Fibronectin modulates the morphology of osteoblast-like cells (MG-63) on nano-grooved substrates

Cell interactions with biomaterials are affected by surface topographic and chemical cues. Although it is well-known that nanometrical grooves/ridges structure modulates cellular spreading, elongation, and alignment, the combinational influence of surface topographic and chemical cues is not well studied. In this study, nano-textured silicon substrata with parallel ridges of 90, 250, or 500 nm wide, separated by grooves with equal width, were fabricated by electron beam lithography and dry etching techniques. Osteoblast-like cells, MG-63, were cultured on the patterned substrata with or without pre-adsorption of fibronectin. The cell morphology was imaged by scanning electron microscopy, and analyzed by image software. We found that FN coating initially modulated cellular spreading, length, and orientation on all types of grooved surfaces. However, after 24 h of culture, the cell morphology was not affected by FN coating on the 250-nm and 500-nm surfaces, while FN decreased cell alignment on the 90-nm surfaces. Our results suggest that surface chemical cues influence the initial cell-substratum contact, while the long-term cellular morphology is dictated by surface topographic cues.     

Novel thin-walled nerve conduit with microgrooved surface patterns for enhanced peripheral nerve repair

Randomly aligned nerve cells in vitro on conventional culture substrata do not represent the complex neuronal network in vivo and neurites growing in uncontrolled manner may form neuroma. It is of great importance to mimic the organised growth pattern of nerve cells in the study of peripheral nerve repair. The aim of this work was to modify and optimize the photolithographic technique in creating a reusable template in the form of a silicon wafer that could be used to produce contact guidance on biodegradable polymer surface for the orientated growth of nerve cells. Micro-grooves (approximately 3 μm in depth) were etched into the silicon template using KOH at increased temperature. The originality of this work lies in the low cost and high efficiency method in producing microgrooves on the surface of biodegradable ultra-thin polymer substrates (50–100 μm), which can be readily rolled up to form clinically implantable nerve conduits. The design of a pattern with small ridge width (i.e., 5 μm) and bigger groove width (i.e., 20 μm) favored the alignment of cells along the grooves rather than on the ridges of the patterns, which minimized the effect of cross growing of neurites between adjacent grooves. Effectively, enhanced nerve regeneration could be anticipated from these patterned conduits.     

Self-assembly of polystyrene microspheres within spatially confined rectangular microgrooves

A convective self-assembly of mono-sized polystyrene spheres with diameters ranging from 262 to 1000 nm was conducted on patterned silicon wafers with one-dimensional, periodic rectangular microgrooves of different widths (0.65–6 μm). The latex beads were driven into the spatially confined microgrooves by the capillary interactions and the confined wall during solvent evaporation, resulting in a range of packing structures. Processing variables including evaporation temperature, particle size (D), groove width (W), and groove height (H) were examined experimentally, and geometrical models were proposed to explain the various packing structures obtained. The degree of spatial freedom for the particles to rearrange themselves in the confined channels is found critical to the assembled particle-packing structure.     

Laser ablation patterning by interference induces directional cell growth

Laser-patterning by interference is a method to introduce micropatterns on the surface of TXL and TXB, which were shown to have an effect on the L929 growth. In this experiment, we have produced collagen-coated and laser-patterned TXL and TXB with different dimensions; the groove width of the line patterns varied approximately from 1.2 /spl mu/m to 9.7 /spl mu/m, ridge depth varied from 0.4 /spl mu/m to 1.3 /spl mu/m, and the groove depth varied between 0.4 /spl mu/m and 1.3 /spl mu/m. Therefore, a homogeneous smooth surface was achieved, and that L929 growth was only affected by the different dimensions of the line patterns. All the laser-patterned TXL and TXB have shown inducing different degrees of directional growth of L929 that the cells grew in the direction aligning the microgrooves. However, the different widths of the microgrooves were demonstrated to play an important role in determining cell morphology and growth orientation. For example, cells were elongated when they grew on the narrower widths, which were 1.26 /spl mu/m, 1.91 /spl mu/m, and 5.04 /spl mu/m while cells tended to be triangular when grew on wider width about 9.76 /spl mu/m. In addition, L929 might grow only on the top of the laser-patterns attaching the ridges when the groove widths were narrow, but might grow into the microgrooves when the width went beyond 5.04 /spl mu/m.     

Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces

This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.     

Nanofibrous modification on ultra-thin poly(e-caprolactone) membrane via electrospinning

Poly(e-caprolactone) (PCL) is a favorable material for tissue engineering. PCL was successfully fabricated into less than 10 μm thin membranes using a 2-roll-heated-mill and biaxial stretching process. However, PCL is known for its poor cellular adhesion and surface modifications are needed for any tissue engineering applications. This paper reports on a novel surface modification technique of the PCL membrane by coating with electrospun nanofibers. The purpose was to mimic the architecture of the natural extracellular matrix and create nanotopography for enhanced cellular attachment. The surfaces were characterized by scanning electron microscopy (SEM), water contact angle and atomic force microscopy. The results showed that uniform nanofibrous topology were successfully achieved on the surface of the PCL membrane, with increased roughness (more than 17 times) and surface area. This nanofibrous topology induced capillary effects after sodium hydroxide (NaOH) treatment, causing the water contact angle to drop to almost zero. Scratch tests revealed a strong interaction of PCL nanofiber coating on the PCL membrane. AlamarBlue assay indicated that 3T3 fibroblast cells proliferated well on the nanofibrous membrane. Confocal Laser Scanning Microscope revealed better cell attachment onto the nanofibrous membranes than the untreated membranes. Results from SEM showed that the cells' spindle-shaped morphology on the NaOH-treated fibrous surface was evident while they remained in isolated spherical shaped entities in the non-treated fibrous surfaces.     

Uptake Kinetics and Nanotoxicity of Silica Nanoparticles Are Cell Type Dependent

In this study, it is shown that the cytotoxic response of cells as well as the uptake kinetics of nanoparticles (NPs) is cell type dependent. We use silica NPs with a diameter of 310 nm labeled with perylene dye and 304 nm unlabeled particles to evaluate cell type‐dependent uptake and cytotoxicity on human vascular endothelial cells (HUVEC) and cancer cells derived from the cervix carcinoma (HeLa). Besides their size, the particles are characterized concerning homogeneity of the labeling and their zeta potential. The cellular uptake of the labeled NPs is quantified by imaging the cells via confocal microscopy in a time‐dependent manner, with subsequent image analysis via a custom‐made and freely available digital method, Particle_in_Cell‐3D. We find that within the first 4 h of interaction, the uptake of silica NPs into the cytoplasm is up to 10 times more efficient in HUVEC than in HeLa cells. Interestingly, after 10 or 24 h of interaction, the number of intracellular particles for HeLa cells by far surpasses the one for HUVEC. Inhibitor studies show that these endothelial cells internalize 310 nm SiO₂ NPs via the clathrin‐dependent pathway. Remarkably, the differences in the amount of taken up NPs are not directly reflected by the metabolic activity and membrane integrity of the individual cell types. Interaction with NPs leads to a concentration‐dependent decrease in mitochondrial activity and an increase in membrane leakage for HUVEC, whereas HeLa cells show only a reduced mitochondrial activity and no membrane leakage. In addition, silica NPs lead to HUVEC cell death while HeLa cells survive. These findings indicate that HUVEC are more sensitive than HeLa cells upon silica NP exposure.     

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330nm deep microgrooves.

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330nm deep and either 10, 25 or 100mum in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100mum wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25mum wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10mum wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.     

