Search

This study reports on the metabolic changes accompanying the differentiation of MC3T3-E1 osteoprogenitor cells induced by mesoporous bioactive glass nanospheres (nMBG) loaded with ipriflavone (nMBG-IP). Ipriflavone (IP) is a synthetic isoflavone known for inhibiting bone resorption, maintaining bone density, and preventing osteoporosis. Delivering IP intracellularly is a promising strategy to modulate bone remodeling at significantly lower doses compared to free drug administration. Our results demonstrate that nMBG are efficiently internalized by pre-osteoblasts and, when loaded with IP, induce their differentiation. This differentiation process is accompanied by pronounced metabolic alterations, as monitored by NMR analysis of medium supernatants and cell extracts (exo- and endo-metabolomics, respectively). The main effects include an early-stage intensification of glycolysis and changes in several metabolic pathways, such as nucleobase metabolism, osmoregulatory and antioxidant pathways, and lipid metabolism. Notably, the metabolic impacts of nMBG-IP and free IP were very similar, whereas nMBG alone induced only mild changes in the intracellular metabolic profile without affecting the cells’ consumption/secretion patterns or lipid composition. This finding indicates that the observed effects are primarily related to IP-induced differentiation and that nMBG nanospheres serve as convenient carriers with both efficient internalization and minimal metabolic impact. Furthermore, the observed link between pre-osteoblast differentiation and metabolism underscores the potential of utilizing metabolites and metabolic reprogramming as strategies to modulate the osteogenic process, for instance, in the context of osteoporosis and other bone diseases., Ministerio de Ciencia e Innovación (España), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

El objetivo principal del proyecto es facilitar el estudio del metabolismo, su regulación y las interrelaciones entre órganos y tejidos con distintos perfiles metabólicos, destacando su importancia en diversos estados fisiopatológicos. El proyecto pretende además poner de manifiesto la importancia de estos conocimientos para poder desarrollar proyectos de investigación en diferentes áreas. Con este objetivo, los estudiantes de los cinco grupos de primero de Grado en Biología, tutorizados por M Teresa Portolés (responsable del proyecto), Begoña Gómez-Miguel, Ana Saborido y Mercedes Echaide, profesoras que imparten Metabolismo en la asignatura de Bioquímica, han realizado las actividades indicadas a continuación, organizados en equipos de trabajo centrados en órganos y tejidos clave como cerebro, hígado, músculo, tejido adiposo y glóbulos rojos. - Elaboración de preguntas de examen, fijando la atención en los aspectos más importantes al finalizar el estudio de cada tema. - Realización de infografías expuestas en la Facultad de Ciencias Biológicas del 13 al 17 de mayo, para representar de forma clara, concisa y atractiva las características metabólicas de tipos celulares especializados y sus interrelaciones, facilitando su comprensión y estudio. - Elaboración de un mapa integrador interactivo del metabolismo en las aulas de informática de la Facultad de Ciencias Biológicas con la pizarra digital interactiva, destacando la interconexión de las diferentes rutas metabólicas en la que se basa el funcionamiento del organismo. Laura Casarrubios (Doctora en Biología) ha participado preparando dos vídeos explicativos de los programas Miro y BioRender. - Asistencia a charlas impartidas por las profesoras del proyecto enfocadas en sus proyectos de investigación y con la participación de Mónica Cicuéndez (PAD) experta en Metabolómica, para transmitir a los estudiantes la importancia del estudio del metabolismo en el desarrollo de actividades investigadoras en diferentes áreas. El

Despite the immense need for effective treatment of spinal cord injury (SCI), no successful repair strategy has yet been clinically implemented. Multifunctional biomaterials, based on porcine adipose tissue-derived extracellular matrix (adECM) and reduced graphene oxide (rGO), were recently shown to stimulate in vitro neural stem cell growth and differentiation. Nevertheless, their functional performance in clinically more relevant in vivo conditions remains largely unknown. Before clinical application of these adECM-rGO nanocomposites can be considered, a rigorous assessment of the cytotoxicity and biocompatibility of these biomaterials is required. For instance, xenogeneic adECM scaffolds could still harbour potential immunogenicity following decellularization. In addition, the toxicity of rGO has been studied before, yet often in experimental settings that do not bear relevance to regenerative medicine. Therefore, the present study aimed to assess both the in vitro as well as in vivo safety of adECM and adECM-rGO scaffolds. First, pulmonary, renal and hepato-cytotoxicity as well as macrophage polarization studies showed that scaffolds were benign in vitro. Then, a laminectomy was performed at the 10th thoracic vertebra, and scaffolds were implanted directly contacting the spinal cord. For a total duration of 6 weeks, animal welfare was not negatively affected. Histological analysis demonstrated the degradation of adECM scaffolds and subsequent tissue remodeling. Graphene-based scaffolds showed a very limited fibrous encapsulation, while rGO sheets were engulfed by foreign body giant cells. Furthermore, all scaffolds were infiltrated by macrophages, which were largely polarized towards a pro-regenerative phenotype. Lastly, organ-specific histopathology and biochemical analysis of blood did not reveal any adverse effects. In summary, both adECM and adECM-rGO implants were biocompatible upon laminectomy while establishing a pro-regenerative microenvironment, which j, European Commission, Depto. de Bioquímica y Biología Molecular, Depto. de Microbiología y Parasitología, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

Extracellular matrix hydrogels are considered one of the most suitable biomaterials for tissue regeneration due to their similarity with the extracellular microenvironment of the native tissue. Their properties are dependent on their composition, material concentration, fiber density and the fabrication approaches, among other factors. The encapsulation of immune cells in this kind of hydrogels, both in absence or presence of a pathogen, represents a promising strategy for the development of platforms that mimic healthy and infected tissues, respectively. In this work, we have encapsulated macrophages in 3D hydrogels of porcine decellularized adipose matrices (pDAMs) without and with the Candida albicans fungus, as 3D experimental models to study the macrophage immunocompetence in a closer situation to the physiological conditions and to mimic an infection scenario. Our results indicate that encapsulated macrophages preserve their functionality within these pDAM hydrogels and phagocytose live pathogens. In addition, their behavior is influenced by the hydrogel pore size, inversely related to the hydrogel concentration. Thus, larger pore size promotes the polarization of macrophages towards M2 phenotype along the time and enhances their phagocytosis capability. It is important to point out that encapsulated macrophages in absence of pathogen showed an M2 phenotype, but macrophages coencapsulated with C. albicans can switch towards an M1 inflammatory phenotype to resolve the infection, depending on the fungus quantity. The present study reveals that pDAM hydrogels preserve the macrophage plasticity, demonstrating their relevance as new models for macrophage-pathogen interaction studies that mimic an infection scenario with application in regenerative medicine research., European Union’s Horizon 2020, Depto. de Bioquímica y Biología Molecular, Depto. de Microbiología y Parasitología, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

