Patterning of the surface termination of ultrananocrystalline diamond films for guided cell attachment and growth

Surface & Coatings Technology 321 (2017) 229–235

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Patterning of the surface termination of ultrananocrystalline diamond films for guided cell attachment and growth Alexandra Voss a, Silviya R. Stateva b, Johann Peter Reithmaier a, Margarita D. Apostolova b, Cyril Popov a,⁎ a Institute of Nanostructure Technologies and Analytics (INA), Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany b Roumen Tsanev Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bontchev Str., bl. 21, 1113 Sofia, Bulgaria

a r t i c l e

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Article history: Received 3 March 2017 Revised 24 April 2017 Accepted in revised form 25 April 2017 Available online 26 April 2017 Keywords: Guided cell attachment Ultrananocrystalline diamond films Surface modification Patterning

a b s t r a c t The surface patterning of a biomaterial, i.e. placing areas with different surface chemistries next to each other is an important step for various biomedical and biotechnological applications, e.g. in biosensor technology, drug screening or tissue engineering. In the current work we investigated the patterning of the surface termination of ultrananocrystalline diamond (UNCD) films prepared by microwave plasma chemical vapor deposition. In order to achieve a high wettability contrast in the neighboring areas O- and F-terminations were selected. The sequence of the modifications and the feasibility of different masking techniques were tested in advance. Based on the achieved results the following process of surface patterning was applied. Initially the whole UNCD surface was modified by UV/O3 treatment rendering the surface O-terminated and strongly hydrophilic (contact angle against water of 11°). The partial masking of this surface before the second modification was achieved with a gold hard mask photolithographically structured and wet etched in form of a grid. After the second modification with CHF3 plasma which provided F-termination and hydrophobic character of the surface (contact angle N 110°), the gold layer was removed. The results of the patterning of the surface termination were visualized by scanning electron microscopy revealing different contrasts of the areas with different terminations. Finally, human SH-SY5Y neuroblastoma cells were plated on the patterned UNCD surfaces and they exhibited preferential attachment and growth on the hydrophilic grid. © 2017 Elsevier B.V. All rights reserved.

1. Introduction The surface patterning is an important step for various biomedical and biotechnological applications where areas with different surface chemistries have to be placed next to each other, e.g. in biosensors, by drug screening or tissue engineering [1–3]. Tailored surface chemistry could be also applied to define channels in microfluidic devices or to guide the outgrowth of cells. The latter one would be of interest, for example, for application in multielectrode array (MEA) for activity studies in order to place neurons directly on top of the electrodes without mechanical manipulation. Additionally the axonal outgrowth could be controlled and predefined network patterns realized. Using the knowledge on the surface properties influencing the cell adhesion, such as roughness, wettability, polarity, zeta potential, etc. [4], there is a possibility

⁎ Corresponding author. E-mail address: [email protected] (C. Popov).

http://dx.doi.org/10.1016/j.surfcoat.2017.04.066 0257-8972/© 2017 Elsevier B.V. All rights reserved.

to guide the cell attachment and outgrowth by defining surface areas with lower or higher cell adhesion ability. Depending on the property to be influenced there is a vast number of possible mask or maskless structuring methods available: micro-contact printing, photolithography, jet patterning, plasma etching, grafting of biomolecules, and nanoparticle deposition to name only a few. The effects achieved differ according to the pattern size [4]. By micro-patterning structures larger or in the range of the cell sizes are created. The pattern effects differ by cell types, the preferential adhesion of one cell type over others can be induced, but also the culturing times vary because the cells have to adjust to the pattern. Nano-patterning is used to control the cell behavior by ligand patterning with different types, amount, spacing or distribution. Especially nano-fibers are considered more advantageous for cell performance than the other irregularities because they bear better resemblance to the natural extracellular matrix (ECM) structure [4]. All above considerations are truly valid and applicable also when diamond in a bulk form or as films of various crystallinity (poly-, nano- or ultrananocrystalline) is used as a biomaterial. Diamond has already revealed its potential for diverse biotechnological and biomedical