Grooved substrata facilitate in vitro healing of completely divided flexor tendons

Multiple grooved substrata with groove depth 5 m were found to facilitate the healing of completely divided rat flexor tendons in vitro. Sections of tendons cultured on plain substrata showed only partial healing with incompletely sealed epitenon layers and immature thin collagen fibres. Tendons cultured on patterned substrata healed with complete restoration of the epitenon layer and reconstitution of the internal structure of collagen fibres. Epitenon fibroblasts isolated from the surface of rat flexor tendons were shown to be more sensitive to topographical features than fibroblasts of the same size BHK fibroblasts. They remained more elongated and better aligned to the groove direction than BHK cells. Multiple grooved substrata facilitated epitenon cell movement. Cells were found to move with higher speed on patterned substrata than on plain substrata. In summary, we conclude that the use of multiple grooved substrata promotes tendon healing in vitro and may find application in clinical practice in tendon repair.     

A dual gradient assay for the parametric analysis of cell-surface interactions

Cellular response to microgrooves is addressed using a new assay format, comprising orthogonal gradients of continuously varied groove pitch and depth. Dual layer etch masks are created using a combination of micropatterning and plasma polymer deposition. A silicon substrate with a constant groove width of 8 μm and with ridge width increasing from 8 μm in 0.5 μm steps across 10 mm is fabricated by photolithography. A plasma-polymerized hexane film which is 120 nm thick at one end of these grooves, and 10 nm at the other, is deposited under a diffusion mask. Reactive etching of the patterned sample transfers a gradient of groove pitch and groove depth into the silicon substrate. A silicon master with a gradient of groove depth spanning more than two orders of magnitude (less than 10 nm to over 1000 nm) is used to create an injection molding inlay for mass replication of the screening topography. Polycarbonate replicas are molded for use in cell culture studies, and the functionality of the topography as a high-throughput screening platform is investigated. The response of MDCK, h-TERT fibroblasts, and LE2 endothelial cells is examined, in terms of attachment and morphological response to the variation in topographical cues, with the aim of pinpointing the optimal combination of groove pitch and depth to elicit a tailored response from each cell type. When the range of topographical features screened on a single substrate is considered, this new assay represents a significant step forward in the parametric design and analysis of topographical cues at the biomaterial interface.     

Cell/surface interactions of human osteo-sarcoma (HOS) cells and micro-patterned polydimelthylsiloxane (PDMS) surfaces

This paper presents the results of an experimental study of the effects of three-dimensional micro-pattern geometry on cell/surface interactions and the adhesion between HOS cells and PDMS surfaces. Micro-grooves with well-controlled ridges and spacings were fabricated by curing poly-di-methy-siloxane (PDMS) in silicon molds produced by photolithography. HOS cells were then cultured onto these surfaces for durations of 6, 12 and 48 h. In cases, where the groove spacing was comparable to the spread cell size, the cells align well in the directions of microgrooves. However, as the ridge separation increases, the cell orientations become more random, and less dependent on ridge height and spacing. The actin cytoskeletal structure and the distribution of focal adhesions are also elucidated by immuno-fluorescence staining.     

Micro-structural geometry of thin films intended for the inner lumen of nerve conduits affects nerve repair

Damage to peripheral nerves can cause significant motor or sensory injuries. In serious cases, a nerve is sacrificed from another part of the body to repair a damaged nerve (autograft). The development of biodegradable polymer conduits may offer an alternative to autografts. This study investigated the surface topography and mechanical properties of smooth, pitted and grooved structures of ultra-thin poly (ε-caprolactone)/poly lactic acid blended, solvent-cast films. We have investigated the effect of the groove shape on cell morphology and alignment. Photolithography and dry/wet etching was used to develop patterned silicon substrates with grooves with accurate geometries (V shaped, sloped walls and square shaped). Using a neural cell line (NG108-15), in vitro experiments confirmed good cell attachment and proliferation on all the polymer scaffolds. Imaging techniques demonstrated that there was different cellular responses and morphology according to the shape of the groove. Studies showed that the geometry, particularly the angle of the slope and the space between grooves, affected cellular responses. In addition, biomechanical studies showed that the patterned films had excellent mechanical properties and were stronger than the natural nerve. The conduit tubes were made by rolling the films around a mandrel and using a thermal welding technique to join the edges. The promising biomechanical and in vitro results demonstrate that nerve cell responses are affected by the shape of longitudinal grooves, and particularly by the angle of the slope of the groove walls.     

