Micro-patterned cell orientation of cyanobacterial liquid-crystalline

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Micro-patterned cell orientation of cyanobacterial liquid-crystalline hydrogels Saranyoo Sornkamnerd, Maiko Kaneko Okajima, Kazuaki Matsumura, and Tatsuo Kaneko ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b15825 • Publication Date (Web): 27 Nov 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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ACS Applied Materials & Interfaces

Micro-Patterned Cell Orientation of Cyanobacterial Liquid-Crystalline Hydrogels Saranyoo Sornkamnerd1,2, Maiko K. Okajima1, Kazuaki Matsumura1 and Tatsuo Kaneko1*

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Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa

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923-1292, Japan

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2Department

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Engineering, Vidyasirimedhi Institute of Science and Technology, (VISTEC), Payupnai, Wang

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Chan, 21210, Thailand

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Corresponding Author: Tatsuo Kaneko (Tel: +81-761-51-1631, Fax: +81-761-51-1635, E-mail:

Energy and Environment Area, Graduate School of Advanced Science and Technology,

of Materials Science and Engineering, School of Molecular Science and

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[email protected])

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ABSTRACT

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Control of cell extension direction is crucial for the regeneration of tissues, which are generally

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composed of oriented molecules. The scaffolds of highly-oriented liquid crystalline polymer

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chains were fabricated by casting cyanobacterial mega-saccharides, sacran, on parallel-aligned

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micrometer bars of polystyrene (PS). Polarized microscopy revealed that the orientation was in

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transverse direction to the longitudinal axes of the PS bars. Swelling behavior of the micro-

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patterned hydrogels was dependent on the distance between the PS bars. The mechanical properties

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of these scaffolds were dependent on the structural orientation; additionally, the Young’s moduli

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in the transverse direction were higher than those in the parallel direction to the major axes of the

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PS bars. Further, fibroblast L929 cells were cultivated on the oriented scaffolds to be aligned along

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the orientation axis. L929 cells cultured on these scaffolds exhibited uniaxial elongation.

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Keywords: Liquid crystal hydrogels, Cyanobacteria, Cell orientation, Micropatterned scaffolds,

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Sulfated polysaccharides

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INTRODUCTION

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Scaffolds are three-dimensional materials used for cell growth in artificial tissue formation and

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regeneration1. They must satisfy basic requirements such as good biocompatibility,

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biodegradability, porosity for material diffusion, and cellular alignment controllability2-8. The

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innate design of native tissues includes orientation of cellular alignment, under which biological

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function is facilitated in the living body. Most tissues such as vasculature9, myocardium10, skeletal

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muscle11, nervous system12-13, and connective tissues14, have anisotropic properties owing to

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structural orientation, yielding asymmetric mechanical properties, which are very important for

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the living cell15. The ability to control cell shape and to engineer tissues with well-controlled

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morphology holds significant potential in many biomedical applications including regenerative

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medicine, tissue regeneration, drug screening, biosensors, and elucidation of cell–cell and cell–

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matrix interactions16-20. Several techniques have been developed to address the orientation control

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in this function. For example, electro-spinning technique21-25 appears to be a simple route for the

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fabrication of filaments but its conditions are generally dependent on the rheological behavior of

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the polymer solution and are difficult to optimize21, 26. Langmuir−Blodgett technique is also

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revealed potential for producing an anisotropic film and applying for cell-oriented scaffolds27-30.

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Surface topography has been introduced as a kind of tool for controlling cell patterns. The specific

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layout can be created through photo crosslinking31-32, chemical introduction,33-34 or patterning of

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the surfaces16, 35.

