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1.
Biomater Adv ; 159: 213794, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38367317

RESUMO

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.


Assuntos
Candida albicans , Hidrogéis , Suínos , Animais , Macrófagos , Pirenos
2.
Nanoscale ; 15(42): 17173-17183, 2023 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-37853851

RESUMO

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.


Assuntos
Células Progenitoras Endoteliais , Nanoestruturas , Animais , Suínos , Células Progenitoras Endoteliais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Nanoestruturas/toxicidade
3.
Int J Mol Sci ; 24(4)2023 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-36834659

RESUMO

Research in nanomaterials with applications in bone regeneration therapies has experienced a very significant advance with the development of bioactive mesoporous nanoparticles (MBNPs). These nanomaterials consist of small spherical particles that exhibit chemical properties and porous structures that stimulate bone tissue regeneration, since they have a composition similar to that of conventional sol-gel bioactive glasses and high specific surface area and porosity values. The rational design of mesoporosity and their ability to incorporate drugs make MBNPs an excellent tool for the treatment of bone defects, as well as the pathologies that cause them, such as osteoporosis, bone cancer, and infection, among others. Moreover, the small size of MBNPs allows them to penetrate inside the cells, provoking specific cellular responses that conventional bone grafts cannot perform. In this review, different aspects of MBNPs are comprehensively collected and discussed, including synthesis strategies, behavior as drug delivery systems, incorporation of therapeutic ions, formation of composites, specific cellular response and, finally, in vivo studies that have been performed to date.


Assuntos
Nanopartículas , Nanoestruturas , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Regeneração Óssea , Preparações Farmacêuticas , Osso e Ossos , Porosidade , Vidro/química
4.
Int J Mol Sci ; 23(18)2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36142540

RESUMO

The activation of T helper (Th) lymphocytes is necessary for the adaptive immune response as they contribute to the stimulation of B cells (for the secretion of antibodies) and macrophages (for phagocytosis and destruction of pathogens) and are necessary for cytotoxic T-cell activation to kill infected target cells. For these issues, Th lymphocytes must be converted into Th effector cells after their stimulation through their surface receptors TCR/CD3 (by binding to peptide-major histocompatibility complex localized on antigen-presenting cells) and the CD4 co-receptor. After stimulation, Th cells proliferate and differentiate into subpopulations, like Th1, Th2 or Th17, with different functions during the adaptative immune response. Due to the central role of the activation of Th lymphocytes for an accurate adaptative immune response and considering recent preclinical advances in the use of nanomaterials to enhance T-cell therapy, we evaluated in vitro the effects of graphene oxide (GO) and two types of reduced GO (rGO15 and rGO30) nanostructures on the Th2 lymphocyte cell line SR.D10. This cell line offers the possibility of studying their activation threshold by employing soluble antibodies against TCR/CD3 and against CD4, as well as the simultaneous activation of these two receptors. In the present study, the effects of GO, rGO15 and rGO30 on the activation/proliferation rate of these Th2 lymphocytes have been analyzed by studying cell viability, cell cycle phases, intracellular content of reactive oxygen species (ROS) and cytokine secretion. High lymphocyte viability values were obtained after treatment with these nanostructures, as well as increased proliferation in the presence of rGOs. Moreover, rGO15 treatment decreased the intracellular ROS content of Th2 cells in all stimulated conditions. The analysis of these parameters showed that the presence of these GO and rGO nanostructures did not alter the response of Th2 lymphocytes.


Assuntos
Ativação Linfocitária , Nanoestruturas , Anticorpos , Antígenos CD4/metabolismo , Citocinas/metabolismo , Grafite , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores , Células Th1 , Células Th17 , Células Th2
5.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206699

RESUMO

Graphene and its derivatives are very promising nanomaterials for biomedical applications and are proving to be very useful for the preparation of scaffolds for tissue repair. The response of immune cells to these graphene-based materials (GBM) appears to be critical in promoting regeneration, thus, the study of this response is essential before they are used to prepare any type of scaffold. Another relevant factor is the variability of the GBM surface chemistry, namely the type and quantity of oxygen functional groups, which may have an important effect on cell behavior. The response of RAW-264.7 macrophages to graphene oxide (GO) and two types of reduced GO, rGO15 and rGO30, obtained after vacuum-assisted thermal treatment of 15 and 30 min, respectively, was evaluated by analyzing the uptake of these nanostructures, the intracellular content of reactive oxygen species, and specific markers of the proinflammatory M1 phenotype, such as CD80 expression and secretion of inflammatory cytokines TNF-α and IL-6. Our results demonstrate that GO reduction resulted in a decrease of both oxidative stress and proinflammatory cytokine secretion, significantly improving its biocompatibility and potential for the preparation of 3D scaffolds able of triggering the appropriate immune response for tissue regeneration.


