The Nano-topology Influence of Osteoblast-like Cell on the Bio-nanostructure Thin Film by Nanoimprint

Jeou-Long Lee Department of Chemical and Material Engineering Lunghwa University of Science and Technology

Taoyuan, Taiwan, R.O.C e-mail: semxrdedx@yahoo.com.tw

Yi Lin Department of Business Administration

Takming University of Science and Technology Taipei, Taiwan, R.O.C

e-mail: linyi@e-strategy.com.tw

Yung-Kang Shen College of Oral Medicine Taipei Medical University

Taipei, Taiwan, R.O.C e-mail: ykshen@tmu.edu.tw

Wei-Ren Chen Department of Mechanical Engineering

National Taiwan University Taipei, Taiwan, R.O.C

e-mail: r98522839@ntu.edu.tw

Abstract—Nanoporous anodic aluminum oxide (AAO) templates are fabricated using an anodization method. The mean diameters of nanoporous anodic aluminum oxide templates are 100 nm and 200 nm by various processing parameters of the anodization method. A molded plastic thin film nanostructure is fabricated by nanoimprinting using the AAO template as a mold insert. The surface properties of the molded plastic thin film are discussed using various nanoimprinting process parameters. Contact angles of the molded plastic thin film with the nanostructure exceed those without the nanostructure. The molded plastic thin films with a nanostructure and a hydrophobic surface are formed, and their contact angles exceed 90°. This study observes the behavior of osteoblast-like cells (MG63) cultured on nanostructure thin films, i.e., AAO, polylactic acid (PLA) and polycarbonate (PC). Cell growth behavior indicates that the AAO template with a 200 nm nanostructure is best. This study shows that cell adhesion and spreading are influenced by surface topography in the nanometer feature.

Keywords-anodic aluminum oxide; surface modification; nanoimprint; nanostructure; cell culture

I. INTRODUCTION Anodic aluminum oxide (AAO) films were formed by

the anodization of elemental aluminum (Al) in suitable acidic or basic electrolytic solutions. The AAO was fabricated with many nano-scaled pores. The diameter of the pores, as well as the cell size measured as the distance between the centers of two neighboring pores, was controlled by applying a pore widening treatment, in which the pores were chemically etched [1, 2]. Because of their unique thermal, mechanical, structure, optical and chemical properties, AAO has attracted much interest for various potential applications in including separation, catalysis, biosensing, adsorption, photonics, energy storage, and drug delivery. Masuda [3] adopted anodization approaches to fabricate porous metallic membranes and nanoimprinting. Puukilainen et al. [4] prepared superhydrophobic polyolefin surfaces by

simultaneous micro- and nanostructuring. Their results determined a static contact angle between polyprolene and water of value of 165°. Koponen et al. [5] achieved patterning using a nanoporous anodized aluminum oxide membrane as a mask in injection molding or imprinting, using cyclo olefin copolymer (COC) and polyvinyl chloride (PVC) as mold materials. The contact angles of the smooth surfaces of PVC and COC were about 89-90°. The contact angles of the microbumps and nanopillers for COC were 120-140°.

The resulting nanotopography combines ordered nanostructures with widely differing surface energies, providing a unique platform to study cell-substrate interactions. Human dermal fibroblasts were cultured on these substrates. Patterning of the surface with nanoscale pillars had a pronounced effect on cell morphology, independent of surface energy. Cell spreading was significantly diminished on both hydrophobic and hydrophilic surfaces that bear nanopillars. This result suggests that surfaces that resist cell spreading can be made by creating the appropriate nanoscale topography, without concern for the effect of surface chemistry on hydrophilicity [6-7].

The motivation of this study is to do the cell culture on the nanostructure of PLA material and PC material. The object of presented research is to find the cell growth on different nanostructures and materials. This study wants to find an effective and quick mass production for previous thin films. In this study, an AAO template (concave, nanoporous) is adopted as a mold insert in nanoimprint to fabricate the plastic thin film with a nanostructure (convex, nanopillar). The aim of this investigation is to elucidate the surface properties and cell cultures of the nanostructure of the plastic thin film for various processing parameters of nanoimprint.