Extracellular matrix hydrogels are considered one of the most suitable biomaterials for tissue regeneration due to their similarity with the extracellular microenvironment of the native tissue. Their properties are dependent on their composition, material concentration, fiber density and the fabrication approaches, among other factors. The encapsulation of immune cells in this kind of hydrogels, both in absence or presence of a pathogen, represents a promising strategy for the development of platforms that mimic healthy and infected tissues, respectively. In this work, we have encapsulated macrophages in 3D hydrogels of porcine decellularized adipose matrices (pDAMs) without and with the Candida albicans fungus, as 3D experimental models to study the macrophage immunocompetence in a closer situation to the physiological conditions and to mimic an infection scenario. Our results indicate that encapsulated macrophages preserve their functionality within these pDAM hydrogels and phagocytose live pathogens. In addition, their behavior is influenced by the hydrogel pore size, inversely related to the hydrogel concentration. Thus, larger pore size promotes the polarization of macrophages towards M2 phenotype along the time and enhances their phagocytosis capability. It is important to point out that encapsulated macrophages in absence of pathogen showed an M2 phenotype, but macrophages coencapsulated with C. albicans can switch towards an M1 inflammatory phenotype to resolve the infection, depending on the fungus quantity. The present study reveals that pDAM hydrogels preserve the macrophage plasticity, demonstrating their relevance as new models for macrophage-pathogen interaction studies that mimic an infection scenario with application in regenerative medicine research., European Union’s Horizon 2020, Depto. de Bioquímica y Biología Molecular, Depto. de Microbiología y Parasitología, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

El objetivo principal del proyecto es facilitar el estudio del metabolismo, su regulación y las interrelaciones entre órganos y tejidos con distintos perfiles metabólicos, destacando su importancia en diversos estados fisiopatológicos. El proyecto pretende además poner de manifiesto la importancia de estos conocimientos para poder desarrollar proyectos de investigación en diferentes áreas. Con este objetivo, los estudiantes de los cinco grupos de primero de Grado en Biología, tutorizados por M Teresa Portolés (responsable del proyecto), Begoña Gómez-Miguel, Ana Saborido y Mercedes Echaide, profesoras que imparten Metabolismo en la asignatura de Bioquímica, han realizado las actividades indicadas a continuación, organizados en equipos de trabajo centrados en órganos y tejidos clave como cerebro, hígado, músculo, tejido adiposo y glóbulos rojos. - Elaboración de preguntas de examen, fijando la atención en los aspectos más importantes al finalizar el estudio de cada tema. - Realización de infografías expuestas en la Facultad de Ciencias Biológicas del 13 al 17 de mayo, para representar de forma clara, concisa y atractiva las características metabólicas de tipos celulares especializados y sus interrelaciones, facilitando su comprensión y estudio. - Elaboración de un mapa integrador interactivo del metabolismo en las aulas de informática de la Facultad de Ciencias Biológicas con la pizarra digital interactiva, destacando la interconexión de las diferentes rutas metabólicas en la que se basa el funcionamiento del organismo. Laura Casarrubios (Doctora en Biología) ha participado preparando dos vídeos explicativos de los programas Miro y BioRender. - Asistencia a charlas impartidas por las profesoras del proyecto enfocadas en sus proyectos de investigación y con la participación de Mónica Cicuéndez (PAD) experta en Metabolómica, para transmitir a los estudiantes la importancia del estudio del metabolismo en el desarrollo de actividades investigadoras en diferentes áreas. El

La osteoporosis es la enfermedad degenerativa con mayor prevalencia en el mundo. Se trata de una enfermedad metabólica ósea, progresiva y crónica. Está causada por un desequilibrio en el proceso de remodelado óseo, donde la actividad resortiva de los osteoclastos se ve favorecida frente a la síntesis de nuevo hueso, llevada a cabo por los osteoblastos. Predispone a un aumento del riesgo de fractura a quien la padece, estimándose que 1 de cada 3 mujeres y 1 de cada 5 hombres sufrirá una fractura osteoporótica a o largo de su vida, con el elevado impacto socioeconómico que esto conlleva. Existen diferentes estrategias para el tratamiento de la osteoporosis que incluyen la utilización de fármacos específicos para frenar la actividad resortiva de los osteoclastos y potenciar la función de los osteoblastos, así como el diseño de biomateriales capaces de inducir los procesos de osteogénesis y angiogénesis, implicados en la regeneración del tejido óseo.., Osteoporosis is the most prevalent degenerative disease worldwide. It is a progressive and chronic metabolic bone disease. It is caused by an imbalance in the bone remodeling process, where the resorptive activity of osteoclasts is favored over the synthesis of new bone, carried out by osteoblasts. It predisposes those who suffer the disease to an increased risk of fracture, estimating that 1 in 3 women and 1 in 5 men will suffer an osteoporotic fracture throughout their lives, with the high socioeconomic impact that this entails.There are different strategies for the treatment of osteoporosis that include the use of specific drugs to slow down the resorptive activity of osteoclasts and enhance the function of osteoblasts, and the design of biomaterials with the ability to induce the processes of osteogenesis and angiogenesis, involved in the regeneration of bone tissue...

Graphene oxide (GO) and reduced graphene oxide (rGO) have been widely used in the field of tissue regeneration and various biomedical applications. In order to use these nanomaterials in organisms, it is imperative to possess an understanding of their impact on different cell types. Due to the potential of these nanomaterials to enter the bloodstream, interact with the endothelium and accumulate within diverse tissues, it is highly relevant to probe them when in contact with the cellular components of the vascular system. Endothelial progenitor cells (EPCs), involved in blood vessel formation, have great potential for tissue engineering and offer great advantages to study the possible angiogenic effects of biomaterials. Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates vascular permeability, mainly activating VEGFR2 on endothelial cells. The effects of GO and two types of reduced GO, obtained after vacuum-assisted thermal treatment for 15 min (rGO15) and 30 min (rGO30), on porcine endothelial progenitor cells (EPCs) functionality were assessed by analyzing the nanomaterial intracellular uptake, reactive oxygen species (ROS) production and VEGFR2 expression by EPCs. The results evidence that short annealing (15 and 30 minutes) at 200 °C of GO resulted in the mitigation of both the increased ROS production and decline in VEGFR2 expression of EPCs upon GO exposure. Interestingly, after 72 hours of exposure to rGO30, VEGFR2 was higher than in the control culture, suggesting an early angiogenic potential of rGO30. The present work reveals that discrete variations in the reduction of GO may significantly affect the response of porcine endothelial progenitor cells., Portuguese Foundation for Science and Technology, European Commission, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

