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Phys. Status Solidi A 206, No. 6, 1348–1351 (2009) / DOI 10.1002/pssa.200881063 p s sapplications and materials science

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The hydrophobic properties of femtosecond laser fabricated spike structures and their effects on cell proliferation

Elena Fadeeva*, 1, Sabrina Schlie1, 2, Jürgen Koch1, Anaclet Ngezahayo2, and Boris N. Chichkov1

1 Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover, Germany 2 Institute of Biophysics, Leibniz University Hannover, Herrenhäuser Str. 2, 30419 Hannover, Germany

Received 18 March 2008, revised 6 January 2009, accepted 8 January 2009

Published online 17 March 2009

PACS 42.62.Be, 79.20.Ds, 81.20.Hy, 87.17.Rt

* Corresponding author: e-mail e.fadeeva@lzh.de

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1 Introduction The interactions between cells and biomaterial surface are of great importance in the field of biomedicine and tissue engineering. Several efforts were undertaken to control and guide cellular behaviour by changes in material properties such as chemistry and sur-face topography in order to create an ‘intelligent’ biomate-rial [1, 2]. With respect to various applications, it would be interesting to have biomaterials which could affect the cell behaviour in a cell specific manner within a tissue with various cell systems. In many tissues, the fibroblasts which participate to scar formation, adhere and proliferate on the implants more quickly than other cells such as neuronal cells [3, 4]. In tissues where the implant should make con-tact to specific cells e.g. neuronal cells, it would be of great importance to generate functionalised implants which would suppress the adhesion and the proliferation of the fib-roblasts while stimulating adhesion and proliferation of the neuronal cells. In the past, many studies have concentrated on the in-fluence of material topography on cellular behaviour. It was shown that different structures such as pores, grooves

and pits in micrometer and nanometer dimensions affect cell morphology, orientation, adhesion, and proliferation of cells [5, 6]. Several studies report that material hydro-philic/hydrophobic character depends on nano- and micro-scale surface topography [7]. Furthermore, it has been demonstrated that hydrophilic/hydrophobic materials in-fluence cellular adhesion and cytoskeletal organization. In assumption that the hydrophilic/hydrophobic character of surface could affect cell adhesion, it should be possible to control the cell type which would adhere on a culture sur-face by changing hydrophilic/hydrophobic properties of the material surface. We fabricated and used Si spike structures to study the influence of topography on cellular behaviour. Such topo-graphies can be easily fabricated by laser ablation of sin-gle-crystalline crystalline silicon (Si) in a reactive gas at-mosphere (SF6) [8, 9]. The produced structures were also used to produce negative replica in soft materials such as silicone elastomer [11]. The structuring is correlated with an increase in hydrophobic properties of material surface as was proposed by [7]. The spike structured surface was

Silicon (Si) spikes with an average tip surface of 1.4 µm2, an

average depth 5.9 µm and a spike-to-spike distance of 4.8 µm

have been produced with a commercial laser system which

delivers sub-30-fs pulses at 800 nm and a repetition rate of

1 kHz. The fabricated spikes can be used for the generation of

negative replica in soft material such as silicone elastomer.

Since the Si spikes were not destroyed during the replication

process, it is possible to reproduce ad libitum soft material

with the same spike configuration rendering comparison pos-

sible. The analysis of the wettability of the surfaces revealed

that the structuring induced an increase in hydrophobic char-

acter of Si as well as of silicone elastomer. This effect is re-

lated to the presence of air trapped in the microstructured sur-

face. Analysis of the effect of the structuring on the prolifera-

tion activity of fibroblasts and SH-SY5Y neuroblastoma cells

showed that the spike structures and its negative replicas in

silicone elastomer are able to suppress the proliferation of the

fibroblasts for 48 h but did not affect the neuroblastoma cells.

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able to reduce the proliferation of fibroblasts while it did not affect the proliferation of SH-SY5Y neuroblastoma cells. These results indicate a possibility to generate sur-faces which could control cellular proliferation in a cell specific manner.