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Sacran, whose molecular weight is approximately 1.6 × 107 g/mol (molecular length over 30

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µm), is a recently discovered megamolecular polysaccharide extracted from cyanobacteria,

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Aphanothece sacrum (structure in Figure 1)36.The functional groups of sacran play a crucial role


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in cell-cell interaction37 especially amino group which is similar to those of glycosaminoglycan

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existing on the cell surface or inside the extracellular matrix, moreover sulfuric acid (~11 mol%)

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and carboxylic acid (~22 mol%) also exist in sacran structure. Biomedical functions of sacran such

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as anti-inflammatory, anti-allergy, and wound-healing properties have already been reported38-41.

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Moreover, a previous report described L929 mouse fibroblasts attached on the specific surface of

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sacran film42-43. Sacran has a sufficiently rigid backbone to show a liquid crystalline phase in a

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very low concentration range over 0.2 wt%44 to exhibit efficient self-orientation behavior. Such

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biological and liquid crystalline functions of sacran motivated us to investigate the orientation

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control of sacran chains and its applicability to scaffolds in order to adjust the cell extension

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direction.

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Here, we show the unique orientation behavior of sacran films casted on a micro-patterned

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polystyrene (PS) substrate (Figure 2), and the orientation of sacran scaffolds is observed to guide

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the cell alignment efficiently. The casting method is simpler than electrospinning17 or scaffold

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stretching18, 45.

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RESULTS

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Hydrogel preparation. Sacran exhibited a liquid crystal (LC) phase at an extremely low

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concentration of approximately 0.2 wt%, which indicated its very high self-orientation ability. In

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previous papers, we reported the in-plane orientation behavior of sacran film cast on an aqueous

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LC solution. The film showed a layer structure with random orientation in the top/bottom surfaces.

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The orientation behavior may be controlled by the morphology of the substrate used for casting.

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Thus, we used PS substrates with micro-patterning on the surface. PS bars with a diameter of 400

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μm were aligned on a PS flat plate using a 3D printer (Figure 2). The distance between bars was

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adjusted to 200, 250, and 300 μm, and the plates are henceforth referred to as P-200, P-250, and

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P-300, respectively. Figure 3 presents the preparation process of the micro-pattered surface

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hydrogels of sacran using these substrates. First, 0.5 % wt/v of sacran LC solution was cast on the

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PS substrate at 60 °C and was maintained at the same temperature for 24 h for complete drying.

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The resulting film obtained on the substrate was thermal cross-linked via annealing at 120 °C for

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6 h. By using this condition which was previously optimized by ourselves,43 the sacran scaffold

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revealed appropriated water contact angle and protein adsorption, which are important properties.

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The films fabricated on P-200, P-250, and P-300 are henceforth referred to as M-200, M-250, and

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M-300, respectively. Figure 4 shows the scanning electron microscope (SEM) images of the films

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where the upper and lower images indicate the film face directly touching the substrates and the

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side face, respectively. From the lower images, one can observe that all the films were undulated

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by molded of the substrate shape, and the parts of the film molded by the gap between bars became

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thick (Region A, Figure 3). The size of Region A was tuned by changing the distance between

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bars, and the parts of M-200 were the sharpest of the three whereas those of M-300 became


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distorted. The film parts molded directly from the bars became concave and thin (Region B). The

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curvature of these parts was independent of the substrate as the bar diameter was the same. The

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close-up images of Figure 4 revealed the layer structure, similar to the result of a previous paper

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for a sacran film cast on a flat substrate (shown in the right images) owing to the LC properties42,

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.

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Swelling. The swelling behavior of micro-patterned hydrogels was monitored by measuring the

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size ratio of swollen hydrogel to dry film under the condition of immersion in deionized water for

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24 h. Figure 5a shows the linear expansion degree (dy) along the longitudinal direction of the bars,

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which is almost independent of the distance between bars, whereas dx, which is perpendicular to

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the bar axes, increased with an increase in the gap between bars (Figure 5b), presumably owing to

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the larger expansion of the bigger region A. The difference between dx and dy indicates sacran

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orientation in the x-y surface. The values of dz in Regions A and B are shown in Figure 5c. The

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values of dz are much higher than those of dx and dy, which is attributed to the layer structures as

