Ceramic nanopatterned surfaces to explore the effects of nanotopography on cell attachment

Surfaces with ordered, nanopatterned roughness have demonstrated considerable promise in directing cell morphology, migration, proliferation, and gene expression. However, further investigation of these phenomena has been limited by the lack of simple, inexpensive methods of nanofabrication. Here, we report a facile, low-cost nanofabrication approach based on self-assembly of a thin-film of gadolinium-doped ceria on yttria-stabilized zirconia substrates (GDC/YSZ). This approach yields three distinct, randomly-oriented nanofeatures of variable dimensions, similar to those produced via polymer demixing, which can be reproducibly fabricated over tens to hundreds of microns. As a proof-of-concept, we examined the response of SK-N-SH neuroblastoma cells to features produced by this system, and observed significant changes in cell spreading, circularity, and cytoskeletal protein distribution. Additionally, we show that these features can be imprinted into commonly used rigid hydrogel biomaterials, demonstrating the potential broad applicability of this approach. Thus, GDC/YSZ substrates offer an efficient, economical alternative to lithographic methods for investigating cell response to randomly-oriented nanotopographical features.     

The effects of colloidal nanotopography on initial fibroblast adhesion and morphology

Colloidal lithography offers a simple, inexpensive method of producing irregular nanotopographies, a pattern not easily attainable utilizing conventional serial writing processes. Colloids with 20- or 50-nm diameter were utilized to produce such an irregular topography and were characterized by calculating the percentage area coverage of particles. Interparticle and nearest neighbor spacing were also assessed for the individual colloids in the pattern. Two-way analysis of variance (ANOVA) indicated significant differences between the number of fibroblasts adhering to planar, 20-, and 50-nm-diameter colloidal topographies, the number of fibroblasts adhering to the substrates at the time intervals studied, namely 20 min, 1 h, and 3 h and significant interaction between time and topography on fibroblast adhesion (P<0.01). Tukey tests were utilized for sensitive identification of the differences between the sample means and compounded ANOVA results. Cytoskeletal and general cell morphology were investigated on planar and colloidal substrates, and indicated cells in contact with irregular nanotopographies exhibit many peripheral protrusions while such protrusions are absent in cells on planar control surfaces. These protrusions are rich in microtubules on 20-nm-diameter colloidal surfaces while microfilaments are prevalent on 50-nm-diameter surfaces. Moreover, by 3 h, cells on the colloidal substrates initiate cell-cell adhesions, also absent in controls.     

The synergistic effect of aligned nanofibers and hyaluronic acid modification on endothelial cell behavior for vascular tissue engineering

The endothelialization of tissue-engineered vascular grafts (TEVGs) is considered to be an effective strategy to prevent the coagulation and restenosis of small-diameter vascular grafts. In this study, we fabricated well aligned nanofibrous scaffolds with PCL using a high speed rotating collector, modified those surfaces with hyaluronic acid (HA) and studied the synergistic effect of the scaffolds on the endothelial cells behavior in vitro. The well-aligned oriented architecture was observed by SEM images in the nanofibrous scaffolds. The contact angle measurements and FTIR-ATR evidenced that HA was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was increased after HA coating. The results of adhesion and morphology of human umbilical vein endothelial cells (HUVECs) showed that the HA-coating aligned PCL (HA-aPCL) nanofibrous scaffolds could highly promote attachment and guide HUVECs bipolar spread with the parallel aligned nanofibers. Furthermore, HUVECs on the HA-aPCL formed a confluent monoendothelial cell layer and exhibited superior protein expression levels of von Willebrand factor (vWF). This study suggested that the combination of aligned nanostructure and HA modification was more capable of promoting the regeneration of functional endothelium for vascular tissue engineering than individual use.     

Tutorial on the biology of nanotopography

The aims of this short tutorial are fourfold: 1) to introduce readers unfamiliar with the field to major concepts in the field; 2) to inform the reader of major unresolved questions; 3) to inform readers of a few major sources of relevant literature; and 4) to place the subject in relation to its relevance to other areas of science and practical application.     