2 Experimental procedures 2.1 Structure fabrication and characterisation

The surface topography for cell studies have been fabri-cated by irradiation of silicon (Si) with femtosecond laser pulses in a reactive gas atmosphere (SF6). This process transformed the flat silicon into an array of quasi-ordered conical spike structures. In our experiment we used an amplified Ti :Sapphire femtosecond laser system (Femtopower Compact Pro, Femtolasers Produktions GmbH, Austria), which delivers sub-30-fs pulses at 800 nm and a repetition rate of 1 kHz. The structures were fabricated on single-crystal p-type Silicon(110) wafers. By varying the laser fluences from 0.36 J/cm2 to 3.6 J/cm2, the number of laser pulses per sur-face area unit from 100 to 500, and the pressure of the re-active gas from 1 mbar to 500 mbar, it was possible to con-trol the spike height and periodicity. We found a spike-to-spike distance of 2 µm to 15 µm, and a spike height of 1 µm to 20 µm (Fig. 1). For the cell culture experiments, structures with an av-erage spike height of 5.9 µm, a spike-to-spike distance of 4.8 µm were produced. The average top flattening of the spikes was 1.4 µm2 (Fig. 2). Additionally, negative replicas of these spikes were produced using silicon as described by [10]. Shortly, a two component silicone elastomer MED-4234 from NuSil Sili-cone Technology was mixed in proportion of 10:1 accord-ing to product description profile. Afterwards a deaeration procedure in vacuum chamber was performed. Negatives were replicated directly from the spike structure described above so that the silicone structure was inverted to the Si relief (Fig. 3). 2.2 Contact angle measurements To investigate the effect of material surface topography on the wetting characteristics, contact angle measurements were per-formed using a video-based optical contact angle measur-ing system (OCA 40 Micro, DataPhysics Instruments GmbH, Germany). We measured the static contact angles for water by the sessile drop method with 2 µl drops at

Figure 1 Scanning electron microscope images of spike struc-

tures fabricated on silicon (Si) at different laser fluences.

Figure 2 Scanning electron microscope images of Si spike struc-

tures as used for cell experiments.

normal atmospheric conditions and a temperature of 20 °C. Every result has been averaged over at least five single measurements. 2.3 Cell proliferation on fabricated spike struc-tures In order to test the influence of the material surface on cellular behaviour, proliferation profiles of fibroblasts and SH-SY5Y neuroblastoma cells grown on the femto-second laser fabricated Si spikes as well as the silicone elastomer negative replica were studied. Non-structured silicone samples and glass slides served as references. Samples were placed in 24 well plates. Each well was filled with 2 ml of Dulbecco modified medium (DMEM; Sigma, Taufkirchen, Germany) supplemented with 10% foetal calf serum. The plates were placed into a cell culture incubator (Heraeus, Hanau, Germany), in which a 95%:5% air: CO2 atmosphere and 80% humidity were maintained. After a cultivation time of 48 h, cells were trypsinized and counted. For a better comparison between the experiments, the cell density was normalised on the seeding density at time 0 h (1.6 × 106 cells/ml). The results are given as average ±SEM for n = 4 experiments in per-cent.

3 Results and discussion 3.1 Structure fabrication The femtosecond laser system allows the generation of quasi-periodical spike structures in Si at a wide size range (Fig. 1). The spike ge-

Figure 3 Scanning electron microscope image showing a nega-

tive relief structure in silicone fabricated by moulding of Si spike

structures.

1350 E. Fadeeva et al.: Hydrophobic properties of femtosecond laser fabricated spike structures

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ometries can be controlled through process parameters. The technique offers the flexible fabrication of structures to test the effects on cell proliferation. Moreover, negative replica could be produced in soft material such as silicone elastomer (Fig. 3). Since the original Si surface is not al-tered by the generation of the replica, the technique offers a possibility to reproduce ad libitum similar surfaces so that comparisons of the effects on different cell systems and cell culture conditions can be undertaken. 3.2 The structure changed the hydrophobic character of the surfaces The wettability of a surface can be measured by estimation of the contact angle θ for a drop of water on surface. For angles between 0° and 90°, the material surface is considered as hydrophilic and for θ > 90° the material surface is considered as hydrophobic. As seen in the Table 1, Si is hydrophilic. The generation of spikes which changed the topography of the surface in-duced an increase of the contact angle (Table 1), rendering Si hydrophobic. The effect is related to the presence of air trapped in the microstructured surface. Silicone elastomer which is originally hydrophobic acquired a more hydro-phobic behaviour when structured by moulding of Si spikes. This, as well, can be explained by air inclusions in the negative replica relief.