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discussed previously42, 47. While the dz values of Region A show no specific trend with a change

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in the gap size, they are higher than those of Region B. These results might be due to the structural

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defects of Region A, as observed from SEM images. The values of dz of Region B showed a slight

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decrease with an increase in the gap size. Figure 5d shows dz/dx values, which are much higher

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than 1 as described above and the corresponding values of the hydrogels formed on the micro-

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patterned PS substrate are higher than those formed on a flat one. The inhomogeneity of the film

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structures formed on the micro-patterned PS substrate might induce higher dz values regardless of

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the region as compared with those formed on a flat one. As demonstrated previously, the cast film

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of sacran formed a layer structure on the flat surface of the substrate46-47. However, on the aligned



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bars in the present case, we speculated that the layers can be formed around region B whereas the

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structure in region A may be complicated. Regarding the x-y face, the LC domains may exhibit

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orientation under the micro-patterned structure of the substrate48.

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Orientation (POM). Sacran solution efficiently exhibited self-orientation behavior in an LC phase

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at a concentration range over 0.2 wt%, which was previously confirmed using a polarizing optical

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microscope (POM)49. Figure 6A shows the POM images of sacran films formed on aligned micro-

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bars, obtained under a first-order retardation plate (530 nm). The blue color indicates sacran chain

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orientation along the first-order retardation plate, whereas the orange color indicates perpendicular

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orientation. The top figures showed negative birefringence, as demonstrated by the additive color

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(orange color) of the film whose line lay from the upper left to the lower right. In addition, the

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bottoms showed the subtractive color (blue color) of the film whose lines lay from the upper right

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to the lower left. The negative birefringence strongly suggested that the orientation of the sacran

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polymer backbones was perpendicular to the micro-bars of the substrates, which is in contrast to

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the pink-colored images of the film cast on the flat substrate. The color contrast of blue/orange

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was qualitatively higher than that of the flat substrate.

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The effects of water swelling during the hydrogelation on the sacran orientation were

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investigated. Figure 7 shows the POM images of sacran hydrogels formed via the immersion of

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sacran LC films in water, obtained under the first-order retardation plate. The orientations of sacran

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chain on the top surfaces of the hydrogels in the water-swollen state were intrinsically same as

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those of the film, as confirmed from the top and middle images. An orientation index line was

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estimated by color decomposition into R, G, and B from POM images shown in Figure 8a-d and

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the ratios of R/B in each substrate was summarized in Figure 8e. From Figure 8e, one can see that


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the R/B ratio of orientation index line was higher in wider grooves (M-300 > M-250 > M-200)

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while flat surface did not show any orientation. From the bottom images (Figure 7i)-7l)) of the

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hydrogel cross-sections, region A can be regarded as a mountain where the left slope showed blue

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color whereas the right one showed orange, regardless of the size of the gap between the microscale

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bars of the substrate. Such coloration indicates the sacran chain orientation along the slope surfaces.

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In region B, sacran chains were oriented horizontally.

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As shown above, region A showed higher degree of swelling than region B. This might be

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attributed to the water intrusion into the vertical boundary around the central axis of region A

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where the orientation direction of sacran chains remarkably changed. In addition, we cannot

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confirm the breaking around the top of hydrogel region A in the bottom images of Figure 7, in

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contrast to the SEM images of the film (lower images of Figure 4).