Uptake pathways and subsequent nucleus targeting of tat-conjugated gelatin-siloxane nanoparticles as nonviral gene vector

Novel hybrid biomaterial of Tat peptide modified gelatin-siloxane nanoparticles (Tat-GS NPs), with positive surface potential was synthesised through a two-step sol-gel process. The particles were subsequently tested in vitro with HeLa cells by fluorescent activitated cell sorter analysis, confocal laser scanning microscopy, and transmission electron microscopy to determine whether the functionalisation with Tat peptide allowed particles to transfer across the cell membrane and locate in the nucleus. Our current results indicated that the internalisation of Tat-GS NPs in HeLa cells is time- and concentration- dependent. Moreover, Tat-GS NPs could penetrate the nucleus membrane and enter into nucleus. The Tat-GS/pDNA nanocomplexes were formulated with higher encapsulation efficiency, and exhibited efficient transfection in vitro. Tat-GS NPs are thus considered as novel non-viral vectors will be widely used in further study.     

Structurally ordered mesoporous carbon nanoparticles as transmembrane delivery vehicle in human cancer cells

A structurally ordered, CMK-1 type mesoporous carbon nanoparticle (MCN) material was successfully synthesized by using a MCM-48 type mesoporous silica nanoparticle as template. The structure of MCN was analyzed by a series of different techniques, including the scanning and transmission electron microscopy, powder X-ray diffraction, and N2 sorption analysis. To the best of our knowledge, no study has been reported prior to our investigation on the utilization of these structurally ordered mesoporous carbon nanoparticles for the delivery of membrane impermeable chemical agents inside of eukaryotic cells. The cellular uptake efficiency and biocompatibility of MCN with human cervical cancer cells (HeLa) were investigated. Our results show that the inhibitory concentration (IC50) value of MCN is very high (>50 microg/mL per million cells) indicating that MCN is fairly biocompatible in vitro. Also, a membrane impermeable fluorescence dye, Fura-2, was loaded to the mesoporous matrix of MCN. We demonstrated that the MCN material could indeed serve as a transmembrane carrier for delivering Fura-2 through the cell membrane to release these molecules inside of live HeLa cells. We envision that further developments of this MCN material will lead to a new generation of nanodevices for transmembrane delivery and intracellular release applications.     

Transmission electron microscopy study of the cell–sensor interface

An emerging number of micro- and nanoelectronics-based biosensors have been developed for non-invasive recordings of physiological cellular activity. The interface between the biological system and the electronic devices strongly influences the signal transfer between these systems. Little is known about the nanoscopic structure of the cell-sensor interface that is essential for a detailed interpretation of the recordings. Therefore, we analysed the interface between the sensor surface and attached cells using transmission electron microscopy (TEM). The maximum possible resolution of our TEM study, however, was restricted by the quality of the interface preparation. Therefore, we complemented our studies with imaging ellipsometry.We cultured HEK293 cells on substrates, which had been precoated with different types of proteins. We found that contact geometry between attached cell membrane and substrate was dependent on the type of protein coating used. In the presence of polylysine, the average distance of the membrane-substrate interface was in the range of 35-40 nm. However, the cell membrane was highly protruded in the presence of other proteins like fibronectin, laminin or concanavalin-A. The presented method allows the nanoscopic characterization of the cell-sensor interface.     

Organization of mesenchymal stem cells is controlled by micropatterned silicon substrates

Mesenchymal stem cells (MSCs) can differentiate into various cellular lineages, including osteoblasts (that deposit hydroxyapatite, the main mineral constituent of bone), and also exhibit a high morphological plasticity. Here we grew for the first time MSCs on micropatterned silicon chips, in order to induce topography-guided alignment. Light microscopy, scanning electron microscopy and atomic force microscopy were used to characterize the cell response on various length scales. A notable alignment and movement of MSCs along the microgrooves on the chips was revealed. The cells were shown to inhabit the grooves rather than ridges and exhibited an elongated shape, with unusually long processes. On these cells, we revealed rhizome structures arranged along the extensions, which may serve as adhesion centers and participate in elongation and locomotion.     

