Simple Inkjet Process To Fabricate Microstructures of Chitinous

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A simple inkjet process to fabricate microstructures of chitinous nanocrystals for cell patterning Shuntaro Suzuki, and Yoshikuni Teramoto Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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A simple inkjet process to fabricate microstructures of chitinous nanocrystals for cell patterning Shuntaro Suzuki† and Yoshikuni Teramoto†,‡,* †

Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University,

Gifu 501-1193, Japan ‡

Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu

University, Gifu 501-1193, Japan KEYWORDS. chitin nanocrystals; deacetylation; inkjet printing; cellular adhesiveness; cellular micropatterning; nonproteolytic cell detachment

ABSTRACT. Structural polysaccharide nanocrystals (NCs) including cellulose nanocrystal have attracted attention. In order to broaden the range of application of the NCs, we can take advantage of their original characteristics by establishing simple and reasonable processing methods. We here demonstrate a micropatterning of animal cellular adhesion by inkjet printing of aqueous dispersions of cytocompatible chitinous NCs onto cellophane films. We display how to regulate the deposition form and two-dimensional shape of the chitinous NC micro-moldings using a research inkjet printer. Adhesive capability of mouse fibroblasts onto the chitinous substrates was greatly improved by alkali deacetylation. The deacetylated products remained

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rod-like nanostructures, but the original chitin crystal form changed to that of chitosan by an intensive deacetylation. The adhered cells could be recovered glycolytically. The chitinous micropatterning substrates can be utilized for biomedical applications such as controlling of cellular shapes, precise monitoring molecular events in biochemistry, and drug screening.

INTRODUCTION Structural polysaccharide nanocrystals (NCs)1 including cellulosic one (CNC) have recently attract considerable attention in the broad field of materials science and technology, because of their well-defined dimensions (~10-nm diameter and ~100-nm length),2,3 excellent mechanical properties,4 and aqueous mesophase formability.5–7 These properties are tempting for utilization as a filler for advanced composite materials, however, we can take advantage of the original characteristics of the NCs themselves by establishing simple and reasonable processing methods. Such an approach will broaden the range of application of the items which are still high-cost at present. We here utilize chitinous NCs as an inkjet ink, taking into consideration that their dimensions are comparable with those of practical inkjet pigments (roughly between 50 and 200 nm). For formulating commercial inkjet inks, in general, pigments have to be dispersed and the dispersion needs to be made colloidally stable, whereas the NCs are essentially suspended in water without any other additives.1,8,9 Even though the inkjet process requires suitable ranges of viscosity and surface tension for inks, these can be satisfied for diluted aqueous systems of NCs. Since minute droplets ejected by inkjet are supposed to be very small and of a size corresponding to aerosol, their drying process occurs rapidly10 and will not cause problems even if the NC dispersion has low concentration. The idea of inkjet-printing NCs as well as nanofibers has been reported to be applied to 3D printers in recent years,11–13 where desired moldings were made by careful viscosity control and combination with other ingredients. These researches are very attractive, but even with 2D inkjet


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printing, if we can fully exploit the inherent characteristics of NCs, we can make it meaningful by accomplishing fine molding. We therefore focus attention on the potential bioactivity of chitinous materials, in particular, their cell adhesiveness and aim to utilize it for micropatterning of animal cells.14,15 Micropatterning cell scaffolds permit fruitful applications such as controlling of cellular shapes, precise monitoring molecular events in biochemistry, and drug screening.16–18

Accurate

processes for fabricating such scaffolds have been performed mainly by photo-lithography and fine laser-ablation. In fact, such micro-processing concept can be acquired by the inkjet printing technology, which is further attractive from the viewpoints of simple contactless procedure, flexibility of choosing inks and substrates, no necessity of using harmful chemicals, suitability for wide-ranging on-demand productions, etc.19,20 In the present study, we first prepared chitin NC (ChNC) according to a conventional method using hydrochloric acid.1,9 Chitinous materials are well known to promote cell adhesion and proliferation21–24 and they have been practically applied for wound healing.25 In this regard, in general, the cell affinity of chitnous materials strongly depends on the degree of deacetylation (DD).21,26 We thus conducted deacetylation of ChNC by alkali treatment of ChNC to obtain deacetylated forms (dChNC). The series of chitinous NC materials were evaluated for the DD, morphology, and crystal forms by means of FT-IR, atomic force microscopy (AFM), and wideangle X-ray diffraction (WAXD), respectively. We then investigated the availability of these NCs to inkjet printing and carried out various micropatterning. Subsequently, mouse fibroblasts L929 were seeded on the micro-patterned chitinous NC and cell adhesion behavior was observed. For in vitro cell culturing, on the other hand, trypsin is commonly added into the system to detach adherent cells from a culture surface via partial degradation of protein. But there is a concern that cells can be damaged to some extent by the ordinary proteolytic procedure.27 Alternatively, we here undertook a decomposition of the micro-patterned chitinous NC scaffold glycolytically and examined the growth behavior of the recovered cells.