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Mechanical properties. The tensile properties of oriented sacran hydrogels prepared on PS

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microscale bars were measured through elongation in the directions both longitudinal (Y) and

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transverse (X) to the longitudinal direction of the bars. The stress–strain curves are shown in Figure

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9 and the data are summarized in Table 1. On the one hand, the stress–strain curves of the hydrogels

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prepared on the flat surface showed that the elastic modulus, E, and tensile strength, σ, were

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independent of the longitudinal (Y) and transverse (X) directions. On the other hand, the hydrogels

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prepared on the micro-patterned surface exhibited anisotropy of the mechanical properties (Table

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1). The elastic modulus of the hydrogels of the M-200, M-250, and M-300 films were 223, 318,

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and 414 kPa, respectively, in the transverse X direction. In the longitudinal Y direction, the elastic

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moduli were 199, 203, and 329 kPa for M-200, M-250, and M-300, respectively. The data in the

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Y direction were lower than those in the X direction probably owing to the orientation, as



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confirmed via the POM observation in Figures 6 and 7. The elastic modulus significantly increased

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with the distance between the micro-bars, indicating that a larger gap induced higher orientation.

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When the distance between micro-bars was large, the sacran chains on the surface of the PS bars

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should be strongly tensioned during drying, for efficient orientation. The strain energy density

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showed no difference between the X and Y directions.

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In a previous paper, we demonstrated that inter-connected pores were produced in the

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hydrogels via the freeze-drying treatment42. Here, we attempted to create pores in the hydrogels

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oriented on the micro-bars using the freeze-drying machine. Through the treatment, the hydrogels

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showed porosity of 45% and the pore size was approximately 19 μm. In particular, the pores were

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aligned in the layered structure of hydrogels, as observed previously42.

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Cell culture. The viability of L929 cells cultured on the porous scaffolds of sacran hydrogel

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oriented on the micro-patterned surface was measured for 24, 48, and 72 h using a CCK-8 assay

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(Figure 10a), to confirm the good proliferation on the porous hydrogels. Figure 10b supported such

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results to show numerous green-colored living cells, where a live/dead assay was performed on

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L929 cells seeded after 72 h. The viability and distribution of L929 cells on the micro-bars were

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observed through the green (calcein AM) color of the live cells whereas the dead cells were

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observed to be red (edthidium homodimer). In addition, we can observe that L929 cells appear to

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extend anisotropically on the micro-patterned scaffolds whereas they were isotropic in the flat

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substrate.

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SEM images were observed to confirm the anisotropic extension (Figure 11a) – 11d)).

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They showed a wrinkling structure on the surface and in the thinner part; however, the flat scaffold

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showed only a wrinkle. The wrinkle appeared perpendicular to the micro-bar axes during water


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sublimation. The Figure 11e) - 11h) showed cell adhesion to the scaffold surfaces. These images

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show that the L929 cells were well spread on the scaffold surfaces after 72 hours of incubation.

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They were extended in a spindle shape on the micro-pattern. Cell extension orientation was

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observed in one direction over the surface. In contrast, on the flat scaffold, cells were extended in

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random directions. The direction of cell extension on the micro-pattern scaffolds was

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perpendicular to the micro-pattern structure, which correlated to the polymer chain orientation.

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To quantify the orientation of cell attachment in response to the micro-patterned surface of

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the substrates, we measured the angle of extension direction of L929 cells to the micro-bar axes

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(Figure 11i) - 11l)). ImageJ software was used to measure the location of cells relative to the

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scaffolds. The results ranged between -90o and 90°. The angle 0° represents the extension of cells

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perpendicular to the micro-bar direction, whereas the angles ±90° represent the cell extension

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along the lines molded by micro-bars grooves. The histogram in Figure 11 showed that 78% of the

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cells were extended at an angle ranging from -50° to 50° for the M-200 scaffold. For M-250 and

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M-300 scaffolds, 90% of the cells were extended between the angles -40° and +40° and between

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-30° and +30°, respectively. In contrast, the cells on the flat surface scaffolds did not show any

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orientation specificity. Considering the micro-pattern size scaffolds, M-200 showed the lowest

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efficiency for controlling the orientation of L929 extension. The cell orientation was quantified by

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orientation order parameter, S, (inset in Figure 11i-f). The S values of the cells on the micro-

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patterned scaffolds were higher than that of flat one and increased with increasing groove widths

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to attain very high value of S = 0.81. The trend corresponds with the R/B ratio for hydrogel scaffold

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orientation (Figure 8e), indicating that the cell extension angle was strongly influenced by the

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surface topology of the scaffolds.