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applications due to a combination of remarkable physical and chemical properties combined with biocompatibility [5–7]. Most studies on surface patterning of diamond for bio-applications rely on structuring of the tailored surface chemistry, i.e. of the surface termination, or of the applied layer of adhesion proteins. The surface of nanocrystalline diamond (NCD) films was patterned using conventional photolithography into H- and O-terminated areas [8]. The subsequent photochemical modification with amine and attachment of green fluorescent protein revealed the successful patterning applied further for immobilization of the enzyme catalase and final realization of a biosensor. Guided outgrowth of cells on diamond was first presented by Specht et al. [9]. Micro-contact printing was applied to prepare defined structures of laminin on the surface of monocrystalline diamond. Cortical mouse neurons seeded on such surfaces attached preferentially on the laminin pattern which was followed by ordered growth of neurites along it. Another approach for patterning of the adhesion protein was using laser micro-machining to structure a poly-Dlysine film on top of boron-doped polycrystalline diamond (PCD) layer. The patterning resulted in successful guidance of rat cortical neurons although some axons extended the designated areas [10]. Further scanning electron microscopy studies showed that the laser beam not only removed the protein layer but also the diamond film exposing partially melted silicon substrate [11]. A different approach applied a monolayer coating of nanodiamonds (NDs) which was lithographically structured and used for growth of patterned neuronal networks [12]. Patterning of the surface chemistry to guide the adhesion of osteoblasts was demonstrated be Rezek et al. using photolithography to create patterns of hydrogen and oxygen termination on nanocrystalline diamond (NCD) films [13]. The cells arranged selectively on the oxidized diamond patterns. Furthermore, preferential adsorption of fetal bovine serum (FBS) from the culture medium on O-terminated areas was shown. Although FBS was adsorbed on the whole NCD surface the protein layer was significantly thicker on the oxidized fields due to their hydrophilic and polar character. This additionally supported the guided cell adhesion. The adhesion properties of human osteosarcoma cells onto boron-doped PCD layers as a function of their surface H- or O-termination patterned by UV/ozone exposure through a copper grid were investigated by Marcon et al. [14]. Also in this case the cells colonized preferentially on the hydrophilic oxidized areas. Recently Mertens et al. presented the surface patterning of ultrananocrystalline diamond (UNCD) films with hydrophilic and hydrophobic areas applying photolithography and plasma techniques [15], however, the influence of this pattern on cell adhesion properties was not studied. All these works reveal the potential for application of diamond and diamond films with patterned surface in fundamental cell biology, tissue engineering, biosensors and biomedical devices. In the current work we demonstrate the lithographic patterning of UNCD surface with O- and F-terminated areas and the application of the obtained grid pattern for guided cell attachment and growth without application of adhesion proteins. The applied human SH-SY5Y neuroblastoma cells can be used as in vitro models of neuronal function and differentiation and their behavior on the patterned UNCD surface can derive information about the possibility for creation of neuronal networks. 2. Experimental 2.1. Deposition and surface modifications of UNCD films Ultrananocrystalline diamond layers (UNCD) were prepared by microwave plasma assisted chemical vapor deposition (MWCVD) on glass substrates (Corning Eagle 2000) from 17 vol% CH4/N2 gas mixture. The output MW plasma power was 800 W, the working pressure 2.5 kPa (25 mbar), the substrate temperature 560 °C and the deposition duration 60 min. In order to enhance the primary nucleation density all substrates were pre-treated ultrasonically prior to the deposition in a