Assuntos
Grafite/metabolismo , Macrófagos/fisiologia , Oxirredução , Estresse Oxidativo , Temperatura , Animais , Biomarcadores , Células Cultivadas , Citocinas/metabolismo , Expressão Gênica , Grafite/química , Mediadores da Inflamação/metabolismo , Camundongos , Microscopia de Força Atômica , Nanoestruturas/química , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Análise Espectral
6.
Int J Mol Sci ; 22(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917732

RESUMO

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.


Assuntos
Tecido Adiposo , Matriz Extracelular , Imunocompetência , Macrófagos/citologia , Macrófagos/fisiologia , Engenharia Tecidual/métodos , Alicerces Teciduais , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Técnicas de Cultura de Células , Matriz Extracelular/química , Coto Gástrico , Humanos , Lipídeos/química , Ativação de Macrófagos , Camundongos , Fagocitose/imunologia , Células RAW 264.7 , Suínos , Alicerces Teciduais/química
7.
Int J Mol Sci ; 21(21)2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33167415

RESUMO

Mesoporous bioactive glass nanospheres (NanoMBGs) have high potential for clinical applications. However, the impact of these nanoparticles on the immune system needs to be addressed. In this study, the biocompatibility of SiO2-CaO NanoMBGs was evaluated on different mouse immune cells, including spleen cells subsets, bone marrow-derived dendritic cells (BMDCs), or cell lines like SR.D10 Th2 CD4+ lymphocytes and DC2.4 dendritic cells. Flow cytometry and confocal microscopy show that the nanoparticles were rapidly and efficiently taken up in vitro by T and B lymphocytes or by specialized antigen-presenting cells (APCs) like dendritic cells (DCs). Nanoparticles were not cytotoxic and had no effect on cell viability or proliferation under T-cell (anti-CD3) or B cell (LPS) stimuli. Besides, NanoMBGs did not affect the balance of spleen cell subsets, or the production of intracellular or secreted pro- and anti-inflammatory cytokines (TNF-α, IFN-γ, IL-2, IL-6, IL-10) by activated T, B, and dendritic cells (DC), as determined by flow cytometry and ELISA. T cell activation surface markers (CD25, CD69 and Induced Costimulator, ICOS) were not altered by NanoMBGs. Maturation of BMDCs or DC2.4 cells in vitro was not altered by NanoMBGs, as shown by expression of Major Histocompatibility Complex (MHC) and costimulatory molecules (CD40, CD80, CD86), or IL-6 secretion. The effect of wortmannin and chlorpromazine indicate a role for phosphoinositide 3-kinase (PI3K), actin and clathrin-dependent pathways in NanoMBG internalization. We thus demonstrate that these NanoMBGs are both non-toxic and non-inflammagenic for murine lymphoid cells and myeloid DCs despite their efficient intake by the cells.


Assuntos
Compostos de Cálcio/química , Células Dendríticas/efeitos dos fármacos , Teste de Materiais/métodos , Nanosferas/química , Óxidos/química , Dióxido de Silício/química , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Feminino , Técnicas Imunológicas , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Porosidade , Baço/citologia
8.
Eur J Pharm Biopharm ; 133: 258-268, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30385420

RESUMO

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 proliferation 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.


Assuntos
Compostos de Cálcio/química , Isoflavonas/farmacologia , Nanosferas/química , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Óxidos/química , Dióxido de Silício/química , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/métodos , Humanos , Isoflavonas/química , Macrófagos/efeitos dos fármacos , Camundongos , Células RAW 264.7
9.
J Cell Physiol ; 232(12): 3611-3621, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28138960

RESUMO

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


Assuntos
Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Glucose/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células 3T3 , Animais , Comunicação Celular/efeitos dos fármacos , Citocinas/metabolismo , Camundongos , Osteoclastos/metabolismo , Osteócitos/metabolismo , Estimulação Física , Células RAW 264.7 , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Colloids Surf B Biointerfaces ; 138: 50-9, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26650079

RESUMO

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


Assuntos
Grafite/química , Osteoblastos/metabolismo , Óxidos/química , Álcool de Polivinil/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Grafite/farmacologia , Humanos , Interleucina-6/metabolismo , Microscopia Eletrônica de Varredura , Nanocompostos/química , Nanocompostos/ultraestrutura , Osteoblastos/efeitos dos fármacos , Óxidos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Análise Espectral Raman , Temperatura , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Difração de Raios X
11.
J Colloid Interface Sci ; 416: 59-66, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24370402

