Nanostructured TiO2 thin films as porous cellular interfaces - UCSB MRL

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Dec 21, 2005 - nano/micro-electro-mechanical systems (N/MEMS) to the ... Address for correspondence: Intel Corp., Assembly Technology ... with either silicon dioxide, silicon nitride or NST. Silicon .... Microsystems Technology Office of the Defence Advanced ... [15] Kumar G, Wang Y C, Co C and Ho C C 2003 Langmuir.
INSTITUTE OF PHYSICS PUBLISHING

NANOTECHNOLOGY

Nanotechnology 17 (2006) 531–535

doi:10.1088/0957-4484/17/2/032

Nanostructured TiO2 thin films as porous cellular interfaces Abu Samah Zuruzi1,3 , Blaine C Butler2 , Noel C MacDonald1 and Cyrus R Safinya2 1 Materials Department and, Mechanical and Environmental Engineering Department, University of California at Santa Barbara, CA 93106, USA 2 Materials Department, Physics Department, Molecular, Cellular, and Developmental Biology Department, Biomolecular Science and Engineering Program, University of California, Santa Barbara, CA 93106, USA

E-mail: [email protected]

Received 9 October 2005 Published 21 December 2005 Online at stacks.iop.org/Nano/17/531 Abstract We report the use of nanostructured TiO2 (NST) as porous cellular adhesion interfaces for microsystems. Integrated NST films and pad arrays with sponge-like morphology were fabricated by aqueous oxidation of Ti films followed by thermal annealing. Cell culture studies indicate significantly (t < 0.02 from the student’s t-test) greater initial attachment of mouse fibroblast cells on porous NST than on silicon dioxide and nitride films for up to 18 h seeding time. Fibroblasts seeded on NST have flat morphology with numerous processes attaching to walls of NST. These results indicate the potential of using NST as porous cell adhesion films and membranes for bioMEMS applications. (Some figures in this article are in colour only in the electronic version)

1. Introduction Currently, there is tremendous interest in applying nano/micro-electro-mechanical systems (N/MEMS) to the life sciences. Devices such as a neural prosthesis [1], muscle-driven devices [2], drug delivery microsystems [3] and immunoisolation biocapsules [4] have been realized using N/MEMS fabrication techniques. In most N/MEMS devices, silicon dioxide and silicon nitride were used to interface with biological systems. With advancements in microfabrication technology and nanomaterial synthesis, novel materials and those with novel morphology are being explored as interfaces to the biological milieu [5]. Porous silicon has been shown to possess compatibility to primary hepatocytes and bioactivity to induce hydroxyapatite growth [6, 7]. Recent work has demonstrated enhanced adhesion of osteoblasts on nanostructured ceramics [8, 9]. Carbon nanotubes were found to support fibroblast growth, and control outgrowth/branching of neuronal processes, and they are being explored as scaffoldings for neuronal circuits and drug delivery devices [10–12]. Also, proteins, 3 Address for correspondence: Intel Corp., Assembly Technology Development, 5000 W Chandler Blvd, Chandler AZ 85226, USA.

0957-4484/06/020531+05$30.00 © 2006 IOP Publishing Ltd

lipid membranes, polyelectrolytes and colloidal particles have been used to control cellular adhesion and growth [13–16]. Following this line of work in exploring novel nanomaterials as cellular interfaces, we report here the initial attachment of fibroblasts on porous sponge-like nanostructured titania (NST) thin films and square pad arrays. Embryonic and L line (L-cells) mouse fibroblast cells have been widely used to study cell attachment and adhesion on substrates [17, 18]. In this work mouse fibroblast cells of the L line were chosen because they spread less than embryonic fibroblasts and hence are a more stringent test of adhesion [17]. The morphology of fibroblast cells and percentage of area covered by cells in a micrograph have been used as an indicator of adhesion [18]. Fibroblast cells adherent to a substrate are flattened and cover a wide area of the surface. In contrast, when fibroblasts are cultured on less biocompatible surfaces, and hence are not adhering, these cells are spheroidal and cover a smaller area. In this study, the attachment of fibroblasts on thin films of silicon dioxide, silicon nitride and NST films for up to 24 h were investigated. Significantly enhanced attachment of fibroblasts on NST relative to silicon dioxide and nitride films were observed, which indicates the potential of using NST in future bioMEMS devices.

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Figure 1. SEM micrographs of (a) unpatterned NST films and (b) patterned NST square pad arrays.

