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Skin aging is a complex biological process affected by a plethora of intrinsic and extrinsic factors that alter cutaneous functions through the modulations of signaling pathways and responses. Expressed in various cell types and skin tissue layers, G protein-coupled receptors (GPCRs) play a vital role in regulating skin aging. We have cataloged 156 GPCRs expressed in the skin and reviewed their roles in skin aging, such as pigmentation, loss of elasticity, wrinkles, rough texture, and aging-associated skin disorders. By exploring the GPCRs found in the skin, it may be possible to develop new treatment regimens for aging-associated skin conditions using GPCR ligands.
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Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm-associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm. Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed. Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer. Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells' health.
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The human body follows a physiological rhythm in response to the day/night cycle which is synchronized with the circadian rhythm through internal clocks. Most cells in the human body, including skin cells, express autonomous clocks and the genes responsible for running those clocks. Melatonin, a ubiquitous small molecular weight hormone, is critical in regulating the sleep cycle and other functions in the body. Melatonin is present in the skin and, in this study, we showed that it has the ability to dose-dependently stimulate PER1 clock gene expression in normal human dermal fibroblasts and normal human epidermal keratinocytes. Then we further evaluated the role of MT-1 melatonin receptor in mediating melatonin actions on human skin using fibroblasts derived from young and old subjects. Using immunocytochemistry, Western blotting and RT-PCR, we confirmed the expression of MT-1 receptor in human skin fibroblasts and demonstrated a dramatic age-dependent decrease in its level in mature fibroblasts. We used siRNA technology to transiently knockdown MT-1 receptor in fibroblasts. In these MT-1 knockdown cells, UV-dependent oxidative stress (H2O2 production) was enhanced and DNA damage was also increased, suggesting a critical role of MT-1 receptor in protecting skin cells from UV-induced DNA damage. These studies demonstrate that the melatonin pathway plays a pivotal role in skin aging and damage. Moreover, its correlation with skin circadian rhythm may offer new approaches for decelerating skin aging by modulating the expression of melatonin receptors in human skin.
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Dano ao DNA/efeitos da radiação , Fibroblastos/metabolismo , Receptor MT1 de Melatonina/metabolismo , Pele/metabolismo , Raios Ultravioleta/efeitos adversos , Envelhecimento , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Fibroblastos/efeitos da radiação , Humanos , Estresse Oxidativo/efeitos da radiação , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Receptor MT1 de Melatonina/genética , Pele/efeitos da radiaçãoRESUMO
Autophagic mechanisms play critical roles in cell maintenance. Damaged organelles that are not removed by autophagosomes, which act by engulfing and degrading these cellular components, have been linked to various pathologies. Recently, the progression of aging has also been correlated to a compromised autophagic response. Here, we report for the first time a significant reduction in autophagic levels in synchronized aged normal human skin fibroblasts as compared to young fibroblasts. We measured a 77.9% reduction in autophagy as determined by reverse transcription-polymerase chain reaction for LC3B expression, a microtubule-associated protein correlated to late stage autophagosome formation. In addition, we visualized these same changes by immunocytofluorescence with antibodies directed against LC3B. By harvesting synchronized, as well as unsynchronized cells over time, we were also able to measure for the first time a nighttime peak in autophagy that was present in young but absent in aged fibroblasts. Finally, since human skin is constantly subjected to environmentally induced oxidative stress from sunlight, we exposed fibroblasts to 10 J/cm2 ultraviolet A and found, in good agreement with current literature, not only that irradiation could partially reactivate autophagy in the aged cells, but also that this increase was phase shifted earlier from its endogenous temporal pattern because of its loss of synchronization with circadian rhythm.
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Autofagia/fisiologia , Senescência Celular , Fibroblastos , Raios Ultravioleta , Idoso , Células Cultivadas , Senescência Celular/fisiologia , Senescência Celular/efeitos da radiação , Ritmo Circadiano/fisiologia , Ritmo Circadiano/efeitos da radiação , Fibroblastos/citologia , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Humanos , Recém-Nascido , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Circadian rhythms, ≈24 h oscillations in behavior and physiology, are reflected in all cells of the body and function to optimize cellular functions and meet environmental challenges associated with the solar day. This multi-oscillatory network is entrained by the master pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus, which directs an organism's rhythmic expression of physiological functions and behavior via a hierarchical system. This system has been highly conserved throughout evolution and uses transcriptional-translational autoregulatory loops. This master clock, following environmental cues, regulates an organism's sleep pattern, body temperature, cardiac activity and blood pressure, hormone secretion, oxygen consumption and metabolic rate. Mammalian peripheral clocks and clock gene expression have recently been discovered and are present in all nucleated cells in our body. Like other essential organ of the body, the skin also has cycles that are informed by this master regulator. In addition, skin cells have peripheral clocks that can function autonomously. First described in 2000 for skin, this review summarizes some important aspects of a rapidly growing body of research in circadian and ultradian (an oscillation that repeats multiple times during a 24 h period) cutaneous rhythms, including clock mechanisms, functional manifestations, and stimuli that entrain or disrupt normal cycling. Some specific relationships between disrupted clock signaling and consequences to skin health are discussed in more depth in the other invited articles in this IJMS issue on Sleep, Circadian Rhythm and Skin.
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Ritmo Circadiano , Fenômenos Fisiológicos da Pele , Núcleo Supraquiasmático/metabolismo , Animais , Relógios Circadianos , Expressão Gênica , Regulação da Expressão Gênica , HumanosRESUMO
Sirtuins are post-translational modifiers that affect transcriptional signaling, metabolism, and DNA repair. Although originally identified as gene silencers capable of extending cell lifespan, the involvement of sirtuins in many different areas of cell biology has now become widespread. Our approach has been to study the temporal variation and also the effect of environmental stressors, such as ultraviolet B (UVB) and ozone, on sirtuin expression in human epidermal keratinocytes. In this report, we measured the variation in expression of several sirtuins over time and also show how a low dose of UVB can affect this pattern of expression. Moreover, we correlated these changes to variations in hydrogen peroxide (H2O2) and ATP levels. Our data show significant variations in normal sirtuin expression, which may indicate a generalized response by sirtuins to cell cycle kinetics. These results also demonstrate that sirtuins as a family of molecules are sensitive to UVB-induced disruption and may suggest a new paradigm for determining environmental stress on aging and provide direction for the development of new cosmetic products.
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Epiderme/efeitos da radiação , Queratinócitos/efeitos da radiação , Sirtuínas/metabolismo , Raios Ultravioleta , Linhagem Celular , Células Epidérmicas , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The endogenous oxidative state of normal human epidermal melanocytes was investigated and compared to normal human epidermal keratinocytes (NHEKs) in order to gain new insight into melanocyte biology. Previously, we showed that NHEKs contain higher levels of hydrogen peroxide (H2O2) than melanocytes and that it can migrate from NHEKs to melanocytes by passive permeation. Nevertheless, despite lower concentrations of H2O2, we now report higher levels of oxidative DNA in melanocytes as indicated by increased levels of 8-oxo-2'-deoxyguanosine (8-oxo-dG): 4.49 (±0.55 SEM) 8-oxo-dG/10(6) dG compared to 1.49 (±0.11 SEM) 8-oxo-dG/10(6) dG for NHEKs. An antioxidant biomarker, glutathione (GSH), was also lower in melanocytes (3.14 nmoles (±0.15 SEM)/cell) in comparison to NHEKs (5.98 nmoles (±0.33 SEM)/cell). Intriguingly, cellular bioavailable iron as measured in ferritin was found to be nearly fourfold higher in melanocytes than in NHEKs. Further, ferritin levels in melanocytes were also higher than in hepatocarcinoma cells, an iron-rich cell, and it indicates that higher relative iron levels may be characteristic of melanocytes. To account for the increased oxidative DNA and lower GSH and H2O2 levels that we observe, we propose that iron may contribute to higher levels of oxidation by reacting with H2O2 through a Fenton reaction leading to the generation of DNA-reactive hydroxyl radicals. In conclusion, our data support the concept of elevated oxidation and high iron levels as normal parameters of melanocytic activity. We present new evidence that may contribute to our understanding of the melanogenic process and lead to the development of new skin care products.
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DNA/metabolismo , Ferro/metabolismo , Queratinócitos/metabolismo , Melanócitos/metabolismo , Células Cultivadas , Dano ao DNA , Glutationa/metabolismo , Humanos , OxirreduçãoRESUMO
Menstruation and desquamation are important routes for humans to excrete iron. Because menstruation is no longer available in postmenopausal women, in the present study, we examined whether iron accumulates more in postmenopausal skin than in premenopausal skin. Skin biopsy samples were obtained from six pre- and six postmenopausal Caucasian women. Iron levels in the form of ferritin were 42% higher, but vascular endothelial growth factor and total antioxidant capacity were 45% and 34% lower in postmenopausal skin (58.8 ± 1.3 years old) than in premenopausal skin (41.6 ± 1.7 years old), respectively. Moreover, in vitro cultured normal human epidermal keratinocytes had surprisingly high levels of ferritin when compared to immortalized human breast epithelial MCF-10A cells or human liver HepG2 cancer cells. Our results indicate that skin is a cellular repository of iron and that menopause increases iron in skin and, thus, may contribute to the manifestation of accelerated skin aging and photo aging after menopause.
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Ferritinas/metabolismo , Menopausa , Pele/metabolismo , Adulto , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Pessoa de Meia-Idade , Pele/citologiaRESUMO
Ozone is a tropospheric pollutant that can form at ground level as a result of an interaction between sunlight and hydrocarbon engine emissions. As ozone is an extremely oxidative reaction product, epidermal cells are in the outer layer of defense against ozone. We exposed normal human epidermal keratinocytes (NHEK) to concentrations of ozone that have been measured in cities and assayed for its effects. Hydrogen peroxide and IL-1α levels both increased while ATP levels decreased. We found a decrease in the NAD-dependent histone deacetylase, sirtuin 3. Lastly, we found that ozone increased DNA damage as evaluated by Comet assay. Taken together, our results show increased damage to NHEK that will ultimately impair normal cellular function as a result of an environmentally relevant ozone exposure.
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Poluentes Atmosféricos/toxicidade , Células Epidérmicas , Queratinócitos/efeitos dos fármacos , Ozônio/toxicidade , Células Cultivadas , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Sirtuína 3/metabolismoRESUMO
Sirtuins (SIRT) are NAD(+) -dependent deacetylases and ADP-ribosyltransferases that play a critical role in metabolism and epigenetics. SIRT3 and SIRT4 are of particular interest because they are localized in the mitochondria where energy is generated and their expression is inversely proportional to each other. Here, we report data, for the first time, demonstrating the presence of SIRT4 in normal human epidermal keratinocytes (NHEK) and confirm that its expression is inversely related to SIRT3 in these cells and that they follow a temporal cycle. Further, UVB radiation modified their expression, as well as ATP and H2 O2 levels. These deviations from the normal sirtuin cycles after UVB exposure can be an epigenetic indicator of lower metabolism levels.
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Metabolismo Energético , Queratinócitos/metabolismo , Proteínas Mitocondriais/metabolismo , Sirtuína 3/metabolismo , Sirtuínas/metabolismo , Metabolismo Energético/fisiologia , Metabolismo Energético/efeitos da radiação , Humanos , Queratinócitos/efeitos da radiação , Estresse Oxidativo , Raios UltravioletaRESUMO
Environmental trauma to human skin can lead to oxidative damage of proteins and affect their activity and structure. When methionine becomes oxidized to its sulfoxide form, methionine sulfoxide reductase A (MSRA) reduces it back to methionine. We report here the increase in MSRA in normal human epidermal keratinocytes (NHEK) after ultraviolet B (UVB) radiation, as well as the reduction in hydrogen peroxide levels in NHEK pre-treated with MSRA after exposure. Further, when NHEK were pre-treated with a non-cytotoxic pentapeptide containing methionine sulfoxide (metSO), MSRA expression increased by 18.2%. Additionally, when the media of skin models were supplemented with the metSO pentapeptide and then exposed to UVB, a 31.1% reduction in sunburn cells was evident. We conclude that the presence of MSRA or an externally applied peptide reduces oxidative damage in NHEK and skin models and that MSRA contributes to the protection of proteins against UVB-induced damage in skin.
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Queratinócitos/efeitos dos fármacos , Metionina Sulfóxido Redutases/metabolismo , Metionina/análogos & derivados , Oligopeptídeos/farmacologia , Protetores contra Radiação/farmacologia , Pele/efeitos dos fármacos , Cultura em Câmaras de Difusão , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Metionina/metabolismo , Metionina/farmacologia , Modelos Biológicos , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Estresse Oxidativo , Pele/citologia , Pele/metabolismo , Pele/efeitos da radiação , Pele Artificial , Técnicas de Cultura de Tecidos , Raios Ultravioleta/efeitos adversosRESUMO
BACKGROUND/PURPOSE: Human skin is constantly exposed to ultraviolet A (UVA), which can generate reactive oxygen species and cause iron release from ferritin, leading to oxidative damage in biomolecules. This is particularly true in post-menopausal skin due to an increase in iron as a result of menopause. As iron is generally released through desquamation, the skin becomes a main portal for the release of excess iron in this age group. In the present study, we examined a strategy for controlling UVA- and iron-induced oxidative stress in skin using a keratinocyte post-menopausal cellular model system. METHODS: Keratinocytes that had been cultured under normal or high-iron, low-estrogen conditions were treated with (2-nitrophenyl) ethyl pyridoxal isonicotinoyl hydrazone (2-PNE-PIH). 2-PNE-PIH is a caged-iron chelator that does not normally bind iron but can be activated by UVA radiation to bind iron. Following incubation with 2-PNE-PIH, the cells were exposed to 5 J/cm² UVA and then measured for changes in lipid peroxidation and ferritin levels. RESULTS: 2-PNE-PIH protected keratinocytes against UVA-induced lipid peroxidation and ferritin depletion. Further, 2-PNE-PIH was neither cytotoxic nor did it alter iron metabolism. CONCLUSION: 2-PNE-PIH may be a useful deterrent against UVA-induced oxidative stress in post-menopausal women.
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Epiderme/metabolismo , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Queratinócitos/metabolismo , Peroxidação de Lipídeos , Pós-Menopausa/metabolismo , Raios Ultravioleta/efeitos adversos , Linhagem Celular , Epiderme/patologia , Feminino , Ferritinas/metabolismo , Humanos , Queratinócitos/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Pessoa de Meia-Idade , Projetos PilotoRESUMO
We have developed a technology to incorporate micronized titanium dioxide (TiO(2)), together with antioxidants, in particles of a UV-visible transparent polymer gel. These particles are coated with silica to avoid clustering and the size of the micronized TiO(2) reduces the back scattering of white light. gel-trapped TiO(2) minimizes the oxidative stress exerted by UV radiation, increases the photo-stability of some accompanying ingredients, such as avobenzone. The size of the particles is in the micrometre range. This favors their permanence on the top of the stratum corneum. Gel-trapped TiO(2)-based sunscreens provide a larger SPF and two-fold larger UVA protection than equal-composition sunscreens that contain larger amounts of untrapped TiO(2).
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Oestrogen deficiency is regarded as the main causative factor in postmenopausal skin ageing and photoageing. While women after menopause experience low levels of oestrogen because of cease of ovarian function, they are also exposed to high levels of iron as a result of cessation of menstruation. In this study, we investigated whether this increase in iron presents a risk to the postmenopausal skin. Because of the lack of appropriate animal models to closely mimic the low oestrogen and high iron conditions, we tested the hypothesis in a high iron and low oestrogen culture model. Here, we showed that primary human dermal fibroblasts exposed to iron did not affect the baseline levels of matrix metalloproteinase-1 (MMP-1) activity. However, the iron-exposed fibroblasts were sensitized to UVA exposure, which resulted in a synergistic increase in MMP-1. UVA activated the three members of MAPK family: ERKs, p38, and JNKs. Additional activation of ERKs by iron contributed to the synergistic increases. Primary normal human epidermal keratinocytes (NHEK) did not respond to iron or UVA exposure as measured by MMP-1, but produced tumor necrosis factor-alpha (TNF-α) in the media, which then stimulated MMP-1 in fibroblasts. Our results indicate that iron and UVA increase MMP-1 activity in dermal fibroblasts not only directly through ERK activation but also by an indirect paracrine loop through TNF-α released by NHEK. We conclude that in addition to oestrogen deficiency, increased iron as a result of menopause could be a novel risk factor by sensitizing postmenopausal skin to solar irradiation.
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MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Ferro/farmacologia , Queratinócitos/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Raios Ultravioleta , Anticorpos/imunologia , Anticorpos/farmacologia , Apoproteínas/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/genética , Citocinas/metabolismo , Desferroxamina/farmacologia , Estradiol/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Ferritinas/farmacologia , Fibroblastos/efeitos da radiação , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Humanos , Ferro/administração & dosagem , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Queratinócitos/efeitos da radiação , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transdução de Sinais/efeitos da radiação , Transferrina/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The extracellular matrix (ECM) is composed of mixed protein fibers whose precise composition affects biomineralization. New methods are needed to probe the interactions of these proteins with calcium phosphate mineral and with each other. Here we follow calcium phosphate mineralization on protein fibers self-assembled in vitro from solutions of fibronectin, elastin and their mixture. We probe the surface morphology and mechanical properties of the protein fibers during the early stages. The development of mineral crystals on the protein matrices is also investigated. In physiological mineralization solution, the elastic modulus of the fibers in the fibronectin-elastin mixture increases to a greater extent than that of the fibers from either pure protein. In the presence of fibronectin, longer exposure in the mineral solution leads to the formation of amorphous calcium phosphate particles templated along the self-assembled fibers, while elastin fibers only collect calcium without any mineral observed during early stage. TEM images confirm that small needle-shape crystals are confined inside elastin fibers which suppress the release of mineral outside the fibers during late stage, while hydroxyapatite crystals form when fibronectin is present. These results demonstrate complementary actions of the two ECM proteins fibronectin and elastin to collect cations and template mineral, respectively.
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Calcificação Fisiológica/fisiologia , Fosfatos de Cálcio/metabolismo , Elastina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Difração de Raios XRESUMO
The effects of exposure of human dermal fibroblasts to rutile and anatase TiO(2) nanoparticles are reported. These particles can impair cell function, with the latter being more potent at producing damage. The exposure to nanoparticles decreases cell area, cell proliferation, mobility, and ability to contract collagen. Individual particles are shown to penetrate easily through the cell membrane in the absence of endocytosis, while some endocytosis is observed for larger particle clusters. Once inside, the particles are sequestered in vesicles, which continue to fill up with increasing incubation time till they rupture. Particles coated with a dense grafted polymer brush are also tested, and, using flow cytometry, are shown to prevent adherence to the cell membrane and hence penetration of the cell, which effectively decreases reactive oxygen species (ROS) formation and protects cells, even in the absence of light exposure. Considering the broad applications of these nanoparticles in personal health care products, the functionalized polymer coating can potentially play an important role in protecting cells and tissue from damage.
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Nanopartículas Metálicas , Pele/efeitos dos fármacos , Titânio/toxicidade , Endocitose , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Espécies Reativas de Oxigênio/metabolismo , Pele/citologia , Pele/metabolismoRESUMO
Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the understanding of bone formation and the development of a successfully engineered bone tissue scaffold. It is still unclear how ECM mechanical properties affect protein-mineral interactions in early stages of bone mineralization. We investigated the longitudinal mineralization properties of MC3T3-E1 cells and the elastic modulus of their ECM using shear modulation force microscopy, synchrotron grazing incidence X-ray diffraction (GIXD), scanning electron microscopy, energy dispersive X-ray spectroscopy, and confocal laser scanning microscopy (CLSM). The elastic modulus of the ECM fibers underwent significant changes for the mineralizing cells, which were not observed in the nonmineralizing cells. On substrates conducive to ECM network production, the elastic modulus of mineralizing cells increased at time points corresponding to mineral production, whereas that of the nonmineralizing cells did not vary over time. The presence of hydroxyapatite in mineralizing cells and the absence thereof in the nonmineralizing ones were confirmed by GIXD, and CLSM showed that a restructuring of actin occurred only for mineral-producing cells. These results show that the correct and complete development of the ECM network is required for osteoblasts to mineralize. This in turn requires a suitably prepared synthetic substrate for bone development to succeed in vitro.
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Osso e Ossos/fisiologia , Calcificação Fisiológica/fisiologia , Matriz Extracelular/metabolismo , Engenharia Tecidual , Animais , Osso e Ossos/citologia , Cálcio/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Forma Celular , Módulo de Elasticidade , Camundongos , Microscopia de Força Atômica , Microscopia Confocal , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Fosfatos/metabolismo , SíncrotronsRESUMO
Nanocomposites of Ethylene Vinyl Acetate (EVA 260) with Cloisite 20A organo clay and Cloisite 20A organo clay impregnated with Fe(CO)(5) were produced in a twin-screw extruder. Dynamic mechanical analysis (DMA) measurements indicated that the moduli increased monotonically for the Cloisite, up to a concentration of 10%, after which the modulus decreased. Adult human dermal fibroblasts (AHDF) were plated on these surfaces and the cell growth was found to be maximal on the nanocomposites containing 10% Cloisite. AHDFs cultured on substrates with higher Cloisite content had low surface area, poor growth curves, and misshaped actin fibers. Compounding EVA with Fe(CO)(5) soaked Cloisite did not enhance the modulus even at a loading of 10%. TEM images indicate nanoparticles form and coat the Cloisite platelet surfaces, possibly interfering with the exfoliation process. On the other hand, cell culture of MC3T3 osteoblasts proliferated on the Fe containing nanocomposites, the largest effect being observed when cultured in a constant magnetic field. These results indicate how the chemical nature of the Cloisite 20A organo clay can also play a major role. Finally, since natural ECM is fibrillar, these EVA nanocomposites were also electrospun into micron thick fibers. MC3T3s proliferated well on these fibers and the MC3T3 proliferation was maximized by culture on electrospun aligned fibers in a constant magnetic field.
Assuntos
Bentonita/farmacologia , Compostos Férricos/farmacologia , Fibroblastos/citologia , Nanopartículas/química , Osteoblastos/citologia , Polivinil/farmacologia , Compostos de Amônio Quaternário/farmacologia , Adsorção , Adulto , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Derme/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Magnetismo , Fenômenos Mecânicos/efeitos dos fármacos , Camundongos , Nanocompostos/química , Osteoblastos/efeitos dos fármacos , Polimetil Metacrilato/farmacologia , TemperaturaRESUMO
A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.
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Enzimas Imobilizadas/química , Compostos de Epóxi/química , Membranas Artificiais , Polímeros/química , Sítios de Ligação , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas , Lipase/metabolismo , Metacrilatos/química , Tamanho da Partícula , Polímeros/síntese química , Propriedades de Superfície , MolhabilidadeRESUMO
Chemical grafting of anti-oxidant molecules with an additional hydrophobic polymer coating directly onto TiO(2) particle surfaces, using sonochemistry, is found to eliminate photocatalytic degradation enabling highly effective screening against UV radiation.