RESUMO

HYPOTHESIS: Synthetic hydroxyapatite (HA) and Si substituted hydroxyapatite (SiHA) are calcium phosphate ceramics currently used in the field of dentistry and orthopaedic surgery. The preparation of both biomaterials as polycrystalline solid pieces or grains formed by nanocrystallites has awakened a great interest to enhance the bioactive behavior due to the microstructural defects and the higher surface area. The study of the macrophage and lymphocyte behavior in contact with nanocrystalline HA and SiHA will allow to elucidate the immune response which conditions the success or rejection of these biomaterials. EXPERIMENTS: HA and SiHA granules (with sizes of tens of microns) have been prepared by controlled aqueous precipitation avoiding subsequent high temperature sintering. HA and SiHA granules were constituted by crystallites smaller than 50 nm. The effects of both nanocrystalline materials on immune system have been evaluated with macrophages (main components of innate immune system) and T lymphocytes (specific cells of adaptive response) after short-term culture as in vitro models of the early immune response. FINDINGS: Significant decreases of macrophage proliferation and phagocytic activity, increased production of inflammatory cytokines (IL-6, TNF-α) and T lymphocyte apoptosis, were induced by these nanocrystalline ceramics suggesting that, after in vivo implantation, they induce significant effects on immune responses, including an early activation of the innate immune system.


Assuntos
Materiais Biocompatíveis/farmacologia , Hidroxiapatitas/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Imunidade Adaptativa , Adsorção , Animais , Materiais Biocompatíveis/química , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Hidroxiapatitas/química , Imunidade Inata , Interleucina-6/biossíntese , Interleucina-6/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Soroalbumina Bovina/química , Silício/química , Propriedades de Superfície , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/metabolismo
12.
Adv Healthc Mater ; 2(8): 1072-90, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23526812

RESUMO

Nano-GO is a graphene derivative with a 2D atomic layer of sp² bonded carbon atoms in hexagonal conformation together with sp³ domains with carbon atoms linked to oxygen functional groups. The supremacy of nano-GO resides essentially in its own intrinsic chemical and physical structure, which confers an extraordinary chemical versatility, high aspect ratio and unusual physical properties. The chemical versatility of nano-GO arises from the oxygen functional groups on the carbon structure that make possible its relatively easy functionalization, under mild conditions, with organic molecules or biological structures in covalent or non-covalent linkage. The synergistic effects resulting from the assembly of well-defined structures at nano-GO surface, in addition to its intrinsic optical, mechanical and electronic properties, allow the development of new multifunctional hybrid materials with a high potential in multimodal cancer therapy. Herein, a comprehensive review of the fundamental properties of nano-GO requirements for cancer therapy and the first developments of nano-GO as a platform for this purpose is presented.


Assuntos
Grafite/química , Nanopartículas/química , Animais , Antineoplásicos/administração & dosagem , Complexos de Coordenação/química , Portadores de Fármacos/química , Grafite/uso terapêutico , Humanos , Lipossomos/química , Lipossomos/uso terapêutico , Nanopartículas/uso terapêutico , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Óxidos/química , Polietilenoglicóis/química , Radiografia
13.
Biomaterials ; 34(5): 1562-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23177613

RESUMO

Graphene oxide (GO) is considered to be a promising nanomaterial for biomedical applications due to its small two-dimensional shape besides its electrical and mechanical properties. However, only a few data concerning the cell responses to this material have been described and the GO biocompatibility has not been yet fully assessed. In the present study, graphene oxide nanosheets (GOs) decorated with 1-arm (1-GOs) and 6-arm (6-GOs) poly(ethylene glycol-amine) (PEG) have been incubated with cultured Saos-2 osteoblasts, MC3T3-E1 preosteoblasts and RAW-264.7 macrophages to analyze several key cell markers for in vitro biocompatibility evaluation. The results demonstrate that, after internalization, GO nanosheets are localized on F-actin filaments inducing cell-cycle alterations, apoptosis and oxidative stress in these cell types. The observed GOs effects must be considered in further studies focused on photothermal cancer therapy as a synergistic factor.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Grafite/farmacologia , Nanopartículas/administração & dosagem , Citoesqueleto de Actina/ultraestrutura , Actinas/ultraestrutura , Óxidos/farmacologia
14.
Acta Biomater ; 8(7): 2770-7, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22487933