2. Experimental procedure Fibroblasts were cultured on 1 cm square Si pieces coated with either silicon dioxide, silicon nitride or NST. Silicon dioxide was deposited using plasma-enhanced chemical vapour deposition using silane and nitrous oxide precursors at 250 ◦ C. Silicon nitride was deposited using silane and ammonia at 250 ◦ C. Porous NST was fabricated by reacting Ti films with aqueous hydrogen peroxide (aq. H2 O2 ) solution as an oxidant [19]. Si pieces were first thermally oxidized at 1100 ◦ C to grow 1 µm thick silicon dioxide layer. Ti film was then deposited on these Si pieces using electron beam evaporation. To form NST pad arrays, a silicon dioxide masking layer was deposited using plasma enhanced chemical vapour deposition. Photo resist (PR) was then spun on the silicon dioxide layer and patterned. The pattern on the PR layer was transferred to the silicon dioxide layer by etching with CHF3 plasma to expose arrays of Ti surfaces. The NST was formed by ageing samples in aqueous H2 O2 solution at 80 ◦ C. To form unpatterned NST thin films, as-evaporated Ti films without the silicon dioxide masking layer were used for oxidation. Mouse fibroblasts cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium, Gibco BRL) supplemented with 10% (v/v) fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin antibiotics (Gibco BRL) in 5% CO2 at 37 ◦ C. Cell cultures were passaged every 3 days. Before seeding, cells were dissociated using trypsin/EDTA, washed and resuspended in supplemented DMEM at a density of ∼105 cells ml−1 . Si tabs were then placed in cell culture dishes (NunclonTM ). Cell seeding was done by adding 4 ml of cell suspension into these dishes. Cells were then incubated for up to 3 days in 5% CO2 at 37 ◦ C. Subsequently, cells were fixed using 3.75% formaldehyde in phosphate-buffered saline (PBS), soaked in ethanol–DI water solutions at increasing ethanol concentration and critical point dried. To reduce charging during SEM studies, samples were coated with an ∼5 nm Pt layer. Confocal laser scanning microscopy was used to examine the morphology of the attached cells on some samples. For these samples, the cells were first washed with Triton-X to permeabilize the cell membrane and then soaked in Texas Red dye conjugated with phalloidin. The cells were then rinsed three times with PBS and fixed in 3.75% formaldehyde in PBS. 532

Figure 2. Percentage average area of surfaces covered by fibroblast cells after various seeding times.

3. Results and discussion 3.1. Cells on unpatterned surfaces SEM micrographs of unpatterned NST films and patterned NST square pad arrays are shown in figure 1. Cracks formed and propagated on unpatterned NST films. In contrast, the NST square pads are crack free when the pad size is ∼20 µm and below. Crack formation occurs when moisture evaporates, generating tensile stress. The NST formed is porous, with sponge-like structure consisting of walls about 25–75 nm thick and pore diameters of about 50–200 nm. XRD and selected area electron diffraction investigations indicate that only the anatase polymorph of titania was formed after annealing [19]. Fibroblast cells exhibit enhanced attachment to NST surfaces after short seeding times. Figure 2 shows the average area of surfaces covered by fibroblast cells after seeding. For NST surfaces, the area covered by cells is consistently greater

Nanostructured TiO2 thin films as porous cellular interfaces

Figure 3. Confocal laser scanning microscopy images of fibroblasts after 6 h seeding time on (a) silicon dioxide and (b) NST.

Figure 4. Scanning electron microscopy images of a fibroblast after 6 h on NST; note the lamella-like structure in (c).

than that of silicon dioxide and silicon nitride for culture times up to 18 h. After 24 h culture time, the area coverage for silicon dioxide and NST surfaces is ∼45%; the corresponding value for silicon nitride is only ∼30%. To determine the level of significance in differences between average values of area coverage, the t-probability value in Student’s t-test is used. It is generally accepted that for a comparison between two data sets a t-probability value less than 0.05 indicates a significant difference in the means. In figure 2, the t-probability values are for comparisons of average values of area coverages between silicon dioxide and silicon nitride with NST. The t-probability values for all seeding times of silicon nitride are less than 0.05. A similar trend was observed for silicon dioxide: the t-probability values from 3 to 18 h seeding time are all less than 0.05; after 24 h the t-probability value is 0.9912. Fibroblast cells on silicon dioxide and silicon nitride surfaces exhibit different morphology from cells on NST. Figure 3 shows confocal scanning microscopy images of fibroblasts after 6 h seeding time. Cells seeded on silicon dioxide have spherical morphology with little contact to the surface. In contrast, cells on NST exhibit a flat morphology and are more spread out on the surface. The flat morphology of fibroblasts on titania surfaces indicates enhanced adhesion relative to silicon dioxide surfaces [17, 18]. After 24 h seeding