Interactions of human blood and tissue cell types with 95-nm-high nanotopography

Two of the major concerns for tissue engineering materials are inflammatory responses from blood cells and fibrous encapsulation by the body in order to shield the implant from blood reaction. A further hurdle is that of vascularization. In order to develop new tissues, or to repair parts of the vascular system, nutrients need to be carried to the basal cell layers. If a material promotes tissue formation, but not vascularization, necrosis will be observed as multilayered cells develop. In this paper, polymer demixed island topography with a 95-nm Z axis was tested using human mononuclear blood cells, platelets, fibroblasts, and endothelial cells. The results showed no difference in blood response between the islands and the flat controls, suggesting that in vivo there would be negligible immunological difference. Fibroblasts reacted by changing morphology into a rounded shape with thick processes and poorly developed cytoskeleton. Retardation of fibroblast growth may be an advantageous, as it is this cell type that forms the fibrous capsule, preventing growth of the required tissue type. Finally, endothelial cells were seen to form arcuate, or curved, morphologies in response to the islands. This is the normal, in vivo, morphology for vascular endothelium. This result suggests that the nano-features are promoting a more phenotypically correct morphology.     

The anatase phase of nanotopography titania plays an important role on osteoblast cell morphology and proliferation

The surface properties of biomaterials play a vital role in cell morphology and behaviors such as cell adhesion, migration, proliferation and differentiation. Three different crystal phases of titania film (rutile, anatase and amorphous titania) with similar roughness were successfully synthesized by DC reactive magnetron sputtering. The surface roughness of each film was about 8-10 nm. Primary rat osteoblasts were used to observe changes in morphology and to evaluate cell behavior at the film surface. The number of the osteoblasts on anatase film was significantly higher than rutile and amorphous films after 36 and 72 h incubation. More importantly, synthesis of alkaline phosphatase was significantly greater by osteoblasts cultured on anatase film than on rutile and amorphous films after 7 and 14 days. In addition, the cells grown on the anatase phase film had the largest spreading area; the actin filaments in cells with regular directions were well defined and fully spreaded. The results indicate that the anatase phase of titania with nanoscale topography yield the best biological effects for cell adhesion, spreading, proliferation and differentiation. There are strong therapeutic prospects for this biomaterial film for osteoblast proliferation, with possible applications for orthopedic and dental implant.     

Synthetic hierarchical nanostructures: growth of carbon nanofibers on microfibers by chemical vapor deposition

In this paper the synthesis of three-dimensional hierarchical nanostructures by pyrolysis of acetylene to grow carbon nanofibers (CNFs) on carbon microfibers (CMFs) and glass microfibers (GMFs) is reported. The morphology and structure of the as-prepared CNFs were studied by scanning electron microscopy and high-resolution transmission electron microscopy. CNFs grown on both substrates typically exhibited two types of morphology: the coil-like CNFs with frequent change in orientation and the relatively straight and long CNFs with parallel graphene sheets. The ethanol pretreatment was effective at improving the yield and distribution of the as-grown CNFs on CMFs, but showed an adverse effect to the CNF growth on GMFs. The influence of different substrates and growth temperatures on CNF morphology and the possible growth mechanism for the observed microstructures was discussed.     

The influence of nanotopography on organelle organization and communication

Cellular differentiation can be affected by the extracellular environment,particularly extracellular substrates.The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vivo.Organelles are major players in various cellular functions;however,the influence of nanotopography on organelles has not yet been elucidated.In the present study,a micropit-nanotube topography (MNT) was fabricated on the titanium surface,and organelle-specific fluorescent probes were used to detect the intracellular organelle organization of MG63 cells.Communication between organelles,identified by organelle-specific GTPase expression,was evaluated by quantitative polymerase chain reaction and western blotting.Transmission electron microscopy was performed to evaluate the organelle structure.There were no significant differences in organelle distribution or number between the MNT and flat surface.However,organelle-specific GTPases on the MNT were dramatically downregulated.In addition,obvious endoplasmic reticulum lumen dilation was observed on the MNT surface,and the unfolded protein response (UPR)was also initiated.Regarding the relationships among organelle trafficking,UPR,and osteogenic differentiation,our findings may provide important insights into the signal transduction induced by nanotopography.     

Plasma-enhanced chemical vapor deposition of multiwalled carbon nanofibers

Plasma-enhanced chemical vapor deposition is used to grow vertically aligned multiwalled carbon nanofibers (MWNFs). The graphite basal planes in these nanofibers are not parallel as in nanotubes; instead they exhibit a small angle resembling a stacked cone arrangement. A parametric study with varying process parameters such as growth temperature, feedstock composition, and substrate power has been conducted, and these parameters are found to influence the growth rate, diameter, and morphology. The well-aligned MWNFs are suitable for fabricating electrode systems in sensor and device development.     