commercially available Opal Seeds slurry (Adámas Nanotechnologies, Raleigh, North Carolina). The latter is a DMSO-based dispersion containing nanodiamonds of 20–30 nm size with a weight portion of 0.5%. The ultrasonic treatment in this slurry was performed for 30 min. The intrinsic hydrogen termination of the as-grown UNCD layers was changed by two processes: • UV/O3 treatment in a fully closed chamber using ambient air at atmospheric pressure without control of the humidity. An UV lamp with an input power of 600 W emitting wavelengths (e.g. 185 nm) necessary for ozone formation was placed 4 cm above the UNCD films. The treatment time was 10 min. • CHF3 plasma modification in an Oxford Plasmalab 100 ICP set-up with the following process parameters: rf power of 50 W, CHF3 flow of 25 sccm, working pressure of 3.33 Pa (25 mTorr). The duration of the modification was 30 s. For different applications, like reusability and patterning, it is of interest to replace an already introduced surface termination with another one. Especially varying between hydrophilic and hydrophobic surfaces is of relevance for the current work. To test the alternation of the surface chemistry UNCD films modified via UV/O3 treatment were subjected to trifluoromethane plasma modification at the parameters presented above and vice versa. In both cases as-grown samples were modified simultaneously and used as controls. Optical microscopy (Leica DMR) was implemented to control the general quality of the films, the transparency of UNCD/glass samples and the results of different lithography steps. AFM measurements were performed using DualScope™ 95 SPM System (DME) in a tapping mode to investigate the topography and the roughness of the as-grown and modified UNCD surfaces; for analysis of the images Gwyddion software was used. Scanning electron microscopy (SEM, Hitachi S-4000) was applied for imaging of the surface of UNCD films after growth and different modifications. Besides the morphology also areas of different surface terminations can be observed by SEM due to their different brightness contrasts resulting from the specific electronic band structure at the diamond surface and different surface electrical properties, respectively. The wettability of UNCD films with different surface terminations was determined by contact angle measurements with a CAM 100 Contact Angle Meter. Volumes of about 1 μl deionized water were placed onto the sample surface and pictures of the resulting drop were taken in an interval of 100 ms. From these pictures the contact angles at the left and right side of the drop profile were calculated by adjusting the parameters of the Young-Laplace equation. For each sample at least four drops were measured. The arithmetic mean of the second to fifth pictures after separation of the drop from the syringe was calculated for each drop. Afterwards the arithmetic mean and standard deviation of all drops per sample were calculated and given in this work. 2.2. Patterning of the surface modification with a gold mask UNCD surfaces with patterned modification were intended for direct contact cell tests in order to investigate guided cell attachment and growth. UNCD layers deposited on Corning Eagle and modified by UV/ O3 treatment were implemented for this purpose. Initially 50 nm of gold were evaporated (Balzers BAK 600) on top of them. In order to obtain the desired patterning, a negative photoresist AZ nLoF 2070 5:1 diluted with a resist thinner PGMA (AZ EBR Solvent, Microchemicals, Ulm, Germany) was applied on the Au film and the surface grid pattern was transferred in it using photolithography (Karl Süss MA4). In the following the gold was etched for 13 min in diluted KI/I2 solution, which resulted in a well-formed grid to be used as a mask for the second modification step after the removal of the photoresist with NMP at 80 °C for about 5 min. However, beginning of under-etching was detected at several places of the grid due to the long etching time. Since we attributed the

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Fig. 1. Flow diagram of the surface modification patterning process with a gold hard mask using a negative photoresist. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

observed problem to any residual photoresist on Au after the development, two additional tests were performed to establish this. In the first one longer resist development time was applied, 2.5 min instead of 2 min, which, however, did not lead to any improvement of the grid quality. In the second one, an additional oxygen plasma ashing step for 1 min at 100 W was included before the wet etching of Au. It significantly improved the hard mask quality; after only 30 s of KI/I2 etch all windows were opened. The second modification step was performed via CHF3 plasma with the process parameters given above. Afterwards the gold mask was removed with a Scotch tape rolling it carefully over the surface without applying any pressure. As a result it was possible to remove the gold mask without significant residues of the tape's glue on the UNCD surface. The process for patterning of the UNCD surface termination with a negative photoresist is shown schematically in Fig. 1.