Osteoblast behavior on TiO2 microgrooves prepared by soft-lithography and sol–gel methods

This study focused on the effects of microgrooved TiO₂ surfaces on osteoblast behavior. Microgrooved TiO₂ surfaces with different widths (12 μm and 40 μm) and flat surfaces were fabricated on glass substrates based on the combination of a sol–gel technique and soft-lithography. Osteoblasts (MC3T3-E1) were cultured on the as-prepared microgrooved and flat TiO₂ surfaces. Optical microscopy and scanning electron microscopy were used to analyze the adherent cell behavior by examining the cell morphology. Orientation angle analysis indicated that the cells tended to align along the microgrooves. This tendency was stronger on the microgrooves with smaller widths and became weak with increasing width. Alamar Blue assay indicated that the microgrooves restricted cell proliferation and the alkaline phosphatase assay revealed that the microgrooves limited the differentiation rate. This restriction increased with decreasing microgroove width. The surface energy of the TiO₂ surfaces was size-dependent and followed the order γ _12 μm < γ _40 μm < γ _{flat surfaces}. Osteoblast proliferation and differentiation on the surface with high surface energy exhibited high proliferation and differentiation rates. These results indicated that surface energy appeared to be a dominant factor for cell activity. Thus, surface energy would be a valuable index for the cell compatibility of a micropatterned surface.     

Nonbleaching fluorescence of gold nanoparticles and its applications in cancer cell imaging

In this paper, we investigated the fluorescent properties of gold nanoparticles (GNPs) with several tens of nanometers by ensemble fluorescence spectrometry, fluorescence correlation spectroscopy (FCS), and fluorescence microscopy. We observed that GNPs synthesized by the citrate reduction of chloroauric acid possessed certain fluorescence, narrow full width at half-maximum (17 nm), and with an increase of particle sizes, the emission intensity showed a gradual increase while the emission wavelength remained almost constant (at 610 nm). Especially, the fluorescence of GNPs possessed the excellent behavior of antiphotobleaching under strong light illumination. Despite their low quantum yields, GNPs exhibited strong native fluorescence under relatively high excitation power. The fluorescence of GNPs could be characterized by fluorescence imaging and FCS at the single particle level. On the basis of this excellent antiphotobleaching of GNPs and easy photobleaching of cellular autofluorescence, we developed a new method for imaging of cells using GNPs as fluorescent probes. The principle of this method is that after cells stained with GNPs or GNPs bioconjugates are illuminated by strong light, the cellular autofluorescence are photobleached and the fluorescence of GNPs on cell membrane or inside cells can be collected for cell imaging. On the basis of this principle, we imaged living HeLa cells using GNPs as fluorescent probes and obtained good cell images by photobleaching of cellular autofluorescence. Furthermore, anti-EGFR/GNPs were successfully used as targeted probes for fluorescence imaging of cancer cells. Our preliminary results demonstrated that GNPs possessed excellent behaviors of antiphotobleaching and were good fluorescent probes in cell imaging. Our cellular imaging method described has potential applications in cancer diagnostics, studies, and immunoassays.     

Single molecular imaging and spectroscopy of conjugated polyelectrolytes decorated on stretched aligned DNA

DNA is the prototype template for building nanoelectronic devices by self-assembly. The electronic functions are made possible by coordinating electronic polymer chains to DNA. This paper demonstrates two methods for fabrication of aligned and ordered DNA nanowires complexed with conjugated polyelectrolytes (CPEs). The complex can be formed either in solution prior to stretching or after stretching of the bare DNA on a surface. Molecular combing was used to stretch the complexes on surface energy patterned surfaces, and PMMA for the bare DNA. Single molecular spectroscopy, in fluorescence, and microscopy, in atomic force microscopy, give evidence for coordination of the short CPE chains to the aligned DNA.