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DISCUSSION 9 ACS Paragon Plus Environment


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We here discuss the orientation mechanism (Figure 3). During drying, the LC domains of

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sacran were pinned at the top of the PS microbars and flowed down toward the valley to become

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oriented perpendicular to the longitudinal direction of the bars. Consequently, sacran chains at the

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top faces of the film and the hydrogels were oriented perpendicular to the ridge lines, in contrast

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to the hydrogels formed on the flat substrates. In swelling, region A plays an important role in

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orientation, i.e., the wider peaks in region A showed higher swelling to yield a stronger drawing

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force of the polymer chains. Therefore, M-300 hydrogels showed the highest R/B ratio and S of

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cell extension. For comparison, we prepared films on the microscale bars using 0.5% xanthan gum

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(Figure 6B), which is a natural extracellular, high-molecular-weight polysaccharide (300–8000

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kDa) produced by Xanthomonas campestris bacteria50. In contrast to sacran, the POM images of

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xanthan gum films showed no uniform orientation. Uniform orientation on microscale bars was

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efficiently achieved in the sacran LC polymer, presumably owing to the low critical liquid

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crystallization concentration of approximately ~0.2 %. Numerous reports showed cell orientation

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control on surface-patterning scaffolds16,

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patterning bars, and the single alignment could not be controlled. All the reports examined bundles

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of cells. S. Chen et al. prepared collagen scaffolds with micro-grooves on the surface, and the

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groove size was between 120 and 380 μm. The scaffolds with the groove sizes of 200 and 380 μm

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revealed very good orientation of the cell bundles in the longitudinal to micro-groove direction35.

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It can be observed that the scaffold with the groove size of 380 μm slightly differed from our

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report; however, the orientation direction of the cells was converse. The cell orientation on our

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materials was influenced by the LC polymer chains. According to the studies by A. McCormick32

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and Y. Hu16, the pattern created using the photolithography technique has a fine structure of

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approximately 10 μm, indicating that the cell attachment was highly oriented in the longitudinal

32, 35

. Otherwise, the cells were longitudinal to the


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to micro-patterned direction. In this report, we establish a simple method using liquid crystalline

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polymer sacran fabricated via the casting technique. By measuring the cell length, the average size

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was 35±7 µm (n=100). Although micro-patterned scale in the present technique ranged 200-300

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µm much larger than cell size, they controlled the orientation of L929 cells very well. The

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phenomena means that the molecular orientation in hydrogels was transferred into the cell

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extension direction.

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CONCLUSIONS

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We successfully prepared micro-patterned surface scaffolds of LC sacran by casting on PS

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micro-bars. The LC domains were highly oriented transverse to the longitudinal direction of the

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micro-bars on the surface. Such well-oriented scaffolds controlled via micro-patterning could

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increase the mechanical moduli. Cultured model cells (L929 fibroblasts) were observed to

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proliferate on the sacran scaffold with micro-patterned surface. Cell extension orientation was

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observed to be perpendicular to the micro-bar axes over the surface. The scaffold without a micro-

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patterned surface showed no orientation of the L929 cells. These results suggested that the micro-

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patterned surface scaffolds could effectively control the LC orientation, mechanical properties,

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and cell attachment, leading to possible applications in tissue engineering.

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EXPERIMENTAL SECTION

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Materials. Sacran was obtained from Green Science Material Inc. (Kumamoto, Japan) and used

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as received. Polystyrene mold was provided by Mutoh Industries Limited, Ikejiri, Setagaya

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Tokyo, 154-8560 Japan.