Electrospinning of nanofibers: Characterization of jet dynamics and humidity effects

Electrospinning is a method to produce submicron polymer fibers for a wide range of applications. In many applications, the average electrospun fiber size and uniformity are important for the product's performance and process economics. Thus, it is desirable for electrospinning to achieve consistent and controllable fiber diameters. However, the current state-of-the-art electrospinning process can result in variable fiber diameters, both run-to-run and during a run. This paper investigates how the operating regime as well as several important process factors affect fiber diameter using a vision-based system. For aqueous polyethylene oxide (PEO) solutions, it is found that the relative humidity has a strong effect on fiber diameter. Correlations between measurable parameters and fiber diameter are also developed to provide the ability to achieve the desired fiber diameters. The jet dynamics are experimentally identified through step response for development of appropriate control strategies.     

Intestinal drug absorption enhancers: synergistic effects of combinations

Although many absorption enhancers have been investigated, very few are used clinically. A need exists therefore for more effective absorption enhancers. The drug-absorption-enhancing effects of combinations of N-trimethyl chitosan chloride (TMC) with degrees of quaternization of 48 and 64%, dicarboxymethyl chitosan oligosaccharide, and chitosan lactate oligomer with monocaprin and melittin were compared to their individual performances using the in vitro Caco-2 cell model. Combining the absorption enhancers showed synergism in both the reduction of the transepithelial electrical resistance (TEER) and the enhancement of the transport of a macromolecular model compound across this intestinal epithelial cell layer. Lower concentrations of the absorption enhancers in the combination groups exhibited greater effects on the epithelial cells compared with the individual absorption enhancers.     

Analytical modeling of wetting dependence on surface nanotopography

An analytical model was developed to describe the mechanism of wetting dependence on surface nanotopography. This model relates the contact angle formation with the asperity geometry for application to a hydrophilic wafer surface, which is derived based on liquid-solid interfacial contact over the contact line. Experimental investigations were performed to verify the model. For much of the examined parameter room in the hydrophilic silicon wafer surface, it was found that the contact angle was strongly dependent on the ratio of asperity height to length, and the sharper asperity led to the higher contact angle. The observations are well consistent with Gibbs' contact-line theory.     

Wet chemical process to enhance osteoconductivity of electrospun chitosan nanofibers

In this study, chitosan/hydroxyapatite (CS/HA) nanofibers were prepared using a wet chemical method. First, CS nanofibers with uniform diameters were fabricated using electrospinning. Then, a wet chemical process was used to mineralize nanofiber surfaces to form a homogeneous HA deposit. Reactions with three cycles were found to optimize biomimetic properties of the HA. The mineralization process required only approximately 3 h, which corresponded to a saving of 98 % in preparation time compared with that needed by the process using a simulated body fluid (SBF). According to the attachment and spreading of UMR (rat osteosarcoma) cells on the CS/HA composite fibers, the deposited mineralization layer significantly enhanced cell affinity of the CS nanofibers and the HA created by the wet chemical method was as effective as that created by the SBF. The composite nanofibrous scaffolds produced by the wet chemical process also promoted osteogenic differentiation by inducing ossification. Thus, expressions of collagen type I, alkaline phosphatase, osteocalcin, bone sialoprotein, and osterix were all enhanced. These results demonstrated that composite electrospun fibers can be efficiently prepared using wet chemical method and the resulting nanofibrous scaffolds have considerable potential in future bone tissue engineering applications.     

Mechanical strength of carbon nanofibers obtained by catalytic chemical vapor deposition

The ultimate strength of carbon nanotips prepared by electrochemical etching of fibers reinforced with multiwall carbon nanotubes has been determined on samples loaded with a strong electric field in a field-ion microscope. The nanostructural fibers were obtained by catalytic chemical vapor deposition of carbon. The phenomenon of strengthening was observed for carbon nanotips loaded to a level close to the theoretical ultimate strength of high-modulus fibers.     