3. Results and discussion 3.1. Brief summary of the surface properties of as-grown and modified UNCD films The deposited UNCD films were smooth, uniform and exhibited the typical surface topography composed of rounded structures with

2.3. Cell culture tests UNCD thin films patterned with a rectangular grid of hydrophilic UV/O3 treated lanes (width of 30 μm) and hydrophobic F-terminated squares (side of 200 μm) were used for plating of SH-SY5Y cells. This cell line is often applied as an in vitro model for neuronal function and differentiation. Human SH-SY5Y neuroblastoma cells were obtained from Sigma-Aldrich (94030304, [16]). The cells were plated on the UNCD samples with a density of 1 × 105 cells/cm2 and cultured in OptiMEM media (Thermo Scientific), supplemented with 10% FBS (Lonza). Afterwards they were washed with PBS before fixation with 3.7% buffered paraformaldehyde for immunofluorescence staining. Single or double fluorescence cell labeling was performed as described by Apostolova et al. [17]. A NeuN (Merck) monoclonal antibody, clone A60, which specifically recognizes the DNA-binding neuron-specific protein NeuN was used. In the final step, the cells were washed three times with PBS for 5 min and incubated for 40 min with an appropriate secondary antibody labeled with AlexaFluor-488 (Invitrogen, USA). Factin was detected using AlexaFlour 568 Phalloidin (Invitrogen, USA). Following three washes with PBS and two with water, the slides were mounted in UltraCruz fluorescence mounting medium with DAPI (CantaCruz Biotechnology, USA). Fluorescence microscopy was performed with a Carl Zeiss AM240 microscope equipped with an Andor (iXon+) camera.

Fig. 2. SEM (top) and AFM (bottom) images of UNCD surface after growth.

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Table 1 Rms roughness (AFM), surface composition (XPS) and wettability (contact angle measurements) of as-grown and modified UNCD films. UNCD surface

Rms roughness

as-grown UV/O3 CHF3 plasma

nm 12.0 11.5 11.0

Surface composition O

N

F

at.% 1.6 7.1 2.6

at.% 0.6 0.7 0.9

at.% – – 22.0

Contact angle

Reference

deg 70° ± 1° 11° ± 1° 112° ± 2°

[19] [19] [20]

diameters of several hundred nanometers consisting of substructures (Fig. 2). As discussed in a previous work this topography combined with respective surface termination is advantageous for fast and strong cell attachment on UNCD [18]. The rms roughness as determined by AFM was in the order 12–13 nm for the as-grown UNCD films. The UV/O3 treatment did not result in a change of the surface topography; the rms roughness values remained in the ranges of the as-grown samples [19]. In case of CHF3 plasma modification an etching rate of 0.08 nm/s was established in a former work which means that several atomic layers were etched during the modification of 30 s [20]. The as-grown UNCD surface is hydrogen-terminated, likewise any CVD diamond surface [21]. In order to alter this stable surface

Fig. 3. Optical image of the gold grid mask on transparent UNCD/glass substrates used for patterning (top) and the respective SEM micrograph of the patterned termination after the second modification (CHF3 plasma) and hard mask removal (bottom). The side of the F-terminated squares is 200 μm, the width of the O-terminated lanes 30 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

termination, processes with high activation energy, e.g. plasma or photochemical, are required. Previous experiments have shown that O-termination can be achieved by O2 plasma or UV/O3 treatment while Ftermination with CHF3 or SF6 plasma. The modifications resulted in a change of the surface composition and wettablility. The surface compositions, rms roughnesses and contact angles against water of as-grown and modified UNCD films are summarized in Table 1. The alternation of the surface termination was investigated by CHF3 plasma modification of UV/O3 treated UNCD films and vice versa. Rendering a hydrophilic surface hydrophobic by applying CHF3 plasma was rather successful resulting in contact angles against water of 118° ± 1° which is even slightly higher than the contact angle after one step fluorination of as-grown films. Starting with fluorine surface termination after UV/O3 treatment the contact angles could be lowered only by about 20° (91° ± 2°) and still were in the hydrophobic regime. These results suggest to start the surface modification patterning with a hydrophilic treatment (UV/O3) followed by a superposed hydrophobic (CHF3 plasma) treatment. 3.2. Patterning of the surface termination of UNCD films The very first experiments with patterning of the surface termination were performed using mechanical masks, such as TEM copper

Fig. 4. Phase contrast images of SH-SY5Y cells grown on UNCD with patterned surface termination (rectangular grid structure with hydrophilic O-terminated lines and hydrophobic F-terminated squares) 3 days after plating (A, C) and 7 days after plaiting (B, D). Magnification 100 × (A, B) and 200 × (C, D). Scale bar: 100 μm. Three independent batches of cultures and coatings were tested.