Graphene oxide (GO) and reduced graphene oxide (rGO) have been widely used in the field of tissue regeneration and various biomedical applications. In order to use these nanomaterials in organisms, it is imperative to possess an understanding of their impact on different cell types. Due to the potential of these nanomaterials to enter the bloodstream, interact with the endothelium and accumulate within diverse tissues, it is highly relevant to probe them when in contact with the cellular components of the vascular system. Endothelial progenitor cells (EPCs), involved in blood vessel formation, have great potential for tissue engineering and offer great advantages to study the possible angiogenic effects of biomaterials. Vascular endothelial growth factor (VEGF) induces angiogenesis and regulates vascular permeability, mainly activating VEGFR2 on endothelial cells. The effects of GO and two types of reduced GO, obtained after vacuum-assisted thermal treatment for 15 min (rGO15) and 30 min (rGO30), on porcine endothelial progenitor cells (EPCs) functionality were assessed by analyzing the nanomaterial intracellular uptake, reactive oxygen species (ROS) production and VEGFR2 expression by EPCs. The results evidence that short annealing (15 and 30 minutes) at 200 °C of GO resulted in the mitigation of both the increased ROS production and decline in VEGFR2 expression of EPCs upon GO exposure. Interestingly, after 72 hours of exposure to rGO30, VEGFR2 was higher than in the control culture, suggesting an early angiogenic potential of rGO30. The present work reveals that discrete variations in the reduction of GO may significantly affect the response of porcine endothelial progenitor cells., Portuguese Foundation for Science and Technology, European Commission, Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Químicas, TRUE, pub

RESEARCH ID B-5081-2017 (Daniel Lozano Borregón) ORCID 0000-0001-5902-9201 (Daniel Lozano Borregón) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí), Zinc-enriched mesoporous bioactive glasses (MBGs) are bioceramics with potential antibacterial and osteogenic properties. However, few assays have been performed to study these properties in animal models.In this study, MBGs enriched with up to 5% ZnO were synthesized, physicochemically characterized, and evaluated for their osteogenic activity both in vitro and in vivo. The ZnO MBGs showed excellent textural properties despite ZnO incorporation. However, the release of Zn2+ ions inhibited the mineralization process when immersed in simulated body fluid. In vitro assays showed significantly highe r values of viability and expression of early markers of celldifferentiation and angiogenesis in a ZnO-content-dependent manner. The next step was to study the osteogenic potential in a sheep bone defect model. Despite their excellent textural properties and cellular response in vitro, the ZnO MBGs were not able to integrate into the bone tissue, which can be explained in terms of inhibition of the mineralization process caused by Zn2+ ions. This work highlights the need to develop nanostructured materials for bone regeneration that can mineralize to interact with bone tissue and induce the processes of implant acceptance, cell colonization by osteogenic cells, and regeneration of lost bone tissue., Unión Europea. Horizonte 2020, Ministerio de Ciencia e Innovación (MICINN), Ministerio de Economía y Competitividad (MINECO), Depto. de Bioquímica y Biología Molecular, Depto. de Química en Ciencias Farmacéuticas, Depto. de Medicina Legal, Psiquiatría y Patología, Fac. de Ciencias Químicas, Fac. de Farmacia, Fac. de Medicina, TRUE, pub

RESEARCH ID B-5081-2017 (Daniel Lozano Borregón) ORCID 0000-0001-5902-9201 (Daniel Lozano Borregón) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí), Zinc-enriched mesoporous bioactive glasses (MBGs) are bioceramics with potential antibacterial and osteogenic properties. However, few assays have been performed to study these properties in animal models.In this study, MBGs enriched with up to 5% ZnO were synthesized, physicochemically characterized, and evaluated for their osteogenic activity both in vitro and in vivo. The ZnO MBGs showed excellent textural properties despite ZnO incorporation. However, the release of Zn2+ ions inhibited the mineralization process when immersed in simulated body fluid. In vitro assays showed significantly highe r values of viability and expression of early markers of celldifferentiation and angiogenesis in a ZnO-content-dependent manner. The next step was to study the osteogenic potential in a sheep bone defect model. Despite their excellent textural properties and cellular response in vitro, the ZnO MBGs were not able to integrate into the bone tissue, which can be explained in terms of inhibition of the mineralization process caused by Zn2+ ions. This work highlights the need to develop nanostructured materials for bone regeneration that can mineralize to interact with bone tissue and induce the processes of implant acceptance, cell colonization by osteogenic cells, and regeneration of lost bone tissue., Unión Europea. Horizonte 2020, Ministerio de Ciencia e Innovación (MICINN), Ministerio de Economía y Competitividad (MINECO), Depto. de Bioquímica y Biología Molecular, Depto. de Química en Ciencias Farmacéuticas, Depto. de Medicina Legal, Psiquiatría y Patología, Fac. de Ciencias Químicas, Fac. de Farmacia, Fac. de Medicina, TRUE, pub

CRUE-CSIC (Acuerdos Transformativos 2021) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), The use of nanoparticles for intracellular drug delivery could reduce the toxicity and side effects of the drug but, the uptake of these nanocarriers could induce adverse effects on cells and tissues after their incorporation. Macrophages play a central role in host defense and are responsible for in vivo nanoparticle trafficking. Assessment of their defense capacity against pathogenic micro-organisms after nanoparticle uptake, is necessary to prevent infections associated with nanoparticle therapies. In this study, the effects of hollow mesoporous SiO2-CaO nanospheres labeled with fluorescein isothiocyanate (FITC-NanoMBGs) on the function of peritoneal macrophages was assessed by measuring their ability to phagocytize Candida albicans expressing a red fluorescent protein. Two macrophage/fungus ratios (MOI 1 and MOI 5) were used and two experimental strategies were carried out: a) pretreatment of macrophages with FITC-NanoMBGs and subsequent fungal infection; b) competition assays after simultaneous addition of fungus and nanospheres. Macrophage pro-inflammatory phenotype markers(CD80 expression and interleukin 6 secretion) were also evaluated. Significant decreases of CD80+ macrophage percentage and interleukin 6 secretion were observed after 30 min, indicating that the simultaneous incorporation of NanoMBG and fungus favors the macrophage non-inflammatory phenotype. The present study evidences that the uptake of these nanospheres in all the studied conditions does not alter the macrophage function. Moreover, intracellular FITC-NanoMBGs induce a transitory increase of the fungal phagocytosis by macrophages at MOI 1 and after a short time of interaction. In the competition assays, as the intracellular fungus quantity increased, the intracellular FITC-NanoMBG content decreased in a MOI- and time-dependent manner. These results have confirmed that macrophages clearly distinguish between inert material and the live yeast in a dynamic intracellular incorporation. Furthe, Unión Europea - Horizonte 2020, Ministerio de Economía, Comercio y Empresa (España)/Fondo Europeo de Desarrollo Regional, Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Depto. de Bioquímica y Biología Molecular, Depto. de Microbiología y Parasitología, Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Biológicas, Fac. de Ciencias Químicas, Fac. de Farmacia, TRUE, pub