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Micro-patterned hydrogels fabrication. Sacran hydrogels with micro-patterned on the surface

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used here were prepared by the procedure shown in Figure 2. First, 5 ml of 0.5 w/v % sacran LC

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solution was casted on polystyrene mold with micro-patterned on surface and dried in an oven at

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60 °C for 24 h to form translucent films with micro structure on the surface. The LC films were

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punched into disk-like samples with a diameter of 5 mm and were thermally treated at 120 °C, in

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order to cross-link the sacran chains in a dry film state. Scanning electron microscopy (SEM, JEOL,

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JCM-6000PLUS) was used to investigate the surface patterned and layered structure. The samples

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were mounted onto metal stubs using carbon tape. The stubs were then coated with gold using a

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sputter coater machine.

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Then the films were immersed in deionized water at room temperature and kept for 24 h,

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the translucent self-standing of LC hydrogels were of an almost constant diameter, whereas the

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thickness was increased. The swelling behavior, LC domains orientation, and mechanical

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properties of these were evaluated.

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To apply these LC hydrogels for scaffolds, porous structure was desired. The precursor LC

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hydrogels were frozen by keeping in liquid nitrogen for about 10 min and then drying in a freeze-

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drying apparatus (EYELA, FDU-1200) for 72 hrs. We were able to confirm their complete drying

263

because the samples spontaneously attached to the glass wall by electrostatic force. As a result of

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freeze-drying, spongy materials were formed. When the sponges were immersed in deionized

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water, self-standing hydrogels were recovered.

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Microscopic observation. Crossed-polarizing microscopy was used to observe the structure

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formation of sacran LC polymer. Microscopic observations were made by a microscope (BX51,

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Olympus) equipped with CCD camera (DP80, Olympus). A specimen of samples was cut to size

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for microscopic observation (ca. 5 mm × 5 mm) and put on the glass plates at room temperature.


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A first order retardation plate (530 nm) was inserted onto the light path to identify the orientations

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direction.

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Swelling behavior The degrees of swelling were measured by monitoring the shape changing.

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The size ratio of swollen to dry samples were measured by the following method. Firstly, the

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pattern-size of dry films measured before hydrogel formation by SEM images. After equilibrium

275

swollen by immersed in de-ionize water for 24 h, the optical microscope was used to measure size

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of pattern hydrogels. The degree of swelling, d, was evaluated by the ratio of the swollen size, ls,

277

to the dry one, ld:

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d= ls/ld

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The swelling in thickness dimension was also calculated same as size swelling method.

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The values of 5 specimens were averaged.

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Mechanical properties of the hydrogels. 51-53 The mechanical properties of the micro-patterned hydrogels

282

were investigated in an elongation test. The elongation probe was set up on an Instron 3365 machine using

283

a 5 kN load cell with a crosshead speed of 1.0 mm/min. Elastic modulus (E) of each sample was calculated

284

using the following neo-Hookean equation applied to unidirectional elongation measurements:

285

τ = 𝐴 = 𝐸(𝜆 − 𝜆−2 )

286

where τ is the stress, F is the applied force, A is the original cross-sectional area of the hydrogels, and E is

287

the elastic modulus. λ = h/h0, where h is the hydrogel length under strain and h0 is the hydrogel length

288

before elongation. Plotting F/A versus (λ − λ−2) resulted in a straight line with a slope of E, which is the

289

modulus of elasticity of the swelling hydrogel. The strain energy density was measured by curve area under

290

stress-strain function.

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Cell Culture: A mouse fibroblast-like cell line (L929) was selected for all biological assays in

292

order to evaluate the effect of the micro-patterned surface on cell adhesion. The L929 fibroblast

293

cell line was obtained from the American Type Culture collection (Manassas, VA). The cells were

𝐹

(1)

(2)



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cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, USA) supplemented with 10%

295

heat-inactivated fetal bovine serum (FBS, Biochrom AG, Germany) incubated at 37 °C in a

296

humidified atmosphere with 5% CO2.

