Article pubs.acs.org/Langmuir
Orthogonal Morphological Feature Size and Density Gradients for Exploring Synergistic Effects in Biology Clément V. M. Cremmel,† Christian Zink,†,# Katharina Maniura-Weber,‡ Lucio Isa,§ and Nicholas D. Spencer*,† †
Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 5, CH-8093 Zurich, Switzerland ‡ Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science & Technology, Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland § Laboratory for Interfaces, Soft matter and Assembly, Department of Materials, ETH Zurich, Vladimir-Prelog-Weg 5, CH-8093 Zurich, Switzerland S Supporting Information *
ABSTRACT: Gradient surfaces enable rapid screening and highthroughput investigations in various fields, such as biology and tribology. A new method is described for the preparation of material-independent morphological gradients, in which the density and height of roughness features are varied along two orthogonal axes. A polystyrene-particledensity gradient was produced by a dip-coating process on titanium-oxide-coated silicon wafers. A controlled exposure to ultraviolet light enabled the generation of a particle-height gradient in the orthogonal direction. These gradients were replicated to generate material-independent morphology gradients. MC3T3 cell proliferation studies were performed on titanium-coated replicas and showed a higher cell density on the high-feature-density region of the gradient. The cell area coverage was found to increase with decreasing particle height.
■
INTRODUCTION Roughness is known to exert a significant influence on cell behavior, such as adhesion, proliferation, and differentiation.1−10 The roughness-feature size is especially of importance for bone-contacting implants8 because different cell types proliferate at different rates in response to specific roughness characteristics.3 In vitro studies can help to understand cell proliferation and to gain a better understanding of the influence of roughness on osseointegration. It is now well known that the micrometer-range topography of the substrate has a strong influence on the cell shape,3 cell migration,11 and tissue organization.1 More recent work has shown that a combination of micrometer-range roughness with nanofeatures enhances osteoblast proliferation, adhesion, and differentiation and that roughness features in different roughness ranges can work synergistically.8,9,12 Such studies have also been performed in vitro9 and confirmed by in vivo studies.13 Recent work on nanotopography described the influence of nanoroughness on cell differentiation,14−17 spreading,18−21 and adhesion.16,22,23 Gradients are helpful in cell-culture studies to reduce sources of error inherent in sample-to-sample variation of experimental conditions.10,24,25 This is a particularly significant issue in the case of cell studies, where, for example, the exact number of initially seeded cells might vastly influence the final number of cells on the sample after a cultivation period. Moreover, in gradient studies, it can be assured that the cell-staining procedure is exactly the same for the entire gradient sample; therefore, no difference in read-out yield is to be expected along © XXXX American Chemical Society
the gradient. Gradient samples decrease the possibility of errors inherent in studies involving multiple individual samples with different values of a surface parameter and can therefore both save significant time and lead to higher reproducibility. Silica particle-density gradients have been fabricated successfully in previous studies.2,4 Negatively charged nanometer-scale silica particles were adsorbed onto a silicon wafer after rendering it positively charged by preadsorbing a monolayer of poly(ethylene imine). The gradient was introduced by varying the residence time via a simple dipcoating process. The creation of a roughness gradient with features in the micrometer range has also been achieved previously,3 by gradual immersion of a sand-blasted aluminum plate into a polishing solution, thus generating a gradient. Other techniques based on diffusion have also been developed, such as the diffusion of a high-concentration buffer into a particle suspension, thereby changing the interparticle distance and thus its adsorption onto a substrate placed in the suspension.26 Microscale, sand-blasted, polished gradients have also been combined with nanoscale particle-density gradients to generate 2-D roughness gradients, incorporating morphological changes on both size scales along orthogonal directions.9 Cells showed different types of behavior with distinct responses to different Received: March 25, 2015 Revised: June 18, 2015
A