Macrophages, cells effective in sensing, internalizing and killing Candida albicans, are intertwined with the extracellular matrix (ECM) through different signals, which include the release of specific cytokines. Due to the importance of these interactions, the employment of in vitro models mimicking a fungal infection scenario is essential to evaluate the ECM effects on the macrophage response. In this work, we have analyzed the effects of human and porcine decellularized adipose matrices (DAMs), obtained by either enzymatic or organic solvent treatment, on the macrophage/Candida albicans interface. The present study has allowed us to detect differences on the activation of macrophages cultured on either human- or porcine-derived DAMs, evidencing changes in the macrophage actin cytoskeleton, such as distinct F-actin-rich membrane structures to surround the pathogen. The macrophage morphological changes observed on these four DAMs are key to understand the defense capability of these cells against this fungal pathogen. This work has contributed to the knowledge of the influence that the extracellular matrix and its components can exert on macrophage metabolism, immunocompetence and capacity to respond to the microenvironment in a possible infection scenario., European Union’s Horizon 2020 Research and Innovation Programme (H2020-FETOPEN-2018-2020, NeuroStimSpinal Project, Grant Agreement No. 829060)., Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage’s functional role., Unión Europea. Horizonte 2020, Depto. de Bioquímica y Biología Molecular, Depto. de Microbiología y Parasitología, Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Químicas, Fac. de Farmacia, TRUE, pub

Due to their specific mesoporous structure and large surface area, mesoporous bioactive glasses (MBGs) possess both drug-delivery ability and effective ionic release to promote bone regeneration by stimulating osteogenesis and angiogenesis. Macrophages secrete mediators that can affect both processes, depending on their phenotype. In this work, the action of ion release from MBG-75S, with a molar composition of 75SiO2-20CaO-5P2O5, on osteogenesis and angiogenesis and the modulatory role of macrophages have been assessed in vitro with MC3T3-E1 pre-osteoblasts and endothelial progenitor cells (EPCs) in monoculture and in coculture with RAW 264.7 macrophages. Ca2+, phosphorous, and silicon ions released from MBG-75S were measured in the culture medium during both differentiation processes. Alkaline phosphatase activity and matrix mineralization were quantified as the key markers of osteogenic differentiation in MC3T3-E1 cells. The expression of CD31, CD34, VEGFR2, eNOS, and vWF was evaluated to characterize the EPC differentiation into mature endothelial cells. Other cellular parameters analyzed included the cell size and complexity, intracellular calcium, and intracellular content of the reactive oxygen species. The results obtained indicate that the ions released by MBG-75S promote osteogenesis and angiogenesis in vitro, evidencing a macrophage inhibitory role in these processes and demonstrating the high potential of MBG-75S for the preparation of implants for bone regeneration., Ministerio de Economía y Competitividad (MINECO)/FEDER, Depto. de Química en Ciencias Farmacéuticas, Depto. de Bioquímica y Biología Molecular, Fac. de Farmacia, Fac. de Ciencias Químicas, TRUE, pub

Unión Europea. Horizonte 2020, Ministerio de Economía y Competitividad (MINECO) / Agencia Estatal de Investigación (AEI) / Fondo Europeo de Desarrollo Regional (FEDER), Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Depto. de Bioquímica y Biología Molecular, Fac. de Ciencias Biológicas, Fac. de Ciencias Químicas, TRUE, pub

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Unión Europea. H2020, Ministerio de Ciencia e Innovación (MICINN), Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Biológicas, Fac. de Farmacia, TRUE, inpress

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Angiogenic biomaterials for bone repair are being designed to promote vascularization and optimize tissue regeneration. The use of nanoparticles of bioactive materials loaded with different drugs represents an interesting strategy to stimulate osteogenesis and angiogenesis and to inhibit bone resorption. Ipriflavone (IP) prevents osteoporosis by inhibiting osteoclast activity and promoting preosteoblast differentiation into mature osteoblasts. Since endothelial progenitor cells (EPCs) are involved in the formation of blood vessels which are necessary for tissue regeneration, the isolation and characterization of porcine EPCs have been carried out in this work to evaluate the in vitro effects of unloaded (NanoMBGs) and IP-loaded nanospheres (NanoMBG-IPs) designed to stimulate osteogenesis. Because different signals between vascular and nonvascular cells are also essential to initiate angiogenic events, the potential modulating role of macrophages has been also evaluated by studying the expression of vascular endothelial growth factor receptor 2 (VEFGR2) as a specific marker for EPC differentiation under different culture conditions: a) EPCs in monoculture treated with NanoMBGs or NanoMBG-IPs, b) EPCs treated with conditioned media from basal, proinflammatory M1 and reparative M2 macrophages previously treated with NanoMBGs or NanoMBG-IPs, c) EPCs cocultured with macrophages in the presence of NanoMBGs or NanoMBG-IPs, and d) EPCs cocultured with M2d angiogenic macrophages. Moreover, the endocytic mechanisms by which these nanospheres are incorporated by EPCs have been identified by using six endocytosis inhibitors (i.e. wortmannin, genistein, cytochalasin B, cytochalasin D, phenylarsine oxide and chlorpromazine) and before the addition of NanoMBGs labeled with fluorescein isothiocyanate. The results evidence the great potential of both NanoMBGs and NanoMBG-IPs to enhance VEFGR2 expression, directly related to angiogenesis, after intracellular incorporation by EPCs thr, Unión Europea. Horizonte 2020, Ministerio de Economía y Competitividad (MINECO)/FEDER, Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Biológicas, Fac. de Farmacia, TRUE, pub

CRUE-CSIC (Acuerdos Transformativos 2021) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), The use of nanoparticles for intracellular drug delivery could reduce the toxicity and side effects of the drug but, the uptake of these nanocarriers could induce adverse effects on cells and tissues after their incorporation. Macrophages play a central role in host defense and are responsible for in vivo nanoparticle trafficking. Assessment of their defense capacity against pathogenic micro-organisms after nanoparticle uptake, is necessary to prevent infections associated with nanoparticle therapies. In this study, the effects of hollow mesoporous SiO2-CaO nanospheres labeled with fluorescein isothiocyanate (FITC-NanoMBGs) on the function of peritoneal macrophages was assessed by measuring their ability to phagocytize Candida albicans expressing a red fluorescent protein. Two macrophage/fungus ratios (MOI 1 and MOI 5) were used and two experimental strategies were carried out: a) pretreatment of macrophages with FITC-NanoMBGs and subsequent fungal infection; b) competition assays after simultaneous addition of fungus and nanospheres. Macrophage pro-inflammatory phenotype markers(CD80 expression and interleukin 6 secretion) were also evaluated. Significant decreases of CD80+ macrophage percentage and interleukin 6 secretion were observed after 30 min, indicating that the simultaneous incorporation of NanoMBG and fungus favors the macrophage non-inflammatory phenotype. The present study evidences that the uptake of these nanospheres in all the studied conditions does not alter the macrophage function. Moreover, intracellular FITC-NanoMBGs induce a transitory increase of the fungal phagocytosis by macrophages at MOI 1 and after a short time of interaction. In the competition assays, as the intracellular fungus quantity increased, the intracellular FITC-NanoMBG content decreased in a MOI- and time-dependent manner. These results have confirmed that macrophages clearly distinguish between inert material and the live yeast in a dynamic intracellular incorporation. Furthe, Unión Europea - Horizonte 2020, Ministerio de Economía, Comercio y Empresa (España)/Fondo Europeo de Desarrollo Regional, Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Depto. de Bioquímica y Biología Molecular, Depto. de Microbiología y Parasitología, Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Biológicas, Fac. de Ciencias Químicas, Fac. de Farmacia, TRUE, pub

Macrophages, cells effective in sensing, internalizing and killing Candida albicans, are intertwined with the extracellular matrix (ECM) through different signals, which include the release of specific cytokines. Due to the importance of these interactions, the employment of in vitro models mimicking a fungal infection scenario is essential to evaluate the ECM effects on the macrophage response. In this work, we have analyzed the effects of human and porcine decellularized adipose matrices (DAMs), obtained by either enzymatic or organic solvent treatment, on the macrophage/Candida albicans interface. The present study has allowed us to detect differences on the activation of macrophages cultured on either human- or porcine-derived DAMs, evidencing changes in the macrophage actin cytoskeleton, such as distinct F-actin-rich membrane structures to surround the pathogen. The macrophage morphological changes observed on these four DAMs are key to understand the defense capability of these cells against this fungal pathogen. This work has contributed to the knowledge of the influence that the extracellular matrix and its components can exert on macrophage metabolism, immunocompetence and capacity to respond to the microenvironment in a possible infection scenario., European Union’s Horizon 2020 Research and Innovation Programme (H2020-FETOPEN-2018-2020, NeuroStimSpinal Project, Grant Agreement No. 829060)., Depto. de Microbiología y Parasitología, Fac. de Farmacia, TRUE, pub

The decellularized extracellular matrix (ECM) obtained from human and porcine adipose tissue (AT) is currently used to prepare regenerative medicine bio-scaffolds. However, the influence of these natural biomaterials on host immune response is not yet deeply understood. Since macrophages play a key role in the inflammation/healing processes due to their high functional plasticity between M1 and M2 phenotypes, the evaluation of their response to decellularized ECM is mandatory. It is also necessary to analyze the immunocompetence of macrophages after contact with decellularized ECM materials to assess their functional role in a possible infection scenario. In this work, we studied the effect of four decellularized adipose matrices (DAMs) obtained from human and porcine AT by enzymatic or chemical methods on macrophage phenotypes and fungal phagocytosis. First, a thorough biochemical characterization of these biomaterials by quantification of remnant DNA, lipids, and proteins was performed, thus indicating the efficiency and reliability of both methods. The proteomic analysis evidenced that some proteins are differentially preserved depending on both the AT origin and the decellularization method employed. After exposure to the four DAMs, specific markers of M1 proinflammatory and M2 anti-inflammatory macrophages were analyzed. Porcine DAMs favor the M2 phenotype, independently of the decellularization method employed. Finally, a sensitive fungal phagocytosis assay allowed us to relate the macrophage phagocytosis capability with specific proteins differentially preserved in certain DAMs. The results obtained in this study highlight the close relationship between the ECM biochemical composition and the macrophage’s functional role., Unión Europea. Horizonte 2020, Depto. de Bioquímica y Biología Molecular, Depto. de Microbiología y Parasitología, Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Químicas, Fac. de Farmacia, TRUE, pub

Due to their specific mesoporous structure and large surface area, mesoporous bioactive glasses (MBGs) possess both drug-delivery ability and effective ionic release to promote bone regeneration by stimulating osteogenesis and angiogenesis. Macrophages secrete mediators that can affect both processes, depending on their phenotype. In this work, the action of ion release from MBG-75S, with a molar composition of 75SiO2-20CaO-5P2O5, on osteogenesis and angiogenesis and the modulatory role of macrophages have been assessed in vitro with MC3T3-E1 pre-osteoblasts and endothelial progenitor cells (EPCs) in monoculture and in coculture with RAW 264.7 macrophages. Ca2+, phosphorous, and silicon ions released from MBG-75S were measured in the culture medium during both differentiation processes. Alkaline phosphatase activity and matrix mineralization were quantified as the key markers of osteogenic differentiation in MC3T3-E1 cells. The expression of CD31, CD34, VEGFR2, eNOS, and vWF was evaluated to characterize the EPC differentiation into mature endothelial cells. Other cellular parameters analyzed included the cell size and complexity, intracellular calcium, and intracellular content of the reactive oxygen species. The results obtained indicate that the ions released by MBG-75S promote osteogenesis and angiogenesis in vitro, evidencing a macrophage inhibitory role in these processes and demonstrating the high potential of MBG-75S for the preparation of implants for bone regeneration., Ministerio de Economía y Competitividad (MINECO)/FEDER, Depto. de Química en Ciencias Farmacéuticas, Depto. de Bioquímica y Biología Molecular, Fac. de Farmacia, Fac. de Ciencias Químicas, TRUE, pub

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Angiogenic biomaterials for bone repair are being designed to promote vascularization and optimize tissue regeneration. The use of nanoparticles of bioactive materials loaded with different drugs represents an interesting strategy to stimulate osteogenesis and angiogenesis and to inhibit bone resorption. Ipriflavone (IP) prevents osteoporosis by inhibiting osteoclast activity and promoting preosteoblast differentiation into mature osteoblasts. Since endothelial progenitor cells (EPCs) are involved in the formation of blood vessels which are necessary for tissue regeneration, the isolation and characterization of porcine EPCs have been carried out in this work to evaluate the in vitro effects of unloaded (NanoMBGs) and IP-loaded nanospheres (NanoMBG-IPs) designed to stimulate osteogenesis. Because different signals between vascular and nonvascular cells are also essential to initiate angiogenic events, the potential modulating role of macrophages has been also evaluated by studying the expression of vascular endothelial growth factor receptor 2 (VEFGR2) as a specific marker for EPC differentiation under different culture conditions: a) EPCs in monoculture treated with NanoMBGs or NanoMBG-IPs, b) EPCs treated with conditioned media from basal, proinflammatory M1 and reparative M2 macrophages previously treated with NanoMBGs or NanoMBG-IPs, c) EPCs cocultured with macrophages in the presence of NanoMBGs or NanoMBG-IPs, and d) EPCs cocultured with M2d angiogenic macrophages. Moreover, the endocytic mechanisms by which these nanospheres are incorporated by EPCs have been identified by using six endocytosis inhibitors (i.e. wortmannin, genistein, cytochalasin B, cytochalasin D, phenylarsine oxide and chlorpromazine) and before the addition of NanoMBGs labeled with fluorescein isothiocyanate. The results evidence the great potential of both NanoMBGs and NanoMBG-IPs to enhance VEFGR2 expression, directly related to angiogenesis, after intracellular incorporation by EPCs thr, Unión Europea. Horizonte 2020, Ministerio de Economía y Competitividad (MINECO)/FEDER, Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Biológicas, Fac. de Farmacia, TRUE, pub

RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), The incorporation and effects of hollow mesoporous nanospheres in the systemSiO2–CaO (nanoMBGs) containing ipriflavone (IP), a synthetic isoflavone that prevents osteoporosis, were evaluated. Due to their superior porosity and capability to host drugs, these nanoparticles are designed as a potential alternative to conventional bioactive glasses for the treatment of periodontal defects. To identify the endocytic mechanisms by which these nanospheres are incorporated within the MC3T3-E1 cells, five inhibitors (cytochalasin B, cytochalasin D, chlorpromazine, genistein and wortmannin) were used before the addition of these nanoparticles labeled with fluorescein isothiocyanate (FITC–nanoMBGs). The results indicate that nanoMBGs enter the pre-osteoblasts mainly through clathrin-dependent mechanisms and in a lower proportion by macropinocytosis. The present study evidences the active incorporation of nanoMBG–IPs by MC3T3-E1 osteoprogenitor cells that stimulate their differentiation into mature osteoblast phenotype with increased alkaline phosphatase activity. The final aim of this study is to demonstrate the biocompatibility and osteogenic behavior of IP-loaded bioactive nanoparticles to be used for periodontal augmentation purposes and to shed light on internalization mechanisms that determine the incorporation of these nanoparticles into the cells., Unión Europea. Horizonte 2020, Ministerio de Economía y Competitividad (MINECO), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Unión Europea. Horizonte 2020, Ministerio de Economía y Competitividad (MINECO)/FEDER, Comunidad de Madrid, Instituto de Salud Carlos III (ISCIII), Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Biológicas, Fac. de Farmacia, TRUE, pub

RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), The incorporation and effects of hollow mesoporous nanospheres in the systemSiO2–CaO (nanoMBGs) containing ipriflavone (IP), a synthetic isoflavone that prevents osteoporosis, were evaluated. Due to their superior porosity and capability to host drugs, these nanoparticles are designed as a potential alternative to conventional bioactive glasses for the treatment of periodontal defects. To identify the endocytic mechanisms by which these nanospheres are incorporated within the MC3T3-E1 cells, five inhibitors (cytochalasin B, cytochalasin D, chlorpromazine, genistein and wortmannin) were used before the addition of these nanoparticles labeled with fluorescein isothiocyanate (FITC–nanoMBGs). The results indicate that nanoMBGs enter the pre-osteoblasts mainly through clathrin-dependent mechanisms and in a lower proportion by macropinocytosis. The present study evidences the active incorporation of nanoMBG–IPs by MC3T3-E1 osteoprogenitor cells that stimulate their differentiation into mature osteoblast phenotype with increased alkaline phosphatase activity. The final aim of this study is to demonstrate the biocompatibility and osteogenic behavior of IP-loaded bioactive nanoparticles to be used for periodontal augmentation purposes and to shed light on internalization mechanisms that determine the incorporation of these nanoparticles into the cells., Unión Europea. Horizonte 2020, Ministerio de Economía y Competitividad (MINECO), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

Ministerio de Economía y Competitividad (España), European Research Council, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

Ministerio de Economía y Competitividad (MINECO), Instituto de salud Carlos III (ISCIII), Fundaçao para a Ciência e a Tecnologia (FCT), Sección Deptal. de Bioquímica y Biología Molecular (Biológicas), Fac. de Ciencias Biológicas, TRUE, pub

RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Silicon-substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to optimize their bone regeneration properties, we have manufactured these scaffolds presenting different microstructures: nanocrystalline and crystalline. Moreover, their surfaces have been decorated with vascular endothelial growth factor (VEGF) to evaluate the potential coupling between vascularization and bone regeneration. In vitro cell culture tests evidence that nanocrystalline SiHA hinders pre-osteblast proliferation, whereas the presence of VEGF enhances the biological functions of both endothelial cells and pre-osteoblasts. The bone regeneration capability has been evaluated using an osteoporotic sheep model. In vivo observations strongly correlate with in vitro cell culture tests. Those scaffolds made of nanocrystalline SiHA were colonized by fibrous tissue, promoted inflammatory response and forested osteoclast recruitment. These observations discard nanocystalline SiHA as a suitable material for bone regeneration purposes. On the contrary, those scaffolds made of crystalline SiHA and decorated with VEGF exhibited bone regeneration properties, with high ossification degree, thicker trabeculae and higher presence of osteoblasts and blood vessels. Considering these results, macroporous scaffolds made of SiHA and decorated with VEGF are suitable bone grafts for regeneration purposes, even in adverse pathological scenarios such as osteoporosis., Unión Europea. H2020, Ministerio de Economía y Competitividad (MINECO), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí), Macroporous scaffolds made of a SiO2-CaO-P2O5 mesoporous bioactive glass (MBG) and ɛpolycaprolactone (PCL) have been prepared by robocasting. These scaffolds showed an excellent in vitro biocompatibility in contact with osteoblast like cells (Saos 2) and osteoclasts derived from RAW 264.7 macrophages. In vivo studies were carried out by implantation into cavitary defects drilled in osteoporotic sheep. The scaffolds evidenced excellent bone regeneration properties, promoting new bone formation at both the peripheral and the inner parts of the scaffolds, thick trabeculae, high vascularization and high presence of osteoblasts and osteoclasts. In order to evaluate the effects of the local release of an antiosteoporotic drug, 1% (%wt) of zoledronic acid was incorporated to the scaffolds. The scaffolds loaded with zoledronic acid induced apoptosis in Saos 2 cells, impeded osteoclast differentiation in a time dependent manner and inhibited bone healing, promoting an intense inflammatory response in osteoporotic sheep., Unión Europea. H2020, Ministerio de Economía y Competitividad (MINECO), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

Macroporous scaffolds made of a SiO2-CaO-P2O5 mesoporous bioactive glass (MBG) and epolycaprolactone (PCL) have been prepared by robocasting. These scaffolds showed an excellent in vitro biocompatibility in contact with osteoblast like cells (Saos 2) and osteoclasts derived from RAW 264.7 macrophages. In vivo studies were carried out by implantation into cavitary defects drilled in osteoporotic sheep. The scaffolds evidenced excellent bone regeneration properties, promoting new bone formation at both the peripheral and the inner parts of the scaffolds, thick trabeculae, high vascularization and high presence of osteoblasts and osteoclasts. In order to evaluate the effects of the local release of an antiosteoporotic drug, 1% (%wt) of zoledronic acid was incorporated to the scaffolds. The scaffolds loaded with zoledronic acid induced apoptosis in Saos 2 cells, impeded osteoclast differentiation in a time dependent manner and inhibited bone healing, promoting an intense inflammatory response in osteoporotic sheep., Ministerio de Economía y Competitividad (España), European Commission, Depto. de Bioquímica y Biología Molecular, Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Químicas, Fac. de Farmacia, TRUE, pub

RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Silicon-substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to optimize their bone regeneration properties, we have manufactured these scaffolds presenting different microstructures: nanocrystalline and crystalline. Moreover, their surfaces have been decorated with vascular endothelial growth factor (VEGF) to evaluate the potential coupling between vascularization and bone regeneration. In vitro cell culture tests evidence that nanocrystalline SiHA hinders pre-osteblast proliferation, whereas the presence of VEGF enhances the biological functions of both endothelial cells and pre-osteoblasts. The bone regeneration capability has been evaluated using an osteoporotic sheep model. In vivo observations strongly correlate with in vitro cell culture tests. Those scaffolds made of nanocrystalline SiHA were colonized by fibrous tissue, promoted inflammatory response and forested osteoclast recruitment. These observations discard nanocystalline SiHA as a suitable material for bone regeneration purposes. On the contrary, those scaffolds made of crystalline SiHA and decorated with VEGF exhibited bone regeneration properties, with high ossification degree, thicker trabeculae and higher presence of osteoblasts and blood vessels. Considering these results, macroporous scaffolds made of SiHA and decorated with VEGF are suitable bone grafts for regeneration purposes, even in adverse pathological scenarios such as osteoporosis., Unión Europea. H2020, Ministerio de Economía y Competitividad (MINECO), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí), Macroporous scaffolds made of a SiO2-CaO-P2O5 mesoporous bioactive glass (MBG) and ɛpolycaprolactone (PCL) have been prepared by robocasting. These scaffolds showed an excellent in vitro biocompatibility in contact with osteoblast like cells (Saos 2) and osteoclasts derived from RAW 264.7 macrophages. In vivo studies were carried out by implantation into cavitary defects drilled in osteoporotic sheep. The scaffolds evidenced excellent bone regeneration properties, promoting new bone formation at both the peripheral and the inner parts of the scaffolds, thick trabeculae, high vascularization and high presence of osteoblasts and osteoclasts. In order to evaluate the effects of the local release of an antiosteoporotic drug, 1% (%wt) of zoledronic acid was incorporated to the scaffolds. The scaffolds loaded with zoledronic acid induced apoptosis in Saos 2 cells, impeded osteoclast differentiation in a time dependent manner and inhibited bone healing, promoting an intense inflammatory response in osteoporotic sheep., Unión Europea. H2020, Ministerio de Economía y Competitividad (MINECO), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

Macroporous scaffolds made of a SiO2-CaO-P2O5 mesoporous bioactive glass (MBG) and epolycaprolactone (PCL) have been prepared by robocasting. These scaffolds showed an excellent in vitro biocompatibility in contact with osteoblast like cells (Saos 2) and osteoclasts derived from RAW 264.7 macrophages. In vivo studies were carried out by implantation into cavitary defects drilled in osteoporotic sheep. The scaffolds evidenced excellent bone regeneration properties, promoting new bone formation at both the peripheral and the inner parts of the scaffolds, thick trabeculae, high vascularization and high presence of osteoblasts and osteoclasts. In order to evaluate the effects of the local release of an antiosteoporotic drug, 1% (%wt) of zoledronic acid was incorporated to the scaffolds. The scaffolds loaded with zoledronic acid induced apoptosis in Saos 2 cells, impeded osteoclast differentiation in a time dependent manner and inhibited bone healing, promoting an intense inflammatory response in osteoporotic sheep., Ministerio de Economía y Competitividad (España), European Commission, Depto. de Bioquímica y Biología Molecular, Depto. de Química en Ciencias Farmacéuticas, Fac. de Ciencias Químicas, Fac. de Farmacia, TRUE, pub

RESEARCHER ID M-3316-2014 (Antonio Salinas Sánchez) ORCID 0000-0002-8408-3389 (Antonio Salinas Sánchez) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez) RESEARCHER ID K-4773-2015 (Blanca González Ortiz) ORCID 0000-0002-0493-6071 (Blanca González Ortiz) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí), Calcium phosphate (CaP) based hybrid materials have been synthesized through a precipitation method in aqueous medium in the presence of the anionic surfactant sodium dodecylbenzene sulfonate (SDBS) as structure directing agent. Parameters of synthesis, such as Ca/P molar ratio, surfactant concentration and initial pH, have been investigated trying to get mesostructured CaP phases. A lamellar-like hybrid mesophase consisting in several wavy layers of apatite or apatite-brushite nanoplates was successfully obtained. Materials have been characterized by several physico- chemical techniques. A potential application of these lamellar calcium phosphate based hybrids can be as matrixes in drug delivery systems with the possibility of loading hydrophobic drugs in the organic interlayers. To evaluate their potential as biomaterials, the biocompatibility of two hybrids has been studied in vitro with human Saos-2 osteoblasts. Cell assays showed that, upon the appropriate synthesis and stabilization conditions, a biocompatible CaP and SDBS mesolamellar hybrid can be prepared., Unión Europea. H2020, Ministerio de Economía, Comercio y Empresa (España), Instituto de Salud Carlos III, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), A mesoporous bioactive glass (MBG) of molar composition 75SiO2-20CaO-5P2O5 (MBG-75S) has been synthetized as a potential bioceramic for bone regeneration purposes. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption studies and transmission electron microscopy (TEM) demonstrated that MBG-75S possess a highly ordered mesoporous structure with high surface area and porosity, which would explain the high ionic exchange rate (mainly calcium and silicon soluble species) with the surrounded media. MBG-75S showed high biocompatibility in contact with Saos-2 osteoblast-like cells. Concentrations up to 1 mg/ml did not lead to significant alterations on either morphology or cell cycle. Regarding the effects on osteoclasts, MBG-75S allowed the differentiation of RAW264.7 macrophages into osteoclast-like cells but exhibiting a decreased resorptive activity. These results point out that MBG-75S does not inhibit osteoclastogenesis but reduces the osteoclast bone-resorbing capability. Finally, in vitro studies focused on the innate immune response, evidenced that MBG-75S allows the proliferation of macrophages without inducing their polarization towards the M1 pro-inflammatory phenotype. This in vitro behavior is indicative that MBG-75S would just induce the required innate immune response without further inflammatory complications under in vivo conditions. The overall behavior respect to osteoblasts, osteoclasts and macrophages, makes this MBG a very interesting candidate for bone grafting applications in osteoporotic patients., Unión Europea. H2020, Ministerio de Economía y Competitividad (MINECO), Comunidad de Madrid, Fondos FEDER, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Mesoporous nanospheres in the system SiO2-CaO (NanoMBGs) with a hollow core surrounded by a radial arrangement of mesopores were characterized, labeled with FITC (FITC-NanoMBGs) and loaded with ipriflavone (NanoMBG-IPs) in order to evaluate their incorporation and their effects on both osteoblasts and osteoclasts simultaneously and maintaining the communication with each other in coculture. The influence of these nanospheres on macrophage polarization towards pro-inflammatory M1 or reparative M2 phenotypes was also evaluated in basal and stimulated conditions through the expression of CD80 (as M1 marker) and CD206 (as M2 marker) by flow cytometry and confocal microscopy. NanoMBGs did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, favoring the M2 reparative phenotype and increasing the macrophage response capability against stimuli as LPS and IL-4. NanoMBG-IPs induced a significant decrease of osteoclast proliferat ion and resorption activity after 7 days in coculture with osteoblasts, without affecting osteoblast proliferation and viability. Drug release test demonstrated that only a fraction of the payload is released by diffusion, whereas the rest of the drug remains within the hollow core after 7 days, thus ensuring the local long-term pharmacological treatment beyond the initial fast IP release. All these data ensure an appropriate immune response to these nanospheres and the potential application of NanoMBG-IPs as local drug delivery system in osteoporotic patients., Unión Europea. H2020, Comunidad de Madrid, Ministerio de Ciencia e Innovación (MICINN), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), A mesoporous bioactive glass (MBG) of molar composition 75SiO2-20CaO-5P2O5 (MBG-75S) has been synthetized as a potential bioceramic for bone regeneration purposes. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption studies and transmission electron microscopy (TEM) demonstrated that MBG-75S possess a highly ordered mesoporous structure with high surface area and porosity, which would explain the high ionic exchange rate (mainly calcium and silicon soluble species) with the surrounded media. MBG-75S showed high biocompatibility in contact with Saos-2 osteoblast-like cells. Concentrations up to 1 mg/ml did not lead to significant alterations on either morphology or cell cycle. Regarding the effects on osteoclasts, MBG-75S allowed the differentiation of RAW264.7 macrophages into osteoclast-like cells but exhibiting a decreased resorptive activity. These results point out that MBG-75S does not inhibit osteoclastogenesis but reduces the osteoclast bone-resorbing capability. Finally, in vitro studies focused on the innate immune response, evidenced that MBG-75S allows the proliferation of macrophages without inducing their polarization towards the M1 pro-inflammatory phenotype. This in vitro behavior is indicative that MBG-75S would just induce the required innate immune response without further inflammatory complications under in vivo conditions. The overall behavior respect to osteoblasts, osteoclasts and macrophages, makes this MBG a very interesting candidate for bone grafting applications in osteoporotic patients., Unión Europea. H2020, Ministerio de Economía y Competitividad (MINECO), Comunidad de Madrid, Fondos FEDER, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID L-6167-2014 (Daniel Arcos Navarrete) ORCID 0000-0002-5367-7272 (Daniel Arcos Navarrete) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez), Mesoporous nanospheres in the system SiO2-CaO (NanoMBGs) with a hollow core surrounded by a radial arrangement of mesopores were characterized, labeled with FITC (FITC-NanoMBGs) and loaded with ipriflavone (NanoMBG-IPs) in order to evaluate their incorporation and their effects on both osteoblasts and osteoclasts simultaneously and maintaining the communication with each other in coculture. The influence of these nanospheres on macrophage polarization towards pro-inflammatory M1 or reparative M2 phenotypes was also evaluated in basal and stimulated conditions through the expression of CD80 (as M1 marker) and CD206 (as M2 marker) by flow cytometry and confocal microscopy. NanoMBGs did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, favoring the M2 reparative phenotype and increasing the macrophage response capability against stimuli as LPS and IL-4. NanoMBG-IPs induced a significant decrease of osteoclast proliferat ion and resorption activity after 7 days in coculture with osteoblasts, without affecting osteoblast proliferation and viability. Drug release test demonstrated that only a fraction of the payload is released by diffusion, whereas the rest of the drug remains within the hollow core after 7 days, thus ensuring the local long-term pharmacological treatment beyond the initial fast IP release. All these data ensure an appropriate immune response to these nanospheres and the potential application of NanoMBG-IPs as local drug delivery system in osteoporotic patients., Unión Europea. H2020, Comunidad de Madrid, Ministerio de Ciencia e Innovación (MICINN), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

RESEARCHER ID M-3316-2014 (Antonio Salinas Sánchez) ORCID 0000-0002-8408-3389 (Antonio Salinas Sánchez) RESEARCHER ID U-1678-2017 (María Teresa Portolés Pérez) ORCID 0000-0002-9681-0184 (María Teresa Portolés Pérez) RESEARCHER ID K-4773-2015 (Blanca González Ortiz) ORCID 0000-0002-0493-6071 (Blanca González Ortiz) RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí), Calcium phosphate (CaP) based hybrid materials have been synthesized through a precipitation method in aqueous medium in the presence of the anionic surfactant sodium dodecylbenzene sulfonate (SDBS) as structure directing agent. Parameters of synthesis, such as Ca/P molar ratio, surfactant concentration and initial pH, have been investigated trying to get mesostructured CaP phases. A lamellar-like hybrid mesophase consisting in several wavy layers of apatite or apatite-brushite nanoplates was successfully obtained. Materials have been characterized by several physico- chemical techniques. A potential application of these lamellar calcium phosphate based hybrids can be as matrixes in drug delivery systems with the possibility of loading hydrophobic drugs in the organic interlayers. To evaluate their potential as biomaterials, the biocompatibility of two hybrids has been studied in vitro with human Saos-2 osteoblasts. Cell assays showed that, upon the appropriate synthesis and stabilization conditions, a biocompatible CaP and SDBS mesolamellar hybrid can be prepared., Unión Europea. H2020, Ministerio de Economía y Competitividad (MINECO), Instituto de Salud Carlos III (ISCIII), Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, inpress

RESEARCHER ID M-3378-2014 (María Vallet Regí) ORCID 0000-0002-6104-4889 (María Vallet Regí) RESEARCHER ID N-4501-2014 (Sandra Sánchez Salcedo) ORCID 0000-0002-1889-2057 (Sandra Sánchez Salcedo), 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., Ministerio de Ciencia e Innovacion (MICINN), Ministerio de Economia y Competitividad (MINECO), Agening Network of Excellence, Depto. de Química en Ciencias Farmacéuticas, Fac. de Farmacia, TRUE, pub