implantation.Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu