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BACKGROUND: Recent evidence indicates that cold atmospheric plasma (CAP) can upregulate the production of extracellular matrix (ECM) proteins in dermal fibroblasts and enhance transdermal drug delivery when applied at a low intensity. OBJECTIVES: The aim of this study was to evaluate the effect of low-intensity CAP (LICAP) on photoaging-induced wrinkles in an animal model and the expression profiles of ECM proteins in human dermal fibroblasts. METHODS: Each group was subjected to photoaging induction and allocated to therapy (LICAP, topical polylactic acid (PLA), or both). The wrinkles were evaluated via visual inspection, quantitative analysis, and histology. The expression of collagen I/III and fibronectin was assessed using reverse transcription-quantitative polymerase chain reaction, western blot analysis, and immunofluorescence. The amount of aqueous reactive species produced by LICAP using helium and argon gas was also measured. RESULTS: Wrinkles significantly decreased in all treatment groups compared to those in the untreated control. The differences remained significant for at least 4 weeks. Dermal collagen density increased following LICAP and PLA application. LICAP demonstrated a hormetic effect on ECM protein expression in human dermal fibroblasts. The production of reactive species increased, showing a biphasic pattern, with an initial linear phase and a slow saturation phase. The initial linearity was sustained for a longer time in the helium plasma (~60 s) than in the argon plasma (~15 s). CONCLUSION: LICAP appears to be a novel treatment option for wrinkles on the photodamaged skin. This treatment effect seems to be related to its hormetic effect on dermal ECM production.
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Gases em Plasma , Envelhecimento da Pele , Animais , Células Cultivadas , Colágeno , Matriz Extracelular , Proteínas da Matriz Extracelular , Fibroblastos , Humanos , Poliésteres , PeleRESUMO
We developed a laser interference lithography (LIL) system for fabrication of period-chirped gratings, which would be useful for sophisticated optical components. Despite its simplicity, the developed LIL system, based on a Lloyd's mirror interferometer with a cylindrically concave mirror, can generate chirped gratings, yet over a large area at high throughput owing to the nature of LIL. We have derived exact theoretical equations needed for system design, built the LIL system, and subsequently realized period-chirped gratings. A fabricated sample whose center period is Λ≈600 nm exhibits a continuous period variation of ΔΛ=92 nm across 17 mm width.
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Interferometria/métodos , Lasers , Fenômenos Ópticos , Impressão/métodos , Microscopia Eletrônica de Varredura , FotografaçãoRESUMO
BACKGROUND: Cold atmospheric plasma (CAP) produces reactive oxygen/nitrogen species (RONS) in the target and can induce cytoprotective effects by activating hormesis-related pathways when its intensity is in the low range. OBJECTIVES: The aim of this study is to evaluate the effect of low-intensified CAP (LICAP) on skin with photoaging-induced hyperpigmentation in an animal model. METHODS: Changes in cell viability and RONS production following LICAP treatment were measured. For the in vivo study, 30 hairless mice underwent antecedent photoaging induction followed by the allocated therapy (i.e., LICAP, topical ascorbic acid (AA), or both). During the first 4 weeks of the treatment period (8 weeks), ultraviolet (UV)-B irradiation was concurrently administered. Visual inspection and measurement of the melanin index (MI) were performed to assess the change in skin pigmentation at Weeks 0, 2, 4, 6, and 8. RESULTS: RONS production increased linearly until the saturation point. Cell viability was not significantly affected by LICAP treatment. At Week 8, MI was significantly decreased in every treatment group compared with the values at Week 0 and Week 4. The treatment effect of the concurrent therapy group was superior to that of the LICAP and AA groups. CONCLUSION: LICAP appears to be a novel modality for photoprotection and pigment reduction in photodamaged skin. LICAP treatment and topical AA application seem to exert a synergistic effect.
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Hiperpigmentação , Envelhecimento da Pele , Animais , Camundongos , Pele , Hiperpigmentação/etiologia , Hiperpigmentação/prevenção & controle , Modelos Animais de Doenças , Raios Ultravioleta/efeitos adversosRESUMO
In the biological microenvironment, cells are surrounded by an extracellular matrix (ECM), with which they dynamically interact during various biological processes. Specifically, the physical and chemical properties of the ECM work cooperatively to influence the behavior and fate of cells directly and indirectly, which invokes various physiological responses in the body. Hence, efficient strategies to modulate cellular responses for a specific purpose have become important for various scientific fields such as biology, pharmacy, and medicine. Among many approaches, the utilization of biomaterials has been studied the most because they can be meticulously engineered to mimic cellular modulatory behavior. For such careful engineering, studies on physical modulation (e.g., ECM topography, stiffness, and wettability) and chemical manipulation (e.g., composition and soluble and surface biosignals) have been actively conducted. At present, the scope of research is being shifted from static (considering only the initial environment and the effects of each element) to biomimetic dynamic (including the concepts of time and gradient) modulation in both physical and chemical manipulations. This review provides an overall perspective on how the static and dynamic biomaterials are actively engineered to modulate targeted cellular responses while highlighting the importance and advance from static modulation to biomimetic dynamic modulation for biomedical applications.
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A lithography technique that combines laser interference lithography (LIL) and photolithography, which can be a valuable technique for the low cost production of microscale and nanoscale hybrid mask molds, is proposed. LIL is a maskless process which allows the production of periodic nanoscale structures quickly, uniformly, and over large areas. A 257 nm wavelength Ar-Ion laser is utilized for the LIL process incorporating a Lloyd's mirror one beam inteferometer. By combining LIL with photolithography, the non-selective patterning limitation of LIL are explored and the design and development of a hybrid mask mold for nanoimprint lithography process, with uniform two-dimensional nanoscale patterns are presented. Polydimethylsiloxane is applied on the mold to fabricate a replica of the stamp. Through nanoimprint lithography using the manufactured replica, successful transfer of the patterns is achieved, and selective nanoscale patterning is confirmed with pattern sizes of around 180 nm and pattern aspect ratio of around 1.44:1.
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The utility of electro-responsive smart materials has been limited by bubble generation (hydrolysis) during application of electrical fields and by biocompatibility issues. Here we describe the design of a device that overcomes these limitations by combining material properties, new design concepts, and microtechnology. 4-hydroxybutyl acrylate (4-HBA) was used as a backbone hydrogel material, and its actuating behavior, bending force, and elasticity were extensively characterized as a function of size and acrylic acid concentration. To prevent bubble generation, the system was designed such that the hydrogel actuator could be operated at low driving voltages (<1.2 V). A microfluidic channel with an integrated electroactive hydrogel actuator was developed for sorting particles. This device could be operated in cell culture media, and the sorting capabilities were initially assessed by sorting droplets in an oil droplet emulsion. Biocompatibility was subsequently tested by sorting mouse embryoid bodies (mEBs) according to size. The sorted and collected mEBs maintained pluripotency, and selected mEBs successfully differentiated into three germ layers: endoderm, mesoderm, and ectoderm. The electroactive hydrogel device, integrated into a microfluidic system, successfully demonstrated the practical application of smart materials for use in cell biology.
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Eletricidade , Hidrogéis/química , Técnicas Analíticas Microfluídicas/instrumentação , Acrilatos/química , Animais , Diferenciação Celular , Separação Celular , Elasticidade , Células-Tronco Embrionárias/citologia , Desenho de Equipamento , Teste de Materiais , Fenômenos Mecânicos , Camundongos , Técnicas Analíticas Microfluídicas/métodosRESUMO
Water-droplet adhesiveness was freely controlled on a single platform of superhydrophobic anodized aluminum oxide (AAO) within the range from highly adhesive to self-cleanable. Changing the structure from nanopore to nanopillar arrays at the surface caused a dramatic increase in the receding angle and a decrease in the hysteresis of water contact angles. The presence of dead-end nanopores but not through nanoholes was recognized as one of the main causes of the adhesiveness of superhydrophobic surfaces. The adhesiveness-controllable superhydrophobic AAO can be an excellent platform on which to elucidate the physical nature of the wetting phenomenon related to the nanostructure and has promising potential in technological applications.
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Óxido de Alumínio/química , Interações Hidrofóbicas e Hidrofílicas , Nanoestruturas/química , Água/química , Adesividade , Microscopia Eletrônica de Varredura , PorosidadeRESUMO
Magnetite (Fe3O4)-gold (Au) core-shell nanoparticles (NPs) have unique magnetic and optical properties. When combined with biological moieties, these NPs can offer new strategies for biomedical applications, such as drug delivery and cancer targeting. Here, we present an effective method for the controllable cellular uptake of magnetic core-shell NP systems combined with biological moieties. Vimentin, which is the structural protein, has been biochemically confirmed to affect phagocytosis potently. In addition, vimentin affects exogenic materials internalization into cells even though under multiple inhibitions of biological moieties. In this study, we demonstrate the cellular internalization performance of Fe3O4-Au core-shell NPs with surface modification using a combination of biological moieties. The photofluorescence of vimentin-tagged NPs remained unaffected under multiple inhibition tests, indicating that the NPs were minimally influenced by nystatin, dynasore, cytochalasin D, and even the Muc1 antibody (Ab). Consequently, this result indicates that the Muc1 Ab can target specific molecules and can control specific endocytosis. Besides, we show the possibility of controlling specific endocytosis in colorectal cancer cells.
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Bioengineering approaches to regulate stem cell fates aim to recapitulate the in vivo microenvironment. In recent years, manipulating the micro- and nano-scale topography of the stem cell niche has gained considerable interest for the purposes of controlling extrinsic mechanical cues to regulate stem cell fate and behavior in vitro. Here, we established an optimal nanotopographical system to improve 3-dimensional (3D) differentiation of pancreatic cells from human pluripotent stem cells (hPSCs) by testing gradient-pattern chips of nano-scale polystyrene surface structures with varying sizes and shapes. The optimal conditions for 3D differentiation of pancreatic cells were identified by assessing the expression of developmental regulators that are required for pancreatic islet development and maturation. Our results showed that the gradient chip of pore-part 2 (Po-2, 200-300â¯nm diameter) pattern was the most efficient setting to generate clusters of pancreatic endocrine progenitors (PDX1+ and NGN3+) compared to those of other pore diameters (Po-1, 100-200 or Po-3, 300-400â¯nm) tested across a range of pillar patterns and flat surfaces. Furthermore, the Po-2 gradient pattern-derived clusters generated islet-like 3D spheroids and tested positive for the zinc-chelating dye dithizone. The spheroids consisted of more than 30% CD200â¯+â¯endocrine cells and also expressed NKX6.1 and NKX2.2. In addition, pancreatic ß- cells expressing insulin and polyhormonal cells expressing both insulin and glucagon were obtained at the final stage of pancreatic differentiation. In conclusion, our data suggest that an optimal topographical structure for differentiation to specific cell types from hPSCs can be tested efficiently by using gradient-pattern chips designed with varying sizes and surfaces. STATEMENT OF SIGNIFICANCE: Our study provides demonstrates of using gradient nanopatterned chips for differentiation of pancreatic islet-like clusters. Gradient nanopatterned chips are consisted of two different shapes (nanopillar and nanopore) in three different ranges of nano sizes (100-200, 200-300, 300-400â¯nm). We found that optimal nanostructures for differentiation of pancreatic islet-like clusters were 200-300â¯nm nano pores. Cell transplantation is one of the major therapeutic option for type 1 diabetes mellitus (DM) using stem cell-derived ß-like cells. We generated 50 um pancreatic islet-like clusters in size, which would be an optimal size for cell transplantation. Futuremore, the small clusters provide a powerful source for cell therapy. Our findings suggest gradient nanopatterned chip provides a powerful tool to generate specific functional cell types of a high purity for potential uses in cell therapy development.
Assuntos
Ilhotas Pancreáticas/citologia , Nanopartículas/química , Células-Tronco Pluripotentes/citologia , Agregação Celular , Diferenciação Celular , Endoderma/citologia , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Células-Tronco Embrionárias Humanas/citologia , Humanos , Proteínas Nucleares , Poliestirenos/química , Esferoides Celulares/citologia , Fatores de TranscriçãoRESUMO
Understanding signals in the microenvironment that regulate endothelial cell behavior are important in tissue engineering. Although many studies have examined the cellular effects of nanotopography, no study has investigated the functional regulation of human endothelial cells grown on nano-sized gradient hole substrate. We examined the cellular response of human umbilical vein endothelial cells (HUVECs) by using a gradient nanohole substrate (GHS) with three different types of nanohole patterns (HP): which diameters were described in HP1, 120-200 nm; HP2, 200-280 nm; HP3, 280-360 nm. In results, HP2 GHS increased the attachment and proliferation of HUVECs. Also, gene expression of focal adhesion markers in HUVECs was significantly increased on HP2 GHS. In vitro tube formation assay showed the enhancement of tubular network formation of HUVECs after priming on GHS compared to Flat. Furthermore, leukocyte adhesion was also reduced in the HUVECs in a hole-diameter dependent manner. To summarize, optimal proliferations with reduced leukocyte adhesion of HUVECs were achieved by gradient nanohole substrate with 200-280 nm-sized holes.
Assuntos
Adesão Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Leucócitos/metabolismo , Membrana Basal/metabolismo , Western Blotting , Citocinas/metabolismo , Imunofluorescência , Humanos , Marcação In Situ das Extremidades Cortadas , Nanoporos/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200â¯nm (GP 120/200), 200-280â¯nm (GP 200/280), and 280-360â¯nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.
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Óxido de Alumínio/química , Técnicas de Cultura de Células/métodos , Células Endoteliais/metabolismo , Nanoestruturas/química , Nanotecnologia/métodos , HumanosRESUMO
Nanotopography plays a pivotal role in the regulation of cellular responses. Nonetheless, little is known about how the gradient size of nanostructural stimuli alters the responses of endothelial progenitor cells without chemical factors. Herein, the fabrication of gradient nanopattern plates intended to mimic microenvironment nanotopography is described. The gradient nanopattern plates consist of nanopillars of increasing diameter ranges [120-200â¯nm (GP 120/200), 200-280â¯nm (GP 200/280), and 280-360â¯nm (GP 280/360)] that were used to screen the responses of human endothelial colony-forming cells (hECFCs). Nanopillars with a smaller nanopillar diameter caused the cell area and perimeter of hECFCs to decrease and their filopodial outgrowth to increase. The structure of vinculin (a focal adhesion marker in hECFCs) was also modulated by nanostructural stimuli of the gradient nanopattern plates. Moreover, Rho-associated protein kinase (ROCK) gene expression was significantly higher in hECFCs cultured on GP 120/200 than in those on flat plates (no nanopillars), and ROCK suppression impaired the nanostructural-stimuli-induced vinculin assembly. These results suggest that the gradient nanopattern plates generate size-specific nanostructural stimuli suitable for manipulation of the response of hECFCs, in a process dependent on ROCK signaling. This is the first evidence of size-specific nanostructure-sensing behavior of hECFCs. SIGNIFICANCE: Nano feature surfaces are of growing interest as materials for a controlled response of various cells. In this study, we successfully fabricated gradient nanopattern plates to manipulate the response of blood-derived hECFCs without any chemical stimulation. Interestingly, we find that the sensitive nanopillar size for manipulation of hECFCs is range between 120â¯nm and 200â¯nm, which decreased the area and increased the filopodial outgrowth of hECFCs. Furthermore, we only modulate the nanopillar size to increase ROCK expression can be an attractive method for modulating the cytoskeletal integrity and focal adhesion of hECFCs.
Assuntos
Células Endoteliais/citologia , Adesões Focais , Nanoestruturas , Células-Tronco/citologia , Actinas/metabolismo , Adulto , Animais , Western Blotting , Células Cultivadas , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Células-Tronco/metabolismo , Vinculina/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Acrylated hyaluronic acid (HA) was used as a scaffold for bone morphogenic protein-2 (BMP-2) and human mesenchymal stem cells (hMSCs) for rat calvarial defect regeneration. HA was acrylated by two-step reactions: (1) introduction of an amine group using adipic acid dihydrazide (ADH); (2) acrylation by N-acryloxysuccinimide. Tetrathiolated poly(ethylene) glycol (PEG-SH(4)) was used as a cross-linker by a Michael-type addition reaction and the hydrogel was formed within 10min under physiological conditions. This hydrogel is degraded completely by 100U/ml hyaluronidase in vitro. hMSCs and/or BMP-2 was added during gelation. Cellular viability in vitro was increased up to 55% in the hydrogels with BMP-2 compared with the control. For in vivo calvarial defect regeneration, five different samples (i.e., control, hydrogel, hydrogel with BMP-2, hydrogel with MSCs, and hydrogel with BMP-2 and MSCs) were implanted for 4 weeks. The histological results demonstrated that the hydrogels with BMP-2 and MSCs had the highest expression of osteocalcin and mature bone formation with vascular markers, such as CD31 and vascular endothelial growth factors, compared with the other samples. This study demonstrated that HA base hydrogel can be used for cell and growth factor carriers for tissue regeneration.
Assuntos
Proteínas Morfogenéticas Ósseas/administração & dosagem , Regeneração Óssea/fisiologia , Portadores de Fármacos/química , Ácido Hialurônico/química , Transplante de Células-Tronco Mesenquimais/métodos , Fraturas Cranianas/tratamento farmacológico , Fraturas Cranianas/cirurgia , Fator de Crescimento Transformador beta/administração & dosagem , Animais , Proteína Morfogenética Óssea 2 , Técnicas de Cultura de Células/métodos , Terapia Combinada , Hidrogéis/química , Masculino , Teste de Materiais , Ratos , Ratos Sprague-Dawley , Fraturas Cranianas/patologia , Resultado do TratamentoRESUMO
Nanoscaled surface patterning is an emerging potential method of directing the fate of stem cells. We adopted nanoscaled pillar gradient patterned cell culture plates with three diameter gradients [280-360 (GP 280/360), 200-280 (GP 200/280), and 120-200 nm (GP 120/200)] and investigated their cell fate-modifying effect on multipotent fetal liver kinase 1-positive mesodermal precursor cells (Flk1+ MPCs) derived from embryonic stem cells. We observed increased cell proliferation and colony formation of the Flk1+ MPCs on the nanopattern plates. Interestingly, the 200-280 nm-sized (GP 200/280) pillar surface dramatically increased cardiomyocyte differentiation and expression of the early cardiac marker gene Mesp1. The gradient nanopattern surface-induced cardiomyocytes had cardiac sarcomeres with mature cardiac gene expression. We observed Vinculin and p-Cofilin-mediated cytoskeleton reorganization during this process. In summary, the gradient nanopattern surface with 200-280 nm-sized pillars enhanced cardiomyocyte differentiation in Flk1+ MPCs.
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Diferenciação Celular , Fatores de Despolimerização de Actina , Citoesqueleto , Células-Tronco Embrionárias , Miócitos Cardíacos , NanoestruturasRESUMO
The topographical environment, which mimics the stem cell niche, provides mechanical cues to regulate the differentiation of mesenchymal stem cells (MSC). Diverse topographical variations have been engineered to investigate cellular responses; however, the types of mechanical parameters that affect cells, and their underlying mechanisms remain largely unknown. In this study, we screened nanotopological pillars with size gradient to activate transcriptional coactivator with PDZ binding motif (TAZ), which stimulates osteogenesis of MSC. We observed that a nanotopological plate, 70 nm in diameter, significantly induces osteogenic differentiation with the activation of TAZ. TAZ activation via the nanotopological plate was mediated by actin polymerization and Rho signaling, as evidenced by the cytosolic localization of TAZ under F-actin or Rho kinase inhibitor. The FAK and MAPK pathways also play a role in TAZ activation by the nanotopological plate because the inhibitor of ERK and JNK blocked nanopattern plate induced osteogenic differentiation. Taken together, these results indicate that nanotopology regulates cell differentiation through TAZ activation.
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Diferenciação Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteogênese/genética , Actinas/metabolismo , Biomarcadores , Células Cultivadas , Quinase 1 de Adesão Focal/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Nanotecnologia , Ligação Proteica , Multimerização Proteica , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador TranscricionalRESUMO
Recently emerging evidence has indicated surface nanotopography as an important physical parameter in the stem cell niche for regulating cell fate and behaviors for various types of stem cells. In this study, a substrate featuring arrays of increasing nanopillar diameter was devised to investigate the effects of varying surface nanotopography on the maintenance of undifferentiated human embryonic stem cells (hESC) colonies in the absence of feeder cells. Single hESCs cultured across gradient nanopattern (G-Np) substrate were generally organized into compact colonies, and expressed higher levels of undifferentiated markers compared to those cultured on the unstructured control substrate. In particular, hESC demonstrates a propensity to organize into more compact colonies expressing higher levels of undifferentiated markers towards a smaller nanopillar diameter range (D = 120-170 nm). Cell-nanotopography interactions modulated the formation of focal adhesions and cytoskeleton reorganization to restrict colony spreading, which reinforced E-cadherin mediated cell-cell adhesions in hESC colonies. Maintaining compact hESC colony integrity revealed to be indispensable for hESC undifferentiated state as the loss of cell-cell adhesions within spreading hESC on the control substrate exhibited morphological and gene expression signatures of epithelial-to-mesenchymal transition-like processes. Findings in this study demonstrated a feasible approach to screen the optimal nanotopographical cues for maintaining undifferentiated hESC colonies in feeder free conditions, which provides a platform for further investigations into developing hESC feeder free culture systems for the purpose of regenerative medicine.
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Materiais Biocompatíveis/química , Células-Tronco Embrionárias/citologia , Caderinas/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Transição Epitelial-Mesenquimal , Adesões Focais/metabolismo , Humanos , Propriedades de SuperfícieRESUMO
Both self-cleanability and antireflectivity were achieved on quartz surfaces by forming heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane self-assembled monolayer after fabrication of nanostructures with a mask-free method. By exposing polymethylmethacrylate spin-coated quartz plates to O2 reactive ion etching (RIE) and CF4 RIE successively, three well-defined types of nanopillar arrays were generated: A2, A8, and A11 patterns with average pillar widths of 33 ± 4 nm, 55 ± 5 nm, and 73 ± 14 nm, respectively, were formed. All the fabrication processes including the final cleaning can be finished within 4 h. All nanostructured quartz surfaces exhibited contact angles higher than 155° with minimal water droplet adhesiveness and enhanced transparency (due to antireflectivity) over a broad spectral range from 350 to 900 nm. Furthermore, A2 pattern showed an enhanced antireflective effect that extends to the deep-UV range near 190 nm, which is a drawback region in conventional thin-film-coating approaches as a result of thermal damage. Because, by changing the conditions of successive RIE, the geometrical configurations of nanostructure arrays can be easily modified to meet specific needs, the newly developed fabrication method is expected to be applied in various optic and opto-electrical areas.PACS codes: 06.60.Ei; 81.65.Cf; 81.40.Vw.