DOI: 10.1021/acs.langmuir.5b01089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. Successive steps in the preparation of a 2-D gradient. (a) Sample immersion into the particle suspension to create the particle-density gradient. (b) UV exposure gradient on a TiO2-coated wafer, creating a particle-height gradient. (c) Replication of roughness features in epoxy, further coated with titanium to mimic a bone implant surface. Preparation of Particle Suspensions. A suspension of polystyrene particles with a diameter of 535 ± 16 nm (Microparticles, Germany, Lot: PS-R- B1266) was ultrasonicated for 10 min and then diluted to 0.002 wt % in purified water (measured resistivity: 83 kΩ· cm−1). Preparation of Particle-Density Gradients. The gradients were prepared by dipping the PEI-coated substrates into the PS particle suspension using a linear-motion drive (Owis Staufen, Germany). (See Figure 1a.) The immersion profile was set to z(t) = 7.72 × 10−7·t2, where z [mm] is the position on the gradient at time t [s]. The maximum immersion time was 3600 s. The settings were such that during the 60 min immersion a gradient with 10 mm length was prepared. The PS particle solution was kept slightly agitated during adsorption. At the end of the process, the samples were immediately removed from the suspension and dipped successively into six beakers containing ultrapure water to rinse off any excess particles. The substrates were then left flat for drying at room temperature. Characterization of Particle-Density Gradients. The particledensity gradients were characterized with a bright-field light microscope (FM Axioscope 2, Zeiss, Germany) in reflection mode. The images were taken using a 20× objective every 300 μm along the gradient direction. The particles were counted using ImageJ (version 1.43r, http://rsb.info.nih.gov/ij), dividing every image into 50 μm wide strips perpendicular to the gradient direction. The particle density was then obtained by dividing the number of particles counted in each strip by the analyzed area. Preparation of Particle-Height Gradients. The samples were exposed to ultraviolet (UV) light, placing them 40 mm away from a 450 W medium pressure quartz mercury arc lamp (Ace Glass, Vineland, NJ). The lamp was maintained at 15 °C to avoid any effect of heat from the lamp on the samples. The samples were placed into a quartz tube in which oxygen was circulated. An aluminum shutter covered with black paper was placed 4 mm above the sample (see Figure 1b) between the lamp and the quartz tube to create a gradient in UV exposure time. Then, the shutter was withdrawn over 1 h at 167 μm·min−1 using a linear-motion drive to create a 10 mm gradient. Characterization of Particle-Height Gradients. The height of the particles was measured by atomic force microscopy (AFM) using a Dimension 3000 with a Nanoscope III controller (Digital Instruments/Veeco Metrology, Santa Barbara, CA). All of the images were recorded using Olympus AC-160 tips in tapping mode (resonant frequency: between 200 and 400 kHz). The data were first flattened using the third-order function from the Nanoscope software (version 5.12 r5, Digital Instruments, Veeco Instruments, CA). The data were then loaded into Matlab (v. 7.9.0.529, Mathworks, USA) and analyzed to obtain a height histogram using steps of 0.1 nm. In the case that artifacts were created during the measurement, they could be manually removed by defining a range of possible particle heights. If any feature did not fall within this range it was not taken into account for the evaluation. The particle height was then measured, looking for local maxima over the whole image. Height average and standard deviations were calculated for at least 20 different particles. Particle-Shape Analysis. The shapes of the particles were characterized by means of scanning electron microscopy with a
length-scale feature sizes on gradients prepared using these techniques. Micrometer-scale roughness particularly enhances the proliferation3 of osteoblasts, while nanoscale roughness inhibits their proliferation.2,4 The combination of both micrometer- and nanometer-roughness into a 2-D orthogonal gradient showed a maximum proliferation rate at high micrometer-scale roughness at an optimum density of nanofeatures.9 This implies a synergistic effect of different roughness-feature sizes on the cells. A new method is presented for the preparation of feature-size gradients. Polystyrene particles deposited on a titanium oxide substrate can be controllably shrunk by means of ultraviolet light exposure. The variation of the UV exposure time thus enables the creation of a gradient in particle height. This technique was combined with a dip-coating process to generate particle-density gradients, yielding 2-D gradients with orthogonal particle-height and density axes. Such gradients show an increase in roughness in one direction due to feature density, as has been previously described,2−4 but also, in the perpendicular direction, an increase in roughness height at constant feature density, which has never been achieved before in this scalerange. Such fabricated structures could then be further replicated27 and post-treated, to generate samples with both controlled roughness and chemistry. The biological potential of such gradients is demonstrated by an example of cellproliferation tests. Such gradients could be further used in various fields in which roughness plays an important role, such as friction or adhesion studies.