297

Cell Adhesion: Prior to biological assays all scaffolds were punched into disklike samples with a

298

diameter of 5 mm and placed on the 24 well tissue culture plate. They were sterilized under UV

299

radiation overnight54, then immersed in ethanol 70% (v/v) for 3 days. Subsequently, the 1 ml of

300

2.0 × 105 cells·ml−1 cell suspension was seeded on each scaffold and cultured for 24, 48 and 72 h

301

at 37 °C. A 24 well tissue culture plate was used as the control. After each incubation period, the

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samples were rinsed with a buffer saline (PBS, Sigma-Aldrich, USA). The number of cells

303

adhering to the scaffold was then counted.

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Live/ Dead assay 55-56: Cell viability on scaffolds was evaluated using live/dead assay. Constructs

305

were harvested, gently rinsed twice with PBS and then incubated with calcein AM and ethidium

306

homodimer-1 (EthD-1) for 15 min to strain live (green) and dead (red), respectively, for 15 min at

307

37 °C and 5% CO2 humidified incubator. Samples were observed using fluorescence microscope (BZ-

308

X700, KEYENCE)

309

CCK-8 assay

310

number according to the manufacturer’s instruction. Each sample (scaffolds with cells adhered),

311

after rinse with PBS, was incubated in 0.1 ml of growth medium supplemented with 10 µl of CCK-

312

8 stock solution for 3 h at 37 °C in a humidified atmosphere of 5% CO2, in air. The optical density

313

(O.D.) was measured by absorbance at 450 nm. The assay was performed in triplicated.

314

Cell morphology: The morphology of the cultured L929 (2.5×105 cell/scaffold) was observed by

315

SEM images. After 72 h of cultured, the cells were fixed by 10% formalin neutral buffer solution

316

(Wako, Japan) for 24 hours. Dehydration process was performed on each specimen in ethanol

55

: Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was applied to evaluate cell


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317

(60%, 70%, 80%, 90%, 100% and 100%) and 2 time of t-Butanol, each for 1 h then dry at room

318

temperature. After that they were sputter-coated with gold and viewed by SEM. The cell alignment

319

measurement was carried out with the ImageJ software to calculate the angle of extension direction

320

of L929 cells to the micro-bar axes. The angle, , represents the extended cell axis to the transverse

321

line across micro-bars. The orientation order parameter of the cells (S) was calculated according

322

to the following equation (3):57

323

S = 1/2 (3 ＜cos2 θ＞ − 1)

(3)

324



325

AUTHOR INFORMATION

326

Corresponding Author

327

*E-mail: [email protected], Tel: +81-761-51-1631, Fax: +81-761-51-1635

328

Author

329

E-mail: [email protected] (S.S.), Tel: +81-761-51-1631, Fax: +81-761-51-1635

330

E-mail: [email protected] (M.K.O.), Tel: +81-761-51-1631, Fax: +81-761-51-1635

331

E-mail: [email protected] (K.M.), Tel: +81-761-51-1680, Fax: +81-761-51-1149

Page 16 of 32

332

ORCID

333

Saranyoo Sornkamnerd

: 0000-0002-1929-5406

334

Kazuaki Matsumura

: 0000-0001-9484-3073

335

Tatsuo Kaneko

: 0000-0001-9794-083X

336

Author Contributions

337

S.S. designed the experiment and wrote the paper; K.M. interpreted the cell culture data; M.K.O.

338

extracted the sacran and supervised the researches; and T.K. also supervised and set the scientific

339

aims. All authors contributed to the results, conception, and presentation of the data.

340

Note

341

The authors declare no competing financial interest.

342

ACKNOWLLEDMENTS

343

The researches were made under the financial supports of a Grant-in-Aid from A-step

344

(AS2915173U) of JST, Japan, and for Challenging Exploratory Research of MEXT (16K14077)

345 16 ACS Paragon Plus Environment

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346 347 348 349 350 351 352 353

Figure 1. Chemical structure of sacran, a liquid crystalline polysaccharide.

