Investigation of size–dependent cell adhesion on nanostructured interfaces
© Kuo et al.; licensee BioMed Central Ltd. 2014
Received: 27 September 2014
Accepted: 18 November 2014
Published: 5 December 2014
Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diameters.
We first checked the viability of cells grown on nanopillars with diameters ranging from 200 nm to 700 nm. It was observed that when cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. We then calculated the average size of the focal adhesions and the cell-spreading area for focal adhesions using confocal microscopy. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200 nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200 nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions.
We have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on the softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate behaviour of cells.
KeywordsNanotopography Cell adhesion Surface topography
The interfacial properties of materials govern the performance of biomaterials because cells are in direct contact with the surfaces of materials. Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix (ECM) molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions -. Therefore, one of the commonly used approaches to improve the performance of biomaterials is surface engineering, whereby a material’s surface properties can be modified by chemical and physical means. In the past few decades, surface engineering techniques have been widely used to improve device biocompatibility, to promote cell adhesion and to reduce unwanted protein adsorption -. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood.
To investigate how cells respond to their nanoenvironments, many techniques, including dip-pen lithography , electron-beam lithography , nano-imprinting , block-copolymer micelle nanolithography -, and nanosphere lithography , have been utilized to create well-defined protein nanopatterns on planar substrates. The dimensional parameters of ECM molecules, including density, spacing, and surface coverage, have been found to be important to cell adhesion and spreading. When cells attach to surfaces, nanometer-scale dot-like focal complexes are formed first . These focal complexes are transient and unstable. Some of the focal complexes will mature into micrometer-scale elongated focal adhesions, which serve as anchoring points for cells. It has been previously shown , that the formation of focal adhesions was dependent on the size of the ECM nanopatterns and that the force experienced by the focal adhesions increased as the pattern size decreased, explaining the instability of smaller focal complexes.
In addition to sensing the protein composition of the nanoenvironment, cells also sense the physical properties around them. It has been demonstrated that by systematically changing the rigidity of microstructures, the regulation of cell functions, such as morphology, focal adhesions and stem cell differentiation, can occur . It was recently observed that the efficiency of drug-uptake by cells was greatly enhanced for cells grown on nanostructured materials, including roughened polymers , nanowires , nanofibers  and nanotubes ,. However, the mechanisms by which the cells interact with these nanostructures have not yet been studied systematically -. To understand how cells interact with nanostructures, we have systematically investigated the interactions between cells and nanostructures using size-tunable polymeric nanopillars with well-defined physical properties.
Results and discussion
Dimensions and apparent Young’s moduli measured for nanopillars
Young’s modulus (MPa)
214 ± 13
781 ± 37
402 ± 130
322 ± 16
999 ± 88
583 ± 128
425 ± 17
814 ± 56
697 ± 118
500 ± 19
971 ± 55
721 ± 164
684 ± 17
663 ± 49
852 ± 249
In summary, we have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. When cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. The size of focal adhesions formed on the nanopillars decreased as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200 nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200 nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on our softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate cell behavior.
Fabrication of polymeric nanopillars
Polymeric nanopillars were fabricated via a combination of nanosphere lithography and nano-molding, as described previously . The schematic for nanopillar fabrication is illustrated in Figure 1. Briefly, a closely packed monolayer of polystyrene beads with a diameter of 870 nm (Bang’s Laboratories, Inc., CV < 5%) was produced on the silicon substrates using nanosphere lithography, and the diameter of the polystyrene beads was reduced to various diameters of interest using oxygen plasma trimming. A layer of chromium was then deposited on the substrate. After removing the polystyrene beads using dichloromethane, a deep-etching process was performed in an inductively coupled plasma system (Samco, RIE-10ip). As a result of the deep-etching, periodic nanohole arrays were obtained on the silicon substrates, which were then used to create the polymeric nanopillars. To remove the chromium, silicon templates were placed in a chromium etchant (Aldrich) solution for 20 minutes. To produce the polymeric nanopillars, an ultraviolet-curable adhesive (NOA 61, Norland, Young’s modulus: 1.2 GPa) was spun onto the silicon-hole substrate at 1000 rpm and subsequently illuminated with UV light in an ELC-500 UV-light chamber for 10 minutes. The polymeric nanopillars were obtained by peeling off the cured films from the silicon-hole templates.