EXPERIMENTAL SECTION


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Materials. An original material of chitin isolated from crab shells was provided as powder from Wako Pure Chemical Industries, Ltd. Chitosan flake (chitosan 10) was purchased from Wako Pure Chemical Industries, Ltd. and used as a reference sample of chitosan. The following reagents for cell culture were used after appropriate sterilizing: Eagle's essential media (MEM) (Nissui Pharmaceutical Co., Ltd.), fetal bovine serum (FBS, Biosera Ltd.), penicillin (Sigma Aldrich Co. LLC), streptomycin (Sigma Aldrich Co. LLC), phosphate buffered saline (PBS, 10 times concentrated, pH 7.4, Nacalai Tesque, Inc.), trypsin (1: 250, manufactured by Life Technologies Co. Ltd.), YatalaseTM (digestive enzyme of cell walls of filamentous fungi, Takara Bio Inc.) ethylenediaminetetraacetic acid (EDTA, manufactured by Dojindo Laboratories, Inc.), Calcein AM solution (1 mM in DMSO, Dojindo Laboratories, Inc.), and ethidium homodimer 1 (Wako Pure Chemical Industries, Ltd.). PBS was diluted ten times with distilled water and used. Other chemicals were purchased mainly from Wako Pure Chemical Industries, Ltd. and used without further purification. ChNC preparation. Chitin powder was hydrolyzed with 3-M HCl for 6 h at 95 °C under stirring. The weight ratio of the aqueous HCl solution to chitin was 15. After the acid hydrolysis, the suspension was refined with the cycle of dilution and centrifugal separation (2500g for 30 min). The suspension was then dialyzed in deionized water until the pH value rose to ~5. After that, to obtain a concentrated ChNC/water suspension, the dialyzing tube was immersed into a 7wt% aqueous solution of poly(ethylene glycol) (number average molecular weight = 20000 ± 5000) for 3 days. The eventual concentration of the ChNC suspension was 21 wt%. A part of the condensed samples was re-dispersed in deionized water with a mechanical homogenizer (Physcotron NS-20G/20P, Microtec Co., Ltd.) and the concentration was fixed at 1.25 wt%. It was provided for the following inkjet printing as the ChNC ink. Deacetylation of ChNC. We adopted two samples of deacetylated ChNC (dChNC) prepared with different hydrolyzing conditions. One dChNC (coded as dChNC41.2; the numeral indicates the degree of deacetylation (DD) estimated by FT-IR as will be mentioned below) was obtained by a single deacetylation procedure: 4.76 g of the condensed ChNC suspension (21 wt%) and 100 mL of 40-w/v% sodium hydroxide aqueous solution were charged into a flask. The flask was placed in an oil bath thermo-regulated at 150 °C and the system was mechanically stirred for 6 h. After the hydrolysis, 300 mL of deionized water was added into the system and the flask


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was left in an ice bath for 15 min. The hydrolyzed mixture was repeatedly centrifuged with deionized water at 2500g for 30 min, and turbid supernatant was collected. The suspension was dialyzed in deionized water until the pH became ~7. The neutralized mixture was diluted by addition of 10 mL of water and centrifuged at 2500g for 15 min.

The supernatant was

homogenized with an ultrasonic homogenizer (US-150T, Nissei Corporation). The resulting suspension was used for the inkjet process. Concentration of the final dispersion was 0.20 wt%. Another dChNC sample (dChNC61.1) was prepared similarly, but the concentration of NaOH aqueous solution was 50 w/v% and the deacetylation step for 7 h was performed twice. A neutralized suspension containing 4.40 wt% of dChNC was provided for the second deacetylation reaction. Characterization of ChNC and dChNC. Fourier-transform infrared (FT-IR) spectroscopy was performed on freeze-dried samples at the Division of Instrumental Analysis, Life Science Research Center, Gifu University. Spectra were recorded using a PerkinElmer Spectrum 100 FT-IR apparatus over the wavenumber range 400–4000 cm−1 at a resolution of 4 cm−1 via the accumulation of 32 scans. A standard KBr-pellet method was used for all measurements and samples. Morphology of the ChNC and dChNC was observed using an atomic force microscope (AFM; AFM5400L, Hitachi Hi-Tech Science Corporation) in a dynamic force mode with a silicon SIDF20 cantilever (Hitachi Hi-Tech Science Corporation).