■
EXPERIMENTAL SECTION
Cleaning Procedures. Silicon wafers (⟨100⟩ orientation, Si-mat, Germany) were coated with 15 nm of TiO2 by magnetron sputtering (PSI Villigen, Switzerland). The substrates were then ultrasonicated in toluene (puriss. p.a., ≥ 99.7%, Fluka Chemicals, Germany) for 20 min, exchanging the solvent after 10 min, and ultrasonicated again for 20 min in 2-propanol (ACS grade, min 99.8%, Fluka Chemicals, Germany), exchanging the solvent after 10 min. The samples were subsequently blown dry using nitrogen (5.0 purity). The samples were oxygen-plasma-treated in a vacuum chamber (PDC-32G, Harrick Scientific, Pleasantville, NY). The substrates were placed in between the coils before the chamber was evacuated down to 2.5 × 10−2 mbar. The chamber was flooded with oxygen and kept at a constant pressure of ∼3 × 10−2 mbar before the plasma was ignited at the highest level (100 W) for 120 s. Surface-Charge Inversion. The cleaned samples were immersed for 1 h in a 1 mg·mL−1 polyethylenimine (PEI) (branched, high molecular weight, Sigma-Aldrich, USA) solution in ultrapure water (TKA GenPure, Germany). The substrates were then rinsed using ultrapure water and blown dry using a jet of nitrogen. The samples were then used within the following few hours. B
DOI: 10.1021/acs.langmuir.5b01089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 2. Particle-density gradient: (a) Light-microscopy images taken every 900 μm along the density gradient. Scale bar is 200 μm. (b) Measured particle density along the gradient. (c) Adsorption kinetics derived from the gradient preparation. The particle adsorption kinetics is clearly proportional to the square root of the immersion time, confirming that the adsorption follows the RSA deposition model. The two significant digits shown represent the number of particles a 10 μm diameter cell would sense at any position on the gradient. Gemini 1530 FEG SEM (Zeiss, Germany) with both InLens and SE2 detectors. The samples were coated with 3 nm of platinum to avoid any charging effects from the electron beam in the top-view images. To take cross-sectional images, we placed the samples in a cross-section holder (holder G301S, Plano, Germany) and coated them for a second time with 3 nm of platinum. For the cross-sectional images the stage was tilted by 5° to improve image quality. Gradient Replication, Ti Coating. Replication of the 2-D gradient was performed, adapting a previously employed technique.27,28 First, a negative of the roughness pattern was formed using a dental-replica material based on polyvinylsiloxane (PVS, PROVIL novo light, Heraeus-Kulzer, Switzerland). Then, the negative was placed overnight in toluene to dissolve any PS particles that had been removed from the substrate and remained trapped inside the replica material and dried until it regained its initial shape. Subsequently, the two components of the epoxy (EpoTek 301, Epoxy Technology, Polyscience AG, Switzerland) were mixed in the ratio 5:1, as described by the manufacturer. The mixture was then degassed in a centrifuge at 4000 rpm for 10 min, cast into the negative, and cured for at least 1 h at 65 °C. (See Figure 1c.) The positives were then separated from the negatives and coated with 60 nm of titanium by magnetron sputtering (PSI Villigen, Switzerland) to mimic a bone-implant surface. Cell Culture. MC3T3-E1 cells were cultured in 75 cm2 flasks in complete Dulbecco’s modified Eagle’s medium (DMEM, 41966-029, Invitrogen, Switzerland), supplemented with 10% fetal bovine serum (heat inactivated, 10500-064, Invitrogen, Switzerland) and 1% antibiotics (Pen/Strep 15240-062, Invitrogen, Switzerland). After reaching 90% confluency, the cells were passaged by changing the media to a 0.25% (w/v) Trypsin−0.53 mM EDTA (Invitrogen, Switzerland) solution and incubated at room temperature until the cells detached from the flask (between 7 and 10 min). A volume of 10 mL of culture medium was subsequently added to block the trypsin, and the cell suspension was then centrifuged for 5 min at 1500 rpm. After the medium was removed, the cells were resuspended in fresh growth medium. The titanium-coated replicas of the 2-D gradients were sterilized and surface-activated in an oxygen plasma (PDC-002, Harrick Scientific, Pleasantville, NY) at a pressure below 3 × 10−2 mbar on the “high” RF setting. The cells were seeded at a density of 3500 cells/ cm2 and incubated for 1, 2, 4, and 7 days at 37 °C in a humidified atmosphere with 5% CO2. Actin/Nuclei Staining of Cells and Fluorescence Microscopy. After cultivation, the cells were rinsed twice with phosphate-buffered saline (PBS, 00-3002, Invitrogen, Switzerland), prewarmed to 37 °C, then fixed with a 4% paraformaldehyde (PFA, 96%, extra pure, Acros Organics, Belgium) solution in PBS for 10 min and rinsed twice with