CHO-K1 cells were seeded in 30-mm plates with Ham’s F-12 K medium supplemented with 10% (v/v) fetal bovine serum (FBS) and passaged every 2 to 3 days. MDCK cells were grown in minimum essential medium with Earle’s BSS supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 1.5 g/L sodium bicarbonate. C2C12 myoblast cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) calf serum, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate and 4.5 g/L glucose. PEN-STREP-AMPHO solution (Biological Industries) was added to all culture media. The cells were incubated at 37°C in a 5% CO2 atmosphere.
Cells were fixed by 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS) for 15 minutes, washed twice with PBS solution, and subsequently permeabilized in a 0.25% Triton X-100 solution for 20 minutes. Before staining, all of the samples were blocked using 1% BSA in PBS solution for 30 minutes. The samples were then incubated with the primary antibody at a concentration of 1 μg/mL for 2 hours. The samples were washed three times with PBS followed by incubation with a fluorescent-labeled anti-mouse secondary antibody for 1 hour. To visualize the cell morphology, actin filaments were labeled with TRITC-conjugated phalloidin. A Focal Adhesions Staining Kit (Chemicon) was used to label the focal adhesions. The fluorescence images were obtained using a Leica SP5 confocal microscope. The area of cell spreading and the size of the focal adhesions on the nanopillars of various diameters were analyzed using MetaMorph software (Universal Imaging).
To measure the apoptosis rates of cells grown on the nanopillars, three different cell lines (CHO, C2C12 and MDCK) were seeded on the substrates at a density of 1 × 105 cells/mL and incubated at 37°C in a 5% CO2 atmosphere. Cells seeded on poly-L-lysine-coated and UV-adhesive-coated cover slips for 24 hours provided experimental controls. After 24 hours of culture, the cells were fixed in 4% paraformaldehyde in PBS for 15 minutes. Next, permeabilization was performed using a 20 minute incubation with 0.25% Triton X-100 followed by two washes with PBS. The TUNEL assay for apoptosis was used to measure DNA-damage fragmentation. The Click-iT TUNEL imaging assay (Invitrogen) utilized a dUTP modification with an alkyne group. Samples were incubated for 30 minutes at room temperature with 100 μL of the Click-iT reaction solution, after which the Click-iT reaction solution was removed and the samples were washed three times for 5 minutes with PBS containing 3% BSA. Finally, 100 μL of Hoechst 33342 solution was then added to the sample and incubated for 15 minutes for DNA staining.
To visualize cells on the nanopillars, the cells were fixed overnight at 4°C using a 2% (wt/vol) glutaraldehyde solution in 0.1 M sodium cacodylate, pH 7.3. Samples were then warmed to room temperature and washed twice with PBS. Before critical-point drying, the samples were incubated in 0.1% aqueous tannic acid for 20 minutes, after which the solution was gradually replaced with a PBS/ethanol mixture in the following progression of ratios: 80:20, 60:40, 20:80 and, finally, pure ethanol. Cells in pure ethanol were dried using a critical-point drying device (Leica, EMCPD030) to preserve the morphology of the cells and the structure of the nanopillars. The SEM images of cells were obtained using a field-emission scanning electron microscope (FEI Nova 200). To investigate the cell-nanostructure interactions, the membranes of the cells on the nanopillars were removed by placing samples in a cytoskeleton buffer (CB; 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 5 mM glucose, 10 mM MES, pH 6.1) at room temperature for a short time. The cells were then fixed and dried by critical-point drying as described above.
To calculate the rigidity of the nanopillar substrates, the apparent Young’s moduli of the nanopillars were measured with an atomic force microscope (AFM; JPK, Nanowizard II) using cantilevers (ULTRASHARP, MIKROMASCH) with a force constant of 0.1-0.4 N/m and a resonance frequency of 17–24 kHz. The rigidity was calculated using K = (3/64 πED4/L3), where K is the rigidity, E is the apparent Young’s modulus of the nanopillar, D and L are the diameter and the height of the nanopillars .
This study was supported in part by the Ministry of Science and Technology (MOST) of Taiwan under contract MOST 103-2113-M-001-008-MY3, NSC-101-2120-M-001-011 and by the Academia Sinica Research Project on Nano Science and Technology. CWK would like to thank the financial support received from NSC funding award NSC-102-2113-M-001-019-MY3.
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