In order to obtain the images of

individual NC, we conducted spin-coating prior to the observation: 80 µL of 0.005-wt% aqueous dispersions of the chitinous NC were spin-coated on a 15 × 15-mm2 surface of a freshly cleaved piece of mica at 3000 rpm for 1 min using a SC4001 spin-coater (Aiden., Ltd.). Wide-angle X-ray diffraction measurements were made for the freeze-dried NC samples and as-provided original chitinous materials with a Rigaku Ultima-IV diffractometer at 20 °C in a reflection mode. Nickel-filtered CuKα radiation was used at 40 kV and 30 mA. The diffraction intensity profiles were collected in the range of 2θ = 5–35 º. Surface tensions of the aqueous suspensions of the chitinous NC were measured according to the Wilhelmy method using an automatic surface tensiometer DY-300 (Kyowa Interface Science Co., Ltd). The viscosities of the suspensions were determined as a function of the shear rate with


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an AR-G2 rotational rheometer (TA Instruments) and a cone fixture (ϕ = 40 mm, 2 ° of cone angle). Micropatterning of NC by inkjet. Inkjet printing was performed using a LaboJet-1000 (Microjet Corporation) with a piezoelectric nozzle (Microjet IJHD-1000; the diameter of spout was 80 µm). The NC inks were filtrated with a 5-µm pore size Nylon syringe filter (Membrane Solutions, LLC.) before injection. The driving signal for inkjet was a rectangular pulse as shown in Scheme 1 including the definitions of the parameters. The pulse sequence was chosen to stabilize droplet formation, which was monitored by a built-in high-speed video camera. For discharging ChNC ink, a representative pulse sequence was given at amplitude of 40 V with pulse widths of t1st = 90 µs, tpause = 30 µs, t2nd = 20 µs, and twaiting = 1860 µs with a prescribed repeating time (m). In the case of dChNC ink, we adopted a set of pulse of t1st = 80 µs, tpause = 0 µs, t2nd = 0 µs, and twaiting = 1920 µs.

Volts repeating m times amplitude

time t1st

tpause t2nd

twaiting

t1st

Scheme 1. Scheme for the rectangular pulse sequence given by the inkjet apparatus used in the present study.

Droplets of the NC suspension were discharged under ambient conditions onto a poly(ethylene terephthalate) film (PET; Toray Industries, Inc., Lumirror T60, 100-µm thickness) or cellophane film (Futamura Chemical Co., Ltd., PL-DG#700, 70 g/m2) placed on a movable stage of the inkjet printer. The distance from the nozzle to the substrate was fixed at 1000 µm. To draw lines, under the continuous stage movement at 50 mm/s, dots composed of m = 10 were overlapped in a linear fashion parallel with the stage movement direction, with a distance p between the dots.


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To measure the dimensions of created inkjet micro-patterns, we used a scanning white light interferometer (WLI) equipped with the above-mentioned AFM apparatus. Cell culture.