PBS. The cells were then permeabilized with a 0.5% Triton X-100 (Fluka Chemicals, Switzerland) solution in PBS. The cells were further exposed to a 3% bovine serum albumin (BSA, Sigma-Aldrich, USA) solution in PBS to block nonspecific binding sites. The cells were further rinsed with 0.1% BSA in PBS and incubated with Phalloidin 488 (Sigma, MO) for 30 min in a dark and sealed atmosphere. The cells were then again rinsed with 0.1% BSA in PBS, and the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 1:1000 dilution in PBS, ≥98%, Sigma-Aldrich, USA) for 15 min. Finally, the samples were rinsed twice with PBS and stored at 5 °C until use. Cell Counting from Fluorescence Images. The samples were placed on microscope slides and mounted with mounting medium (ibidi, Germany) to stabilize the fluorophores and prevent the sample from drying out. Images were taken with a fluorescence microscope (FM Axioscope 2, Zeiss, Germany) using a 10× objective (Archoplan, 10×, 0.25, Zeiss, Germany) and the filter sets for DAPI and Phalloidin (filter set no. 49, with λex= 365 nm and λem= 450 nm and filter set no. 10, with 450 < λex < 490 nm and 515 < λem < 565 nm, respectively). The gradient was measured taking an image every 2 mm, with the exposure time being held constant for each of the channels, keeping all of the other parameters constant throughout the whole experiment. The images taken with the DAPI channel were then further processed in ImageJ for cell counting. The nuclei of the cells were counted over the entire image, using the “Particle Analysis” tool of ImageJ, and the result and standard deviation were calculated for three different samples. The cell number divided by the area of the image will be further referred to as “cell density”. Cell Preparation for SEM. The gradients incubated for 4 and 7 days were prepared for SEM analysis. The samples were first washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich, USA) before fixing them in a modified Karnovsky solution (4% paraformaldehyde (PFA, Acros Organics, Belgium) and 2.5% glutaraldehyde (25% in water, Sigma-Aldrich, USA) solution in PBS) for 1 h. The samples were further washed twice with PBS before dehydration in ethanol/water mixtures. Steps for dehydration involved incubations for 30 min each in 50, 70, and 80% ethanol, followed by 1 h each in 90% and pure ethanol. Then, the samples were transferred into hexamethyldisilazane (HMDS, reagent grade, ≥99.9%, SigmaAldrich, USA) for 30 min and afterward left for drying overnight. The samples were finally coated with 3 nm of platinum before SEM imaging. The samples were further imaged, taking images at 1000× and 10 000× magnification over a grid spaced at 2 mm intervals along the two gradient axes. SEM Image Analysis. The images were analyzed with ImageJ. The 1000× magnification images were used to quantify the cell-area coverage. First, a band-pass filter was applied to remove features C
DOI: 10.1021/acs.langmuir.5b01089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 3. Particle-height gradient: (a) SEM micrographs showing the cross-section of the particles. The height decrease is clearly visible. Scale bar is 400 μm. (b) AFM measurement of the height H of the particles. The gradient region is located in the middle of the sample, with both the sides being homogeneous in particle height. Both insets show AFM images at 4 and 17 mm on the gradient, from which the particle height was extracted. (c) Volume of the particle Vpart as a function of UV exposure time according to Eq. 1. The particle was considered as a sphere without a cap; H was measured by AFM. The initial sphere diameter Hi was obtained from the regions where the sample was not exposed to UV. smaller than 5 pixels and bigger than 500 pixels. Then, the Smooth algorithm from ImageJ was applied and a threshold was manually set according to the cell’s shape. Then, the coverage area was measured using the “Particle Analysis” tool. Only features larger than 50 pixels, equivalent to a circle with 3 μm in diameter, were taken into account, to discard any feature originating from the roughness of the samples. (See Figure S1 in the SI.) The obtained area divided by the total image area will be further referred to as “cell-area coverage”. The 10 000× magnification images were used to monitor the actual feature density on each of the image spots. Particles were counted using ImageJ with similar settings as for the cell analysis but taking into account features down to 5 pixels for the “Particle Analysis” tool.