354 355 356 357 358 359 360 361 362 363

Figure 2. PS substrate with micro-patterning on the surface. (a) PS bars with a diameter of 400 μm aligned on a PS flat plate. b) Distance between the bars is adjusted to 200, 250, and 300 μm, where the corresponding substrates are referred to as P-200, P-250, and P-300, respectively.

364



Page 18 of 32

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pinned and oriented

368 369

Sacran LC

370 371

z PS mold

372 373 374 375

drying

60 °C

PS mold

378

PS mold

x annealed at 120 oC

Sacran film

peeled

freeze-dry

hydrogel

in water

A

376 377

dry up

oriented toward valleys

Porous hydrogel

B

cells

upside-down

379

Porous scaffold

380 381 382 383 384 385 386 387 388

Figure 3. Schematic illustration of the preparation process of porous scaffolds on the micropatterned surface of PS substrate. Porous hydrogels were prepared by casting sacran solution with a concentration of 0.5 % wt/v on PS at 60 °C for 12 h and annealing at 120 °C for 6 h. Films were immersed in deionized water for 24 h to form hydrogels whose shape was molded by the PS substrate. Region A of hydrogels sticks out through the molded of the gap between bars whereas region B was concaved by the molded from the convex surface of the bars. The hydrogels were freeze-dried to form pores and the porous freeze-dried film can be used for cell-cultivation scaffold after setting it upside down.

389


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390

M-200

M-250

M-300

Flat

392

Top

391

393 394

Side

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


500 µm

500 µm

500 µm

500 µm

500 µm

500 µm

500 µm

500 µm

395 396 397 398 399 400 401 402 403 404 405

Figure 4. SEM images of sacran films M-200, M-250, and M-300, with a micro-pattern on the surface after annealing at 120 °C for 6 h on the PS substrates of P-200, P-250, and P-300, respectively. The samples were set upside down w.r.t. the film illustration of Figure 3. Flat: the sacran film formed on the PS flat substrate was shown for comparison. Close-up of M-250 film image at the bottom for viewing layer structure (arrow) clearly.

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Figure 5. Linear swelling degree, d, of hydrogels under the condition of immersion in deionized water for 24 h, given as the length ratios of swollen hydrogel to those of the dry film. Values of d for the hydrogels in the x, y, and z directions (dx, dy, and dz) are shown in a), b), and c), respectively. In c) and d), A and B in the legend refer to the swelling degrees in regions A and B indicated in inset a), respectively. The anisotropy of swelling defined as the ratio, dz/dx, is shown in d).

427 428


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429


A) Sacran

430 431 432 433 434 435 436 437 438 439

B) Xanthan gum

440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

Figure 6. Crossed POM images under the first-order retardation plate (530 nm) of LC polysaccharide films formed by casting on the micro-patterned PS substrates on the surface, A) Sacran, B) Xanthum gum. Lower microscopic images of each polysaccharide were obtained after rotating 90° w.r.t the upper images. Blue color indicates the polysaccharide chain orientation along the first-order retardation plate, whereas the orange color indicates perpendicular orientation Inset a) and e) revealed the coordinate directions of films.

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Figure 7. Crossed POM images under the first-order retardation plate (530 nm) of the top and side faces of sacran hydrogels formed via the immersion of sacran LC films in water. Lower microscopic images were obtained after rotating 90° w.r.t. the upper images. Blue color indicates the sacran chain orientation along the first-order retardation plate, whereas orange color indicates perpendicular orientation. Orientations were well confirmed in the water-swollen state. Coordinate directions of films shown as white arrows in a), e) and i).

478


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Figure 8. RGB profile of POM images of sacran LC hydrogels taken under the first-order retardation plate (530 nm), which were presented in Figure 7. a)-h) Decomposition data of Figure 7. a)–d) into Red (>530nm), Green (~530nm), and Blue lines (

Micro-patterned cell orientation of cyanobacterial liquid-crystalline

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