II. EXPERIMENTAL METHOD Fig. 1 presents the structure of AAO. A pure aluminum

sheet (99.99%, 100*20*10 mm3, Wako Pure Chemical Industry, Ltd.) was electrochemically polished in a mixed

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2010 International Conference on Nanotechnology and Biosensors IPCBEE vol.2 (2011) © (2011) IACSIT Press, Singapore

solution of 95% C2H5OH and 60% HClO4 at 4:1. This specimen was employed to fabricate a conical AAO template following ultrasonic cleaning in ethanol and pure water. The specimen and a carbon electrode were used as the anode and cathode, respectively. The aluminum was anodized at 40 V in a 0.3 M oxalic acid solution at 16°C. To fabricate the AAO template, a long anodization of ten hours was used to generate a hexagonally ordered array of pores. The AAO film was dissolved in a mixed solution of 6 volume % phosphoric and 1.8 wt % chromic acid. To fabricate conical pores in this substrate, alternating anodization and pore widening treatments were performed. The substrate was then anodized using the same solution and voltage to produce uniformly sized pores. The pores were then widened by chemical etching, and the substrate was once again anodized under the same operating conditions. At this time, the pores were tapered: the interior of each pore was a structure with two steps. To obtain the final inverted conical structure, each step of the anodization and pore widening process was performed twice more. Each anodization step was conducted at 9°C at 40 V in the same solution. The anodizing time was 25 s in the first step and 20 s in each subsequent step. In the pore widening treatment, the specimen was dipped in a 5 volume % phosphoric acid solution at 30°C for 12 min. The morphology of the specimen was observed using a field emission scanning electron microscope (JOEL JSM-6700F, Japan). The cross-sections of the AAO templates were prepared by bending the aluminum until the substrate fractured to reveal the cross-section of the template. The AAO film specimen was coated by platinum sputtering before observation. The details of morphology of the specimen were observed using atomic force microscope (Nanosurf Mobile S). Prior to replication, the AAO template was self-assembled to form an anti-adhesive monolayer (1H, 1H, 2H, 2H-perfluorodecyltrichlorosilane, FDTS) by vapor phase deposition, and the minimization of the surface energy on AAO template, which is required for easy de-molding between the plastic thin film and the AAO template.

Figure 1. The illustration of structure of AAO.

After the AAO template was formed, a nanoimprint machine (NIL-3.0 Imprinter, Obduct AB, Sweden) was adopted to perform the imprinting. Fig. 2 displays the process from the fabrication of the AAO template to nanoimprint. The imprint processing parameters are the imprinting temperature, the imprinting pressure, the imprinting time and the de-molding temperature. Table 1 presents the values of the processing parameters. To identify the relative significance of these four parameters, various

experiments were performed, for a total of 34 runs. A statistics-based experimental design method, the Taguchi method [8], was utilized to reduce the number of experimental runs. The contact angle is the property of major concern on nanoimprint of molded plastic thin film with nanostructure.

The PC (T2FOQ, GE, USA) and PLA (Mitsubishi, Japan) were used as the molded materials in the nanoimprint process. The thickness of the plastic thin films was 1.0 mm. A contact angle meter (CA-051, UK) was employed to measure the contact angle of the molded plastic thin film.

Figure 2. The full process for AAO template fabrication and nanoimprint.

TABLE I. LEVELS OF PROCESSING PARAMETERS FOR NANOIMPRINT( PLA/PC).