spherical upon UV exposure (See Figure 3 a.), a spherical cap being removed from the bottom (i.e., the part in contact with the titanium dioxide). The kinetics of this reaction were studied, and the SEM cross-section images enabled the construction of a shrinking model. The AFM data could be used to calculate the actual volume of the particles, considering that a spherical cap had been lost over time. (See Figure 3a.) The volume of the particle is the given by the formula
RESULTS AND DISCUSSION Particle-Density Gradient. The particle density along the gradient was shown to vary linearly when the sample was immersed with a dipping profile z(t) = 7.72 × 10−7 ·t2, as expected from diffusion-driven adsorption. (See Figure 2 a,b.) The immersion profile had previously been developed to produce linear gradients with other negatively charged particles. 2 From the particle-density measurement, the particle-adsorption kinetics could be extracted. The rate of adsorption is proportional to √t, where t is the immersion time. (See Figure 2c.) Such kinetics correspond to the “random sequential adsorption” (RSA) model,29,30 in which the particles adsorb electrostatically onto the surface and stick there tightly. A jamming limit is observed that is lower than the full coverage because the particles cannot reorganize after adsorption. This jamming limit depends not only on the size of the particles but also on the ionic double layer around the particles, which is influenced by the pH and ionic strength of the solution; therefore, the maximum coverage can be controlled by tuning the experimental conditions. The obtained particle densities range from 40 × 103 particles/mm2 down to 0, which means that cells with a diameter of 10 μm would actually cover 3.5 particles on average at the rough end of the gradient and none at the smooth end of the gradient. Particle-Height Gradient. The PS particles shrink when exposed to UV light for longer than a critical length of time. (See Figure 3.) The shape of the particle does not remain
where Hi is the initial height of the particles prior to shrinking and H is their height after a given UV exposure time. The linear relationship between the remaining volume and the UV exposure time can clearly be seen on Figure 3c. The shrinking of the PS particles on TiO2 is due to the generation of radicals by the TiO2 layer under UV exposure.31 The radicals oxidize all adjacent organic material and therefore oxidize the base of the particles, causing them to decrease in height. The short range of the radicals in air prevents isotropic shrinkage of the particles. The delay in particle shrinking could be due to an initial period during which first the PEI layer is oxidized, prior to the particle being affected. The steepness and the extension of the gradient can be influenced by adjusting the speed at which the shutter is withdrawn. Replication. It is important to be able to replicate the roughness gradient, in order to conduct biological experiments on a large number of samples that have exactly the same roughness properties and constant surface chemistry. Such techniques have been previously introduced,9,27,28 where masters with long-term stability were used for negative fabrication. In the case of PS particles, the electrostatic interactions and van der Waals forces that are responsible for the particles sticking to the surface are not sufficiently strong to prevent particle transfer from the master to the negative material. Epoxy cast into a PVS mold incorporating transferred particles would not replicate all roughness features from the master. To remove the particles from the PVS, we introduced one further step to dissolve the remaining particles and thus to
Vpart =
■
D
3 2 4 ⎛⎜ Hi ⎞⎟ π⎛H H⎞ ⎛ H⎞ π − ⎜ i − ⎟ · ⎜H i + ⎟ 3 ⎝2⎠ 3⎝ 2 2⎠ ⎝ 2⎠
(1)
DOI: 10.1021/acs.langmuir.5b01089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 4. Particle-feature replication. (a,b) Light microscopy images of a master and its epoxy replica at the same spot. The features are clearly visible on the epoxy replica and coincide with the particles on the master. The arrows show the exact same features on both pictures, with two neighboring particles being replicated and resolved in the replica. (c) SEM micrographs of the epoxy replica before titanium coating along the particle-height gradient showing the shape of the features after replication. This technique does not allow the replication of a completely spherical feature. Scale bar is 500 nm.