RESUMO

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


Assuntos
Durapatita/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Proteínas Imobilizadas/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Proteína Relacionada ao Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Materiais Biocompatíveis/farmacologia , Calcificação Fisiológica/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Osteoblastos/enzimologia , Sus scrofa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
15.
J Biomed Mater Res A ; 100(1): 64-71, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21972012

RESUMO

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


Assuntos
Cerâmica/farmacologia , Vidro/química , Hipertermia Induzida/métodos , Osteossarcoma/terapia , Temperatura , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Eletroforese em Gel Bidimensional , Humanos , Espaço Intracelular/metabolismo , L-Lactato Desidrogenase/metabolismo , Peso Molecular , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Proteômica , Espécies Reativas de Oxigênio/metabolismo
16.
J Mater Sci Mater Med ; 22(2): 405-16, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21132351

RESUMO

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


Assuntos
Osso e Ossos/metabolismo , Durapatita/química , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Silício/química , Engenharia Tecidual/métodos , Apatitas/química , Adesão Celular , Proliferação de Células , Citometria de Fluxo/métodos , Humanos , Microscopia Confocal/métodos , Neovascularização Patológica , Osteoblastos/citologia , Oxigênio/química , Pós , Espécies Reativas de Oxigênio
17.
Acta Biomater ; 5(6): 2045-53, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19332384

RESUMO

Poly(epsilon-caprolactone) (PCL) is a biodegradable polyester whose biocompatibility has been widely demonstrated both in vivo and in vitro. In the last few years, our group has confirmed that NaOH-treated PCL films can serve as a suitable biomaterial for vascular tissue engineering by supporting the culture of primary vascular cells and, more recently, endothelial-like EC(2) cells derived from endothelial progenitor cells (EPC). In the present study, NO production in basal conditions and after stimulation with different agents has been evaluated and related to the reactive oxygen species (ROS) content and the intracellular calcium levels on EC(2) cells cultured on NaOH-treated PCL films. The results obtained demonstrate that EC(2) seeded on NaOH-treated PCL films enhance the basal NO levels and show a faster, more intense response to physiological stimuli such as VEGF, bradykinin and thrombin than vein endothelial cells (ECv). This result could be indicative of a better capacity of EC(2) cells to maintain their endothelial functionality when seeded on polymers. On the other hand, the culture of both EC(2) and ECv cells on NaOH-treated PCL films induces a significant increase in both ROS content and intracellular calcium that is balanced out through the stimulation of NO production in these cells. In conclusion, these results demonstrate the ability of NaOH-treated PCL films to support endothelial cell production of nitric oxide and reinforce the idea of considering the endothelial-like EC(2) cells derived from blood progenitors as an adequate source of endothelial cells to functionalize vascular grafts. Furthermore, NaOH-treated PCL films could be considered as a promising cellular NO production-inducing biomaterial for vascular tissue engineering applications.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Óxido Nítrico/metabolismo , Poliésteres/química , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Teste de Materiais , Membranas Artificiais , Espécies Reativas de Oxigênio/química , Hidróxido de Sódio/química , Propriedades de Superfície , Suínos
18.
Biomaterials ; 30(1): 45-51, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18838165

RESUMO

Biphasic calcium phosphate (BCP), a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has attracted attention as an excellent bone graft substitute. Mixtures of ceramics with agarose, as natural biodegradable binder, have been recently performed in order to increase the flexibility of the ceramic component and to facilitate the biomaterial preparation. In previous studies we have evaluated the response of both L929 fibroblasts and Saos-2 osteoblasts to hydroxyapatite-betaTCP/agarose disks observing a higher sensitivity of osteoblasts to this biomaterial. In the present study, the use of specific fluorescent probes and antibodies has allowed to evaluate different cell function parameters as biocompatibility markers for the cell/biomaterial interaction of Saos-2 osteoblasts cultured for 7 days on hydroxyapatite-betaTCP/agarose disks. The cell cycle subG(1) fraction, the exposition of phosphatidylserine on the outside surface of the plasma membrane and the analysis of plasma membrane integrity versus cell size, indicate that the interaction with the biomaterial induces a light increase of apoptosis in osteoblasts without producing cell necrosis. The high percentage of viable cells on the biomaterial and the preservation of endothelial nitric oxide synthase (eNOS) expression, eNOS activity and mitochondrial membrane potential (Deltapsi(m)), demonstrate the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.


Assuntos
Materiais Biocompatíveis/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Necrose , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Osteoblastos/enzimologia , Osteoblastos/patologia , Fosfatidilserinas/metabolismo
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