3.3 The proliferation was effected by the struc-tures To analyse the effects of material topography on cellular proliferation behaviour, growth profiles of cells cultivated on different control samples, as well as on spike structures of Si and its replica in silicone elastomer were compared after 48 h (Table 1). The growth behaviour of the cells is given as the cell density in percent normalised on the seeding density at time 0 h (1.6 × 106 cells/ml). As the experiments were performed with different cell lines such as fibroblasts and SH-SY5Y neuroblastoma cells, it could be tested whether material topography affected the proliferation in a cell specific manner. For neuroblastoma cells it was observed that none of the tested Si and silicone elastomer samples (non-struc- Table 1 Contact angle and cell proliferation results (given as av-

erage ±SEM of four independent measurements for each type of

surface)

sample contact

angle

fibroblast

proliferation

(%)

neuroblastoma

proliferation

(%)

glass (control) 40° 283.32 ± 57.74 230.86 ± 47.23

Si

(non-structured)

62° 290.36 ± 22.08 257.12 ± 52.20

Si

(spike structures)

130° 155.52 ± 14.90 251.62 ± 28.05

silicone elastomer

(non-structured)

110° 224.01 ± 8.96 243.42 ± 26.03

silicone elastomer

(spikes replica)

159° 95.94 ± 12.15 258.91 ± 28.76

tured, structured by moulding) significantly affected cell proliferation. The cells proliferated at the same rate as those cultivated under control conditions on glass slides. On the contrary, for the fibroblasts a tendency to reduction of proliferation on Si spikes and on its replica in silicone elastomer was observed (Table 1). On the spikes replica in silicone elastomer the reduction of proliferation was sig-nificant compared to the control glass surface. Using the student’s-t-test, a significance p < 0.05 was evaluated (Ta-ble 1). It could be argued that the reduction of the surface for the cells to attach would reduce the proliferation activ-ity of the cells. However, if this were the case, the prolif-eration of fibroblasts on Si spike would be significantly different to the proliferation observed on the control. The spikes of Si offer a more reduced surface than the spikes of silicone elastomer which are the negative replica of the conic spikes of Si. Our results introduce the parameter of hydrophobic behaviour to partly explain the effects of the structuring of the surface to the proliferation of the cells as was observed by other authors [11, 12]. We observed that the contact angle of the surface was different, increasing in the following order: glass control < non-structured Si < non-structured silicone elastomer < Si spikes structured < spikes replica in silicone elastomer (Table 1). As discussed above, two parameters are impor-tant to explain the hydrophobic behaviour of the materials used in the experiments. (i) The material: we observed that Si and glass are very hydrophilic while silicone elastomer is hydrophobic. (ii) The creation of the spike increases the contact angle of both type of material by about 47°. This increase of hydrophobic character of the material is mainly related to air trapped under the water drop during the con-tact angle measurements. In cell culture conditions the air bubbles under the culture media could escape more or less quickly depending on the interactions between material, air and the culture media. Since the silicone elastomer is natu-rally hydrophobic, it can be assumed that the increased hy-drophobic character of the silicone elastomer material would be maintained for a relatively long period. Maintain-ing a large hydrophobic character for a long period would reduce the proliferation activity of the fibroblast on the sur-face. The observation that the hydrophobic surface effects the proliferation of cells can be explained by the considera-tion that for adherent cells such as fibroblast or neuroblas-toma cells, the attachment of the cells is the first step to proliferation. The attachment to adequate surface is con-sidered as a first messenger which stimulates intracellular signalling pathways via Ras, Rho, MAPK cascades in-volved in organisation of the cytoskeleton, cell growth pro-liferation and differentiation [13, 14]. In this report, it is observed that fibroblast and neuroblastoma react differ-ently to the hydrophobic surfaces. This may reflect differ-ences in the molecular structures involved in adhesion mechanism that are used by these different cell systems to attach onto the surfaces. Adhesion is mediated by compo-nents of the extracellular matrix such as fibronectin and

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collagen that associate with material surface in a non spe-cific manner. Members of these extracellular molecules serve as ligands which specifically bind to adhesion recep-tors in the cell membrane such as different types of in-tegrins [15]. Different cells systems express different type of adhesion receptor which interact with different members or epitopes of molecules within the extracellular matrix. The conformation of the ligands in extracellular matrix can be modulated by the association with the culture surfaces in dependence of topopography and hydrophobic level. It was demonstrated that the fibronectin on hydrophobic sur-faces presented a conformation which did not sustain the binding of integrins. Fibroblast cells adhere on the surface by the use of interin [15–17]. It is therefore tempting to propose that the effect observed by fibroblast is related to the fibronectin-integrin mediated adhesion in particular. Other adhesion factors like neuronal cell adhesion mole-cule (NCAM’s) are known to serve as adhesion receptors, which are expressed in SH-SY5Y neuroblastoma cells, but few is known about their interactions with hydrophobic surfaces [18]. It can be suggested that other adhesion mechanisms involved do not react sensitively to material hydrophobicity in comparison with fibronectin-integrin. Further studies are needed to clarify this specific topic.

Acknowledgements This work was supported by the Son-

derforschungsbereich 599 “Zukunftsfähige bioresorbierbare und

permanente Implantate aus metallischen und keramischen

Werkstoffen” of the Deutsche Forschungsgemeinschaft (DFG).

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