Figure 5. Cell density after 7 days of culture. (a−e) Fluorescence microscopy of the MC3T3 cells stained with DAPI and for the actin fibers. The images shown were taken every 4 mm along the particle-density gradient marked in panel f. The increase in cell density is clearly visible and representative of the increase in cell density along this direction of the gradient. Scale bar is 500 μm and is similar for all images. (f) Normalized cell density over the full 2-D gradient. There is a clear effect of particle density on the cell density after 7 days. Little effect of the particle height on the number of cell is visible. The line along which the fluorescence microscopy images in panels a to e were taken is denoted in red.
generate exact replicas of the master. (See Figure 4 a,b.) Such a replication process does not allow the replication of under-cut structures and therefore generates circular bases on the roughness features. (See Figure 4 c.) This replication process allows for a limited number of replicas, as the PVS negative starts to degrade after more than 12 epoxy replicas. Cell Response − Cell Density and Area Coverage. The cell density after 2 days was found to be homogeneous over the
whole area, with no significant variation. (See Figure S2 in the SI.) After 2, 4 (see Figure S3 in the SI), and 7 days, the cell density was monitored with fluorescence microscopy and DAPI staining and showed a clear increase in cell density with particle density. (See Figure 5.) Few cells were counted in the area with low particle density, while a cell density of 79 800 ± 4500 cells per cm2 was observed in the high-particle-density areas after 7 days. This behavior, using 500 nm diameter particles, is similar E
DOI: 10.1021/acs.langmuir.5b01089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 6. (a−d) SEM images along the particle-height gradient. The images were taken every 4 mm, with increasing particle height going from top to bottom. The SEM images are representative of cell coverage along this direction of the gradient. The images were taken along the red line shown in panel e, at high particle density, the exact intersections being shown with the corresponding letters. Scale bar is 200 μm. (e) Cell area coverage on the 2-D gradient. A clear trend is visible showing higher cell area coverage in lower particle-height areas, independently of the feature density.
■
to that observed by Künzler et al.3 in previous studies on micrometer-range roughness. The cell area coverage after 7 days was assessed by SEM and is shown in Figure 6. The cell-area coverage seems to depend much more strongly on feature height than on feature densitycompletely opposite behavior to that of the cell density. While the previous graph showed that few cells were observed at low particle density, the cells still covered a high surface area according to the SEM measurement. This shows that the MC3T3 cells spread differently, depending on substrate roughness. While high roughness-feature density was necessary to achieve high cell density, the low height of the roughness features enabled the cells to spread more extensively over the surface.
ASSOCIATED CONTENT
S Supporting Information *
Cell-coverage estimation from SEM images and normalized cell density after 2 and 4 days of culture. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b01089.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: +41 44 633 10 27. Present Address #
C.Z.: Sonova AG, Laubisrütistrasse 28, 8712 Stäfa, Switzerland.
■
Notes
CONCLUSIONS We have presented a new method for the production of novel roughness feature-size gradients, which has the potential to facilitate high-throughput studies. These gradients combine both a change in feature density as well as a change in feature size within a single sample. An adapted method to generate replicas was also developed, which enables the production of samples with identical roughness gradients, whose chemistry can further be controlled through coating. Such gradients can be useful in many areas where roughness plays an important role, such as biology, friction, and adhesion. Primary results with bone cells showed a significant increase in cell proliferation toward the high-particle-density end of the gradient, while the cell-area coverage increased when the particle height was reduced. This implies that both feature density and height influence cellular development.
