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Research Papers: Engineering Cell Microenvironments Using Novel Hydrogels

The Micro/Nanohybrid Structures Enhancing B35 Cell Guidance on Chitosan

[+] Author and Article Information
Ying-Ting Lin

Graduate Institute of Biomedical Engineering,
National Chung-Hsing University,
250 Kuo Kuang Road,
Taichung 40227, Taiwan
e-mail: jeff0213@gmail.com

Ching-Wen Li

Ph.D. Program in Tissue Engineering and
Regenerative Medicine,
National Chung-Hsing University,
250 Kuo Kuang Road,
Taichung 40227, Taiwan
e-mail: fayeshin2@hotmail.com

Gou-Jen Wang

Mem. ASME
Department of Mechanical Engineering,
Graduate Institute of Biomedical Engineering,
National Chung-Hsing University,
250 Kuo Kuang Road,
Taichung 40227, Taiwan
e-mail: gjwang@dragon.nchu.edu.tw

1Corresponding author.

Manuscript received September 16, 2015; final manuscript received January 4, 2016; published online March 17, 2016. Assoc. Editor: Feng Xu.

J. Nanotechnol. Eng. Med 6(3), 031005 (Mar 17, 2016) (9 pages) Paper No: NANO-15-1082; doi: 10.1115/1.4032602 History: Received September 16, 2015; Revised January 04, 2016

A novel chitosan scaffold with micro- and nano-hybrid structures was proposed in this study. The hemispheric array of the barrier layer of an anodic aluminum oxide (AAO) film was used as the substrate. Microelectromechanical systems and nickel electroforming techniques were integrated for fabricating chitosan scaffolds with different micro/nanohybrid structures. Nerve cells were then cultured on the conduits. It was demonstrated that the scaffold with pure microstructures can guide the nerve cells to grow along the ridges of the microstructure and some cells to grow across the groove in between two ridges of the microstructure. It was also shown that the scaffold with microscale ridges and nanopatterns on the groove between two ridges can more effectively guide the cells to grow along the ridges, thus enhancing the proliferation of nerve cells.

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Figures

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Fig. 1

Schematic of fabrication process of chitosan scaffold with micro/nanohybrid patterns: (a) AAO barrier-layer template preparation, (b) spinning coating of photoresist, (c) ultraviolet exposure, (d) development, (e) nano-electroforming, and (f) chitosan casting and demoding

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Fig. 2

Surface morphology of barrier-layer surface of AAO membrane: (a) SEM image of the ordered nanohemisphere array of fabricated AAO barrier layer and (b) measurement of height and diameter of nanohemisphere using AFM

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Fig. 3

Micropatterns with different groove/ridge combinations transferred onto AAO barrier-layer and silicon substrates via photolithography. The ridge/groove combinations are (a) and (d) 30/30, (b) and (e) 30/50, and (c) and (f) 30/100 (unit: μm).

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Fig. 4

SEM and AFM images of micro/nanohybrid pattern on AAO barrier-layer substrate: (a) SEM image of micropattern defined on AAO barrier-layer substrate via photolithography and (b) AFM image showing complete removal of photoresist

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Fig. 5

Nickel molds with different micro/nanopattern combinations fabricated via electroforming using AAO barrier-layer template. The ridge/groove combinations are (a) and (d) 30/30, (b) and (e) 30/50, and (c) and (f) 30/100 (unit: μm).

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Fig. 6

Chitosan scaffolds with different micro/nanopattern combinations. (a)–(c) Scaffolds with nanostructures. The ridge/groove combinations are (a) and (d) 30/30, (b) and (e) 30/50, and (c) and (f) 30/100 (unit: μm).

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Fig. 7

Proliferation rates of B35 cells cultured on different chitosan scaffolds. Note that the asterisk symbol indicates that p < 0.05 when compared with pure nanostructure and cell proliferation rate (number of cells on micro, nano, micro/nanohybrid structured chitosan scaffold or flat chitosan membrane/number of cells on flat chitosan membrane). The values are expressed as mean value ± standard deviation.

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Fig. 8

Comparison of cell morphologies of B35 cells cultured on a chitosan membrane of pure nanosphere array and a flat chitosan membrane (scale bar: 100 μm)

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Fig. 9

Cell morphologies of B35 cells cultured on different chitosan scaffolds with pure micropatterns. The cells tend to grow along the ridges of the micropattern. Some cells, especially those cultured on 30/50 and 30/100 micropatterns, grow from one ridge across the groove to the neighboring ridges. White arrows: cell crossed to another pattern (indicated by rectangular boxes) (scale bar: 100 μm).

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Fig. 10

Cell morphologies of B35 cells cultured on chitosan scaffolds with micro/nanohybrid structures (nanostructure on the groove). The conduits can more effectively guide cells to grow along the ridges (scale bar: 100 μm).

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Fig. 11

Cell morphologies of B35 cells cultured on reversed micro/nanohybrid structured chitosan scaffolds (nanostructure on the ridges). Note that no obvious guidance of cell growth direction can be observed (scale bar: 100 μm).

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