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meshes. Starting from an as-grown surface the UNCD film was modified via UV/ozone treatment using a Cu mesh as a mask. A negative image of the mesh was observed by SEM due to the different surface electric properties of areas with different terminations. As-grown hydrogen termination appeared bright whereas the oxidized surface was less surface conductive and therefore dark. However, parts of the pattern were blurry due to partial modification beneath the mask. This was due to one of the major drawbacks of the mechanical masks – they bend easily leading to some areas standing away from the surface. By the application of a mechanical mask care has to be taken for its flat positioning very close to the surface, together with the exact placement with respect to the sample. As a second approach photolithography was applied with photoresist AZ 1518 as a mask followed by a plasma modification second step. This approach also showed itself not feasible because the resist hardened by the plasma to an extent where subsequent removal was not possible without using aggressive chemicals. The chemical treatment corrupted the just achieved surface termination. The above preliminary tests showed that thin film hard masks were needed for patterning of the surface modification of UNCD. Literature research came up with a process using a gold film and potassium iodide/iodine (KI/I2) etchant [22]. Because gold adheres only weakly to UNCD an additional titanium interlayer was applied to improve the gold adhesion. However, this introduces a second etching step since titanium is not etched by KI/I2. To remove the thin titanium adhesion layer concentrated hydrochloric acid (conc. HCl) has to be used. The main goal of the process developed in the current work for masking specific areas of the UNCD surface during the second modification was to decrease (or avoid completely) the contact of the freshly modified surface with chemical agents, especially with etchants, such as HCl. Organic solvents, e.g. acetone, i-propanol and ethanol, do not induce significant changes of the modified UNCD surfaces, as shown earlier [23]. The approach applied in the current work used the low adhesion of Au layers on UNCD surfaces if there is no titanium interlayer below. After processing and second modification the gold can be removed mechanically with a Scotch tape. The UV/O3 treated UNCD on Corning Eagle 2000 were coated with thin Au film (50 nm) on top of it. After the lithographic step the hard

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mask was structured by etching of the gold film with KI/I2. The etching time was stepwise increased starting with 80 s which were sufficient for gold etch of test UNCD on Si samples. In the current case, however, even after 3 min etching only few windows of the grid were opened. After additional 10 min of KI/I2 etch almost all windows were opened but under-etching has already started (not shown graphically). The main reason for the longer etch time might be any residual photoresist after the lithography. Due to the very low thermal conduction coefficient of Corning Eagle 2000 (or of glass in general) the post-exposure baking step was not as efficient as on other substrate materials, like silicon. This baking step is important for negative photoresists such as the AZ 2070 resist used in the current work. During this step the remaining solvent is evaporated and the cross-linking initiated by the UV exposure is completed. To eliminate the problems occurring due to the inefficient baking there are two possibilities: longer development or introduction of an additional plasma asher step to etch the residual resist. Two experiments were performed testing the influence of both parameters separately. A sample was treated with the described above recipe with a slightly longer resist development time of 2.5 min instead of 2 min. However, the increased development time did not lead to reduction of the etch time and to any improvement of the grid quality. The second sample was additionally treated with an oxygen plasma in a TePla plasma asher for 1 min at 100 W after photolithography and 2 min of resist development. This step dramatically improved the hard mask quality. After only 30 s of KI/I2 etch all windows were opened and there were only few defects visible over the whole sample (Fig. 3, top). The further processing lead to nice and clean surfaces with patterned termination after removal of the Au grid mask as revealed by SEM images (Fig. 3, bottom). 3.3. Guided cell growth on patterned surfaces UNCD thin films on Corning Eagle 2000 glass substrates patterned with a rectangular grid of hydrophobic F-terminated squares and hydrophilic O-terminated lanes were used for plating of SH-SY5Y neuroblastoma cells. The morphology of the cells growing on the patterned surfaces was analyzed by phase contrast and fluorescent microscopy.