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The Electron Microscopy Center, ETH Zurich, and especially Peter Wägli are greatly acknowledged for their skillful SEM support. Stefanie Guimond-Lischer is acknowledged for her help with cell-preparation for SEM imaging. The Swiss National Science Foundation is gratefully acknowledged for funding.
■
REFERENCES
(1) Brunette, D. M.; Chehroudi, B. The effects of the surface topography of micromachined titanium substrata on cell behavior in vitro and in vivo. J. Biomech. Eng. 1999, 121 (1), 49−57. (2) Huwiler, C.; Kunzler, T. P.; Textor, M.; Vörös, J.; Spencer, N. D. Functionalizable Nanomorphology Gradients via Colloidal SelfAssembly. Langmuir 2007, 23 (11), 5929−5935.
F
DOI: 10.1021/acs.langmuir.5b01089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
human mesenchymal cell responses. J. Biomed. Mater. Res., Part A 2012, 100A (10), 2695−2709. (22) Dalby, M. J.; Gadegaard, N.; Oreffo, R. O. C. Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate. Nat. Mater. 2014, 13 (6), 558−569. (23) Low, S. P.; Williams, K. A.; Canham, L. T.; Voelcker, N. H. Evaluation of mammalian cell adhesion on surface-modified porous silicon. Biomaterials 2006, 27 (26), 4538−4546. (24) Genzer, J.; Bhat, R. R. Surface-Bound Soft Matter Gradients. Langmuir 2008, 24 (6), 2294−2317. (25) Ross, A. M.; Lahann, J. Surface engineering the cellular microenvironment via patterning and gradients. J. Polym. Sci., Part B: Polym. Phys. 2013, 51, 775. (26) Hulander, M.; Lundgren, A.; Faxälv, L.; Lindahl, T. L.; Palmquist, A.; Berglin, M.; Elwing, H. Gradients in surface nanotopography used to study platelet adhesion and activation. Colloids Surf., B 2013, 110 (0), 261−269. (27) Wieland, M.; Chehroudi, B.; Textor, M.; Brunette, D. M. Use of Ti-coated replicas to investigate the effects on fibroblast shape of surfaces with varying roughness and constant chemical composition. J. Biomed. Mater. Res. 2002, 60 (3), 434−444. (28) Schuler, M.; Kunzler, T. P.; de Wild, M.; Sprecher, C. M.; Trentin, D.; Brunette, D. M.; Textor, M.; Tosatti, S. G. P. Fabrication of TiO2-coated epoxy replicas with identical dual-type surface topographies used in cell culture assays. J. Biomed. Mater. Res., Part A 2009, 88A (1), 12−22. (29) Evans, J. W. Random and cooperative sequential adsorption. Rev. Mod. Phys. 1993, 65 (4), 1281. (30) Hinrichsen, E. L.; Feder, J.; Jøssang, T. Geometry of random sequential adsorption. J. Stat. Phys. 1986, 44 (5), 793−827. (31) Sato, Y.; Uebayashi, A.; Ito, N.; Kamiyama, T.; Shigesato, Y. High Rate Deposition of Photocatalytic TiO2 Films by dc. J. Vac. Sci. Technol. 2008, 26, 903−907, DOI: 10.1116/1.2870226.