Parameter Level 1 Level 2 Level 3

A. Imprinting temp. (℃) 150/180 160/190 170/200

B. Imprinting press. (MPa) 5/5 6/6 7/7

C. Imprinting time (Sec.) 90/90 120/120 150/150

D. De-molding temp. (℃) 30/50 40/60 50/70

Human osteogenic sarcoma (MG63, ATCC CRL-1427)

was maintained in Dulbecco’s modified Eagle Medium (DMEM; HyClone) with 10% heat-inactivated Fetal Bovine Serum (FBS; Sigma). Prior to reaching confluence, the osteoblasts were harvested from monolayer culture with 0.25% trypsin/EDTA (Sigma). Trypsin was neutralized with 10% FBS in DMEM. Each substrate was sterilized with 70% ethanol. Osteoblasts (<15 doublings) were seeded onto the substrates at a density of 5000 cells ml-1 in DMEM supplemented with 10% FBS. After 24 h, the cells were fixed using 4% paraformaldehyde. The cells were then processed for fluorescent staining of actin filaments and nuclei with TRITC-conjugated Phalloidin and DAPI using a staining kit (part # FAK100, Chemicon International) according to the manufacturer’s protocol. Substrates were washed with buffer to remove loosely bound cells before imaging. This study observes the morphology results of cell culture for 4 hours, 1 day and 4 day by scanning electron microscope (SEM, JSM-6700F, JOEL). This study also does the microculture tetrazolium test (MTT) assay to measure the cell activity. The MTT assay measures the optical density (OD) Value.

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The OD value indicates the activity of particle glanda that it means the numbers of live cell. This study uses the ELISA reader (Anthos 2020) to measure the OD value on 4 hours, 1 day and 4 day.

III. RESULTS AND DISCUSSION Fig. 3 depicts the nanostructure (nanohole) in AAO that

was employed in this study (SEM, AFM). The mean diameters of the nanoholes in AAO were 100 nm and 200 nm. The results indicate that the shape of nonohole is very favorable. The results demonstrate that the AAO template included nanoholes with a high aspect ratio. The height of the nanoholes of the sample was 24000 nm. The aspect ratios of the nanoholes in the AAO template were 120 and 240. Fig. 4 shows that the nanostructure (containing nanopillars) of the molded plastic thin films (convex) formed using the AAO template was employed in nanoimprint. The shape of each nanopillar markedly favored nanoimprint. Fig. 5 indicates the contact angle of the PLA thin film with the nanopillar (L7) and without the nanopillar. The mean original contact angle of the PLA thin film (without nanopillar) was about 80.2°. That with the nanopillar were around 129.1 ﾟ (100 nm) and 126.5 ﾟ (200 nm). The mean original contact angle of the PC thin film (smooth plate) was about 82.1°. That with the nanopillar were around 133.7° (100 nm) and 120.7° (200 nm). Figure 7-10 show that the variation of the S/N ratio with factor level for contact angle of different nanopillars (100 nm, 200 nm) on different materials (PLA, PC). The results appear that the optimal processings for contact angle of nanopillar (100 nm) on PLA material are A3B1C3D2. From the previous result, it is shown that the imprinting temperature is the most important factor for contact angle of nanopillar on PLA. The optimal processings are A3B2C1D3 for contact angle of nanopillar (200 nm) on PLA material. The imprinting temperature is the most important factor. The results reveal that the optimal processings are A1B2C2D1 for contact angle of nanopillar (100 nm) on PC material. From the previous result, it is shown that the de-molding temperature is the most important factor for contact angle of nanopillar on PC. The optimal processings are A1B2C2D1 for contact angle of nanopillar (200 nm) on PC material. The de-molding temperature is the most important factor and the imprinting time is the unimportant factor.

(a) Φ=100 nm. (Ra=10.83 nm)

(b) Φ=200 nm. (Ra=18.07 nm)

Figure 3. SEM images and surface roughnesses of AAO.

(a) Φ=100 nm, PLA (b) Φ=200 nm, PLA

(c)Φ=100 nm, PC (d) Φ=200 nm, PC

Figure 4. SEM images of nanopillar of different molded plastic thin films using AAO template.

(a) θ= 80.2° (PLA without nanostructure)

(b) θc=129.1° (Φ=100 nm)

(c) θc =126.5°(Φ=200 nm)

Figure 5. Contact angles of PLA on different situations.

(a) θ= 82.1° (PC without nanostructure)

(b) θc=133.7° (Φ=100 nm)

(c) θc=120.7° (Φ=200 nm)

Figure 6. Contact angles of PC on different situations.