Mouse L929 fibroblast cells (European Collection of Cell Cultures) were

subcultured in the Eagle’s MEM at 37 °C in a humidified atmosphere of 95-% air and 5-% CO2. 500 mL of the Eagle’s MEM was supplemented with 50 mL of FBS, 10 mL of 3-wt% Lglutamine aqueous solution, 5 mL of 7.5-wt% sodium hydrogen carbonate aqueous solution, 38.4 mg of penicillin, and 55.7 mg of streptomycin. Prior to the cell adhesion studies, the films (20 mm × 20 mm) printed with the chitinous NC were sterilized by immersing in Eagle’s MEM containing penicillin and streptomycin for 5 h. 2 mL of cell suspension (5.0 × 104 cells/mL) was seeded onto the sterilized specimen. The cells were cultured at 37 °C under a humidified atmosphere of 5- % CO2 and 95-% air. After 24 h, the cell suspension was removed and the surfaces of specimens were washed with PBS (phosphate buffer saline, pH 7.4) twice. Further culturing for the cells attached on film specimens was continued for prescribed period in the Eagle’s MEM medium in the similar manner mentioned above. A two-color fluorescence cell viability test was performed, which permits the simultaneous determination of live and dead cells with two probes. For this, we added 0.2 mL of 2-µM Calcein AM (for living cells) and 0.2 mL of 4-µM Ethidium Homodimer 1 (for dead cells) into the culturing systems and coincubated for 15 min. Samples were imaged with a Keyence Fluorescence Microscope BZ-X700 (Keyence Corporation) with excitation/emission filters at 470/525 nm for Calcein AM and 545/605 nm for Ethidium Homodimer 1, respectively. Recovery of patterned cells and re-culture. A 0.25 w/v% trypsin solution in PBS was prepared: 10 mL of the solution contains 0.5 mL of 5.0 w/v% trypsin and 0.1 mL of 2.0 w/v% ethylenediaminetetraacetic acid (EDTA) solutions in PBS. To prepare 0.25 or 1.0 w / v% Yatalase solution (pH 6.5), Yatalase was added to PBS which had been brought to pH 6.5 with citric acid and sterilized by filtration. After 48 h from seeding the cells on the micropatterning scaffolds, the suspension was removed and washed twice with 3 mL of PBS and the cellophane substrate was transferred to a new 6-well plate. Thereafter, 3 mL of 0.25 w/v% trypsin solution was added thereto, and then the 6-well plate was placed in an incubator at 37 °C under the 5-% CO2 and 95-% air for 5 min.


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Cells were detached from the scaffold by pipetting and collected in a centrifuge tube. After centrifugation at 1000 rpm for 2 min, the supernatant was removed and 5 mL of medium was added to obtain a cell suspension. In the same manner, cell detachment was carried out using 0.25 and 1.0-w/v% Yatalase solution at pH 6.5 to obtain a cell suspension. The obtained cell suspension was diluted with a medium to a concentration of 1.0 × 104 cells/mL and seeded in a volume of 100 µL in a 96-well plate. The seeded cells were subjected to viable cell count measurement and fluorescence microscopy observation. In the measurement of the number of viable cells, 10 µL of a solution of Cell Counting Kit-8 (CCK-8, manufactured by Dojindo Laboratories, Inc.) was added and allowed to perform a color reaction of 1-4 h in an incubator, where

WST-8

(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-

tetrazolium, monosodium salt) was reduced by dehydrogenase activities in cells to give a yellowcolor formazan dye. Thereafter, the absorbance at 450 nm was measured with a plate reader (Tecan Spark 10 M), and the number of viable cells was calculated from the calibration curve. The amount of the formazan dye was directly proportional to the number of living cells (Mean ± SD, n = 5). We also performed cell detachment experiments using human lysozyme (plant expression recombinant, Wako Pure Chemical Industries, Ltd.).

RESULTS AND DISCUSSION In the present study, we try to realize micropatterning cells with the aim of converting chitinous NCs to functional materials by a simple and rational processing method, inkjet printing. For this purpose, we characterize a series of NCs in detail, evaluate their inkjet printability, and examine the behavior of cells on the printed matters. Characterization of ChNC and dChNC. FT-IR spectra of the original chitin powder, ChNC, and two dChNC products are shown in Figure 1. Mutual comparison of the spectra demonstrates that the deacetylation gave rise to a suppression in the relative intensities of amide bands. The degree of deacetylation (DD), representing the molar fraction in percent of the N-acetyl-Dglucosamine units within the chitinous polysaccharides, can be calculated by the following equation:28 DD = 100 − (A1655/A3450 × 100/1.33)


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where A1655 and A3450 were the absorption intensities of amide I (1655 cm−1, C=O of NHCOCH3) and hydroxyl (3450 cm−1) bands, respectively. Two lines connecting 1854–4000 cm−1 and 1277–1854 cm−1 were adopted as the baselines to determine the respective band intensities. The DD values were calculated to be 1.7 and 22.2 % for the original chitin and ChNC, and 41.2 and 61.1 % for the two dChNC products, respectively. Using these DD values, the dChNC products shall be coded as dChNC41.2 and dChNC61.1, respectively. Not small increase in the DD (22.2 %) of ChNC probably resulted from the strong acid treatment (3-M HCl at 95 °C for 6 h) for its preparation procedure. (a)

Transmittance/%

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OH

amide I

(b) (c)

(d)

4000 3500 3000 2500 2000 1500 1000 500 Wavenumber/cm-1

Figure 1. FT-IR spectra of (a) chitin, (b) ChNC, (c) dChNC41.2, and (d) dChNC61.1.

Figure 2 shows typical topographic