(3) Kunzler, T. P.; Drobek, T.; Schuler, M.; Spencer, N. D. Systematic study of osteoblast and fibroblast response to roughness by means of surface-morphology gradients. Biomaterials 2007, 28 (13), 2175−2182. (4) Kunzler, T. P.; Huwiler, C.; Drobek, T.; Vörös, J.; Spencer, N. D. Systematic study of osteoblast response to nanotopography by means of nanoparticle-density gradients. Biomaterials 2007, 28 (33), 5000− 5006. (5) Flemming, R. G.; Murphy, C. J.; Abrams, G. A.; Goodman, S. L.; Nealey, P. F. Effects of synthetic micro- and nano-structured surfaces on cell behavior. Biomaterials 1999, 20 (6), 573−588. (6) Morgenthaler, S.; Zink, C.; Spencer, N. D. Surface-chemical and -morphological gradients. Soft Matter 2008, 4 (3), 419−434. (7) Faia-Torres, A. B.; Guimond-Lischer, S.; Rottmar, M.; Charnley, M.; Goren, T.; Maniura-Weber, K.; Spencer, N. D.; Reis, R. L.; Textor, M.; Neves, N. M. Differential regulation of osteogenic differentiation of stem cells on surface roughness gradients. Biomaterials 2014, 35 (33), 9023−9032. (8) Bruinink, A.; Bitar, M.; Pleskova, M.; Wick, P.; Krug, H. F.; Maniura-Weber, K. Addition of nanoscaled bioinspired surface features: A revolution for bone related implants and scaffolds? J. Biomed. Mater. Res., Part A 2014, 102 (1), 275−294. (9) Zink, C.; Hall, H.; Brunette, D. M.; Spencer, N. D. Orthogonal nanometer-micrometer roughness gradients probe morphological influences on cell behavior. Biomaterials 2012, 33 (32), 8055−8061. (10) Michelmore, A.; Clements, L.; Steele, D. A.; Voelcker, N. H.; Szili, E. J. Gradient Technology for High-Throughput Screening of Interactions between Cells and Nanostructured Materials. J. Nanomater. 2012, 2012, 7. (11) Kolind, K.; Leong, K. W.; Besenbacher, F.; Foss, M. Guidance of stem cell fate on 2D patterned surfaces. Biomaterials 2012, 33 (28), 6626−6633. (12) Zhao, G.; Raines, A. L.; Wieland, M.; Schwartz, Z.; Boyan, B. D. Requirement for both micron- and submicron scale structure for synergistic responses of osteoblasts to substrate surface energy and topography. Biomaterials 2007, 28 (18), 2821−2829. (13) Lavenus, S.; Trichet, V.; Le Chevalier, S.; Hoornaert, A.; Louarn, G.; Layrolle, P. Cell differentiation and osseointegration influenced by nanoscale anodized titanium surfaces. Nanomedicine 2012, 7 (7), 967− 980. (14) McNamara, L. E.; McMurray, R. J.; Biggs, M. J. P.; Kantawong, F.; Oreffo, R. O. C.; Dalby, M. J. Nanotopographical Control of Stem Cell Differentiation. J. Tissue Eng. 2010, 1 (1), 120623. (15) Tsimbouri, P.; Gadegaard, N.; Burgess, K.; White, K.; Reynolds, P.; Herzyk, P.; Oreffo, R.; Dalby, M. J. Nanotopographical Effects on Mesenchymal Stem Cell Morphology and Phenotype. J. Cell. Biochem. 2014, 115 (2), 380−390. (16) Wang, P.-Y.; Clements, L. R.; Thissen, H.; Jane, A.; Tsai, W.-B.; Voelcker, N. H. Screening Mesenchymal Stem Cell Attachment and Differentiation on Porous Silicon Gradients. Adv. Funct. Mater. 2012, 22 (16), 3414−3423. (17) Fu, J.; Wang, Y.-K.; Yang, M. T.; Desai, R. A.; Yu, X.; Liu, Z.; Chen, C. S. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 2010, 7 (9), 733−736. (18) Sjöström, T.; Dalby, M. J.; Hart, A.; Tare, R.; Oreffo, R. O. C.; Su, B. Fabrication of pillar-like titania nanostructures on titanium and their interactions with human skeletal stem cells. Acta Biomater. 2009, 5 (5), 1433−1441. (19) Kim, D.-H.; Han, K.; Gupta, K.; Kwon, K. W.; Suh, K.-Y.; Levchenko, A. Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients. Biomaterials 2009, 30 (29), 5433−5444. (20) Yim, E. K. F.; Darling, E. M.; Kulangara, K.; Guilak, F.; Leong, K. W. Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells. Biomaterials 2010, 31 (6), 1299−1306. (21) Mozumder, M. S.; Zhu, J.; Perinpanayagam, H. Titaniapolymeric powder coatings with nano-topography support enhanced G
DOI: 10.1021/acs.langmuir.5b01089 Langmuir XXXX, XXX, XXX−XXX