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Figure 7. Variation of the S/N ratio with factor level for contact angle of

nanopillar(PLA, Φ = 100 nm)

Figure 8. Variation of the S/N ratio with factor level for contact angle of

nanopillar(PLA, Φ = 200 nm)

Figure 9. Variation of the S/N ratio with factor level for contact angle of

nanopillar(PC, Φ = 100 nm)

Figure 10. Variation of the S/N ratio with factor level for contact angle of

nanopillar(PC, Φ = 200 nm)

The results of cell culturing are shown in Fig. 11. The OD values increase as the cell time increases whether different materials or different nanostructures. The results show that the OD values of different nanopillars of PC material are larger then them of flat PC material. The OD value on nanopillar (100 nm) is smaller than it on nanopillar (200 nm). The OD value of flat PLA is smaller than it of different nanopillars of PLA material. The OD value of nanopillar (100 nm) of PLA is larger than it of nanopillar (200 nm). The results of Fig. 11 appear that the OD values

with nanostructure of different materials are larger than them without nanostructure of different materials. This meaning indicates that a nanostructured bio-materials membrance can be more contributive to the cell growth.

Figure 11. The OD values of different materials and nanostructures on

culture time (MTTassay).

To investigate the effect of topography on the cell-substratum interaction, nanopillars of widely varying aspect ratios and wettabilities were prepared. Human osteogenic sarcoma was cultured on these substrates and their morphology was analyzed. Fig. 12-13 show that the cell behaviors on different structures of PLA and PC materials. The results also reveal that the cell behaviours are adhesion at 4 hours, spreading at day 1 but filopodia group together at day 4 whether different nanopillars or different plastic materials. The cells on nanopillars displayed a fundamentally different morphology than cells on a flat control surface. They restricted themselves to a significantly smaller surface area, appearing spheroidal or having a highly polarized morphology of long protrusions with little spreading on the surface. In contrast, cells on flat control were polygonal and well spread. In addition, cells on the nanopillars seemed to aggregate, indicated by clusters of nuclei. This result addresses the possibility that poor cell spreading on the nanopillar surfaces is simply an effect of the surface energy, with the highly hydrophobic surface resisting cell spreading. An alternative interpretation is that cell spreading depends upon surface adsorption of extracellular proteins which itself requires an intermediate surface energy. Another possibility is that the imprinting process itself induces a chemical change in the polymer that results in reduced support for cell spreading. Thus we favor the interpretation that the high aspect ratio nanopillar topography itself resists spreading of adherent cells. Although the aspect ratios of these surface features differ by an order of magnitude and their surface energies are appreciably different, we observe no difference in cell spreading between tall and short nanopillars.

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Figure 12. The cell behavior on different nanopillars of PLA.

Figure 13. The cell behavior on different nanopillars of PC.

IV. CONCLUSIONS In this study, an AAO template is applied as a mold insert

in fabricating a nanostructure on the plastic thin film for nanoimprint. The results indicate that the contact angle of the plastic thin film changes from approximately 80° to 130°. The surface property of the plastic thin film is changed from hydrophilic to hydrophobic. The results also demonstrate that the imprinting temperature and de-molding temperature are the most important factors in determining the contact angle of the molded PLA and PC thin film for nanoimprint. In this investigation, the nanostructure of the plastic thin film is formed and its surface is modified in a high-speed mass production process.

The highly hydrophobic nanopillar surfaces was used as cell culture substrates, and cell morphology was assessed. Cells spread poorly on the hydrophobic nanopillars. The results can be rationalized in terms of the ability of the cells to form effective adhesion complexes on the restricted area at the tops of the nanopillars. This study demonstrates that nanostructuring is a feasible way to make surfaces that resist cell spreading and adhesion, which can be useful for cell culture, bio-MEMs, and implant devices where biological and abiological interfaces are involved.

ACKNOWLEDGMENT The authors would like to thank the National Science

Council of the Republic of China, Taiwan, for financially

supporting this research under Contract No. NSC 98-2221-E-038-001.

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