Improved cell viability of biocompatible nutrient-contained polymeric nanofibers

Nanofibers containing cell nutrients (PGDs) were fabricated by mixing 5 wt% poly(epsilon-caprolactone) (P), 4 wt% gelatin (G), and 0-2.4 wt% Dulbecco's Modified Eagle's Medium (D). The contact angles showed a considerable decrease from 118.4 degrees on the P scaffold to 17.6 degrees on PGD1.6 (containing 1.6 wt% D). The weight loss ratios between PGD1.6 and the P nanofiber, and between PGD1.6 and the PG nanofiber by degradation after 28 days were approximately 3.1 and 1.4, respectively. The rate of cell proliferation on PGD1.6 was greater than that on the PG nanofiber by 14% and 38% for the exchanged and unexchanged culture media, respectively. The physicochemical measurement results showed that the PGDs exhibited enhanced hydrophilic properties and rapid biodegradation. The PGD nanofibers with increasing D content showed better conditions for long-term cell viability. The growth mechanism of the cells on the PGDs was explained by an attachment and growth process.     

The synergistic effect of nanocrystal integration and process optimization on solar cell efficiency

This paper investigates the roles of semiconducting single-walled carbon nanotubes (SWNTs) and metallic SWNTs in the SWNT/poly(3-hexylthiophene) (P3HT)-based photovoltaic conversion system. SWNTs containing different fractions of semiconducting nanotubes were conjugated with P3HT by virtue of π-π interaction. The energy transfer and carrier transport mechanisms in the photovoltaic composites were experimentally investigated by optical absorption spectroscopy, photoluminescence spectroscopy and carrier mobility measurements. At low loading of SWNTs, a high percentage of semiconducting nanotubes result in diminished non-radiative decay of exciton and lower carrier mobility, causing higher open circuit voltage and lower photocurrent. At an optimized morphology, SWNT/P3HT/phenyl-C61-butyric acid methyl ester (PCBM) hybrid-based solar cells demonstrated much higher photocurrent than a reference solar cell (P3HT:PCBM) due to the improved carrier mobility. Further thermal annealing of the devices significantly increased the open circuit voltage to 610?mV, resulting in an 80% increase of power conversion efficiency in comparison to the reference solar cell. These results are expected to lay a foundation for the integration of various nanocrystals into solar cells for efficient photovoltaic conversion.     

Fabrication of nanocomposite membranes from nanofibers and nanoparticles for protection against chemical warfare stimulants

Nanoparticles of MgO were synthesized by Aero gel method. These MgO nanoparticles were then mixed with various polymer solutions (poly(vinyl chloride) (PVC), poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF copolymer), polysulfone (PSU)) and then subjected to electrospinning to produce nanocomposite membranes. The hydrolysis of paraoxon, a never agent stimulant, in presence of these membranes was studied using UV. The order of the reactivity of the membranes are found to be PVC-MgO < PVDF < PSU < PVDF-MgO < PSU-MgO. After selecting PSU as the supportive candidate, relative rates of hydrolysis were compared for nanoparticles or charcoal with nanocomposite membranes. The order is as follows; Charcoal (1) < PSU-Al₂O₃ (1.5) < PSU-MgO (2.1) < Al₂O₃ nanoparticles (2.8) < MgO nanoparticles (5.4). The amount of hydrolysis of PSU-MgO composite membrane was 60% less when compared to MgO nanoparticles as such usage. The loading percentage of MgO into nanofiber is 35 %. The fabricated composite membrane (containing 5% MgO) was tested for chemical warfare agent stimulant, paraoxon, and found to be about 2 times more reactive than currently used charcoal.     

Controlled growth of carbon nanofibers using plasma enhanced chemical vapor deposition: Effect of catalyst thickness and gas ratio

The characteristics of carbon nanofibers (CNFs) grown, using direct current plasma enhanced chemical vapor deposition system reactor under various acetylene to ammonia gas ratios and different catalyst thicknesses were studied. Nickel/Chromium-glass (Ni/Cr-glass) thin film catalyst was employed for the growth of CNF. The grown CNFs were then characterized using Raman spectroscopy, field emission scanning electron microscopy and transmission electron microscopy (TEM). Raman spectroscopy showed that the Ni/Cr-glass with thickness of 15 nm and gas ratio acetylene to ammonia of 1:3 produced CNFs with the lowest I_D/I_G value (the relative intensity of D-band to G-band). This indicated that this catalyst thickness and gas ratio value is the optimum combination for the synthesis of CNFs under the conditions studied. TEM observation pointed out that the CNFs produced have 104 concentric walls and the residual catalyst particles were located inside the tubes of CNFs. It was also observed that structural morphology of the grown CNFs was influenced by acetylene to ammonia gas ratio and catalyst thickness.