Fig. 5. Ordered human SH-SY5Y neuroblastoma cells outgrowth on O-terminated diamond lines. The cells were fixed after 7 days in culture and stained for DNA (A, blue), specific neuronal marker Neu N (B, green) and F-actin (C, red). An overlay of the three images (D) shows, that the cells and neurites grow along the lines of the O-terminated grid. Scale bar: 100 μm. Three independent batches of cultures and coatings were tested. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Spontaneous neuronal differentiation of SH-SY5Y cells grown on UNCD surface with O-patterned surface modification on day 45 (A, B) and day 55 (C) after plating. The cell clusters (1 and 2) are mounds of undifferentiated cells (A, B, C). Immunostaining reveals neurons via the specific neuronal marker Neu N (B and C, green), while the cytoskeletal filaments of F-actin are stained in red (C) and the DNA in blue (C). Scale bars: 100 μm (A) and 50 μm (B and C). Three independent batches of cultures and coatings were tested. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In the case of O-terminated surfaces a different adhesion with respect to F-terminated ones was observed. The cell densities on O-terminated UNCD were higher than those on the F-terminated, which can be explained by the role played by the hydrophilic surface, promoting the cell adhesion. The cells tend to follow a preferential orientation when grown on hydrophilic O-terminated lanes. SH-SY5Y cells adhered and grew successfully, adopting a characteristic elongated morphology with extending processes in bipolar or tripolar configurations (Fig. 4A, C). On day 7 after plating a good arrangement of cells was observed on the hydrophilic O-terminated lanes (Fig. 4B, D). Notable neuronal outgrowth (day 7) could be detected via neuronalspecific immunoreactivity (Neu N) (Fig. 5, green). On the patterned substrates most of the cells exhibited a directional growth providing a network of parallel aligned axons. Based on all fluorescent images examined, the fraction of axons that followed the linear path was estimated to be ca. 75%. Axonal outgrowth and path finding were primarily governed by dynamic microtubule/F-actin interactions and corresponding filopodia/lamelipodia dynamics in response to the favorable O-terminated lanes (Fig. 5, red). The SH-SY5Y neuroblastoma cells cultured for 45 days on these substrates led to the ordered growth of mounds of undifferentiated cells on the UNCD surfaces, which expressed Neu N (Fig. 6A, B). In our experiments the cells were maintained in the presence of OptiMEM with 10% FBS, and the proteins present in this medium together with the expressed and released ECM proteins may adsorbed onto the surface and influence further the cell adhesion and survival, thus supporting the formation of mounds of undifferentiated cells much larger than the pattern. Furthermore, the axons tended to form direct connections which might be a result of their shortening as already observed in early studies on substrates with patterned protein coatings [24]. A longer culturing of the cells (day 55) enhanced the spontaneous differentiation of SH-SY5Y cells from the mounds, which then spread into the surrounding area (Fig. 6C). Those results for longer incubation revealed that the guidance patterns have to be designed for specific cell cultures. In the case of SH-SY5Y larger areas for the mounds would be preferable with narrow interconnections for the axons. Also non-rectangular shapes, like honeycomb structures, could be of interest because many cells extended in only three directions. 4. Conclusions In the current work we demonstrated the patterning of the surface termination of UNCD films applying lithography, a gold hard mask and two modification steps, namely UV/O3 treatment and CHF3 plasma. Special attention was paid to the reduction of the chemical steps for structuring and removal of the hard mask which could affect the surface termination already achieved on the UNCD surface. As a result areas with different surface chemistries and wettabilities were placed next to each other. Hydrophilic grid structures of O-terminated UNCD with hydrophobic F-terminated squares were applied for direct contact cell tests with SH-SY5Y cells which arranged during the first days after

plating predominantly on the grid pattern extending their axons along the hydrophilic lanes. Longer incubation (N45 days) showed that the UNCD is a favorable culture surface for spontaneous neural differentiation.