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Anchorage-dependent cells growing over a substratum require stable adhesion areas on the surface for the next cellular activities. The adhesion is achieved by some contact points called focal adhesions. Because focal adhesions were distributed randomly, a trial to control the positions of focal adhesion with a specific order may cause interesting effects like as cytoskeleton rearrangement, which may induce and transfer new signals to the nucleus. Here, we cultured human osteoblasts over two sorts of nanopatterned surfaces with different pattern densities fabricated by using laser interference lithography and the nanoimprinting technique. Of the two nanopatterns, cells over the nanopattern with low pattern density showed relatively higher adaptation to the topography with guided filopodia protrusion. However, cells over the dense nanopattern showed difficulty in finding suitable paths for migration, as judged from the activities of filopodium formation and the presence of a shovel-like feature at the tip of each filopodium.
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Nanotecnologia/métodos , Osteoblastos/citologia , Pseudópodes/metabolismo , Técnicas de Cultura de Células , Citoesqueleto/metabolismo , Humanos , Lasers , Impressão , Propriedades de SuperfícieRESUMO
A mask-free, cost-effective dry-etching method for the fabrication of height- and spacing-controlled, pillar-like nanostructures was established in order to detect DNA molecules. The height and spacing of the quartz nanostructure were regulated by successive O(2) and CF(4) reactive ion etching times. The height and spacing of the nanostructures were tuned between 118 and 269 nm and between 107 and 161 nm, respectively. Probe DNA was immobilized on the structure and hybridized with fluorescently-labeled target DNA. Increases in the height and spacing of the nanopillar structure positively correlated with the fluorescence intensity of bound DNA. Usage of the nanostructure increased the DNA detection limit by up to 100-fold.
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Técnicas Biossensoriais/instrumentação , DNA/genética , Sistemas Microeletromecânicos/instrumentação , Nanoestruturas/química , Nanotecnologia/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Quartzo/química , DNA/análise , Desenho de Equipamento , Análise de Falha de Equipamento , Nanoestruturas/ultraestrutura , Tamanho da PartículaRESUMO
Hyaluronic acid is a natural glycosaminoglycan involved in biological processes. Low-molecular-weight hyaluronic acid (10 and 50 kDa)-based hydrogel was synthesized using derivatized hyaluronic acid. Hyaluronic acid was acrylated by two steps: (1) introduction of an amine group using adipic acid dihydrazide, and (2) acrylation by N-acryloxysuccinimide. Injectable hyaluronic acid-based hydrogel was prepared by using acrylated hyaluronic acid and poly(ethylene glycol) tetra-thiols via Michael-type addition reaction. Mechanical properties of the hydrogel were evaluated by varying the molecular weight of acrylated hyaluronic acid (10 and 50 kDa) and the weight percent of hydrogel. Hydrogel based on 50-kDa hyaluronic acid showed the shortest gelation time and the highest complex modulus. Next, human mesenchymal stem cells were cultured in cell-adhesive RGD peptide-immobilized hydrogels together with bone morphogenic protein-2 (BMP-2). Cells cultured in the RGD/BMP-2-incorporated hydrogels showed proliferation rates higher than that of control or RGD-immobilized hydrogels. Real-time RT-PCR showed that the expression of osteoblast marker genes such as CBFalpha1 and alkaline phosphatase was increased in hyaluronic acid-based hydrogel, and the expression level was dependent on the molecular weight of hyaluronic acid, RGD peptide, and BMP-2. This study indicates that low-molecular-weight hyaluronic acid-based hydrogel can be applied to tissue regeneration as differentiation guidance materials of stem cells.