time, cells seeded on silicon dioxide surfaces also exhibit the flat spread-out morphology, which results in the increased average area covered; see figure 2. Further evidence for adhesion is the formation of numerous processes as shown in figures 4(a)–(c). These processes are about 75 nm in diameter and can be up to ∼7 µm long. Recent studies have reported that processes of human osteoblast-like cells are able to penetrate porous alumina with 200 nm average pore diameter [20]. Hence, it was expected that these processes would penetrate these pores as the pore diameters range in size from about 50 to 250 nm. However, the tips of processes were observed to expand into lamella-like structures (∼400 nm diameter) that span the opening of large pores. Figure 4(c) shows a single lamella-like structure at the tip of a process. 3.2. Cells on patterned NST features SEM micrographs of L-cells cultured for 3 days on arrays of patterned NST pad are shown in figure 5. Generally, the cells have flat and spread-out morphology similar to those on blanket layers. However, it was observed that cell shape can be modulated by the patterned NST pads. It was found that fibroblast cells may attach to and take the shape of NST pads: 533

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Figure 5. SEM micrographs of L-cells cultured for 3 days on arrays of patterned NST pad.

figures 5(b) and (c) are SEM micrographs of a cell that has taken the shape of a 20 µm pad. It may be observed that the cell is flattened with lamellapodia wrapping the periphery of the pad. Figure 5(d) shows a cell that has completely taken the shape of a square pad. Such a strong interaction between cells and NST pads was not expected. The shape of single cells on synthetic surfaces has been shown to be modulated by organic cues on surfaces [13, 21]. Attachment of cultured cells to synthetic surfaces is mediated by the adsorption of proteins from serum in the culture medium [8, 13, 22, 23]. Hence by directing adsorption of proteins to spatially defined regions using organic cues that have been deposited on surfaces prior to cell seeding, it has been demonstrated that cell shape could be controlled [13, 21]. In the present study, it is speculated that there is enhanced adsorption of protein molecules because of the nanoscale nature of the titania pore walls. Recent work by Zhang et al showed significant increase in the adsorption coefficient on nanocrystalline titania particles due to the higher molar surface energy associated with smaller-sized particles [24]. Similarly, Webster et al reported that greater quantities of vitronectin were adsorbed on nanocrystalline ceramics compared to conventional ones, with micrometre-sized particles, which resulted in enhanced cellular adhesion on the nanocrystalline ceramics [8]. Due to the large surface area of the NST, it could be expected that greater quantities of proteins would be adsorbed on NST pads than on a smooth silicon dioxide mask. This could result in the enhanced attachment of fibroblasts on NST. Furthermore, the enhanced cellular attachment suggests that adsorbed proteins do not lose any of their activity. Protein 534

adsorption is not specific to the NST pads, however, and hence over time cells also attached and spread on the silicon dioxide mask, as in the case after 24 h seeding time. It must also be noted that the topography of the pads may also affect the attachment of cells.

4. Conclusions In conclusion, we have investigated the attachment of mouse fibroblast cells on silicon dioxide, silicon nitride and nanostructured titania (NST). It was found up to about 18 h, significantly (t < 0.02) more fibroblast cells attached on NST as indicated by area coverage of cells on these surfaces. However, after 24 h, the area coverage of cells on silicon dioxide and NST is similar. This enhanced initial attachment of cells is suggested to be due to enhanced adsorption of proteins on titania surfaces. It is speculated that adsorbed proteins on titania surfaces do not lose their activity. The cells on titania surfaces have a flat and spread-out morphology. On patterned NST arrays with silicon dioxide masking layer, some fibroblast cells preferentially adhered to patterned square NST pads.

Acknowledgments The authors acknowledge support for this work from the Microsystems Technology Office of the Defence Advanced Research Projects Agency. We also gratefully acknowledge support by NIH GM-59288, NSF DMR-0503347 and CTS-0404444. This work made use of MRL Central

Nanostructured TiO2 thin films as porous cellular interfaces

Facilities supported by the MRSEC Program of the National Science Foundation under Award No DMR-00-80034. ASZ acknowledges an International Fellowship (National Science Scholars Program) from the Agency for Science, Technology and Research, Singapore.

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