Acknowledgements The authors would like to acknowledge the financial support of the Deutsche Forschungsgemeinschaft (DFG) under the project PO 789/31. They are also very grateful to the German Academic Exchange Service (DAAD, Project ID 57085200) and the Bulgarian National Science Fund (DNTS - Germany 01/7, 03.09.2014) for the support of the bilateral cooperation. They thank Gergina Encheva for her technical support and assistance. References [1] R.S. Kane, S. Takayama, E. Ostuni, D.E. Ingber, G.M. Whitesides, Patterning proteins and cells using soft lithography, Biomaterials 20 (1999) 2363–2376. [2] T. Betancourt, L. Brannon-Peppas, Micro- and nanofabrication methods in nanotechnological medical and pharmaceutical devices, Int. J. Nanomedicine 1 (2006) 483–495. [3] X. Zhou, F. Boey, F. Huo, L. Huang, H. Zhang, Chemically functionalized surface patterning, Small 7 (2011) 2273–2289. [4] L. Bacakova, E. Filova, M. Parizek, T. Ruml, V. Svorcik, Modulation of cell adhesion, proliferation and differentiation on materials designed for body implants, Biotechnol. Adv. 29 (2011) 739–767. [5] L. Tang, C. Tsai, W. Gerberich, L. Kruckeberg, D. Kania, Biocompatibility of chemicalvapour-deposited diamond, Biomaterials 16 (1995) 483–488. [6] C. Nebel, B. Rezek, D. Shin, H. Uetsuka, N. Yang, Diamond for bio-sensor applications, J. Phys. D. Appl. Phys. 40 (2007) 6443–6466. [7] O. Auciello, P. Gurman, M.B. Guglielmotti, D.G. Olmedo, A. Berra, M.J. Saravia, Biocompatible ultrananocrystalline diamond coatings for implantable medical devices, MRS Bull. 39 (2014) 621–629. [8] A. Härtl, E. Schmich, J.A. Garrido, J. Hernando, S.C.R. Catharino, S. Walter, P. Feulner, A. Kromka, D. Steinmüller, M. Stutzmann, Protein-modified nanocrystalline diamond thin films for biosensor applications, Nat. Mater. 3 (2004) 736–742. [9] Ch.G. Specht, O.A. Williams, R.B. Jackman, R. Schoepfer, Ordered growth of neurons on diamond, Biomaterials 25 (2004) 4073–4078. [10] E.M. Regan, A. Taylor, J. Uney, A.D. Dick, P.W. May, J. McGeehan, Spatially controlling neuronal adhesion and inflammatory reactions on implantable diamond, IEEE JETCAS Conf. 1 (2011) 557–565. [11] P.W. May, E.M. Regan, A. Taylor, J. Uney, A.D. Dick, J. McGeehan, Spatially controlling neuronal adhesion on CVD diamond, Diam. Relat. Mater. 23 (2012) 100–104. [12] R.J. Edgington, A. Thalhammer, J.O. Welch, A. Bongrain, P. Bergonzo, E. Scorsone, R.B. Jackman, R. Schoepfer, Patterned neuronal networks using nanodiamonds and the effect of varying nanodiamond properties on neuronal adhesion and outgrowth, J. Neural Eng. 10 (2013), 056022. [13] B. Rezek, L. Michalíková, E. Ukraintsev, A. Kromka, M. Kalbacova, Micro-pattern guided adhesion of osteoblasts on diamond surfaces, Sensors 9 (2009) 3549–3562. [14] L. Marcon, C. Spriet, Y. Coffinier, E. Galopin, C. Rosnoblet, S. Szunerits, L. Heliot, P.-O. Angrand, R. Boukherroub, Cell adhesion properties on chemically micropatterned boron-doped diamond surfaces, Langmuir 26 (2010) 15065–15069. [15] M. Mertens, M. Mohr, K. Brühne, H.J. Fecht, M. Lojkowski, W. Swieszkowski, W. Lojkowski, Patterned hydrophobic and hydrophilic surfaces of ultra-smooth nanocrystalline diamond layers, Appl. Surf. Sci. 390 (2016) 526–530. [16] Sigma-Aldrich, SH-SY5Y cell line human, www.sigmaaldrich.com/catalog/product/ sigma/94030304 (09/12/2016). [17] M.D. Apostolova, M.G. Cherian, Delay of M-phase onset by aphidicolin can retain the nuclear localization of zinc and metallothionein in 3T3-L1 fibroblasts, J. Cell. Physiol. 183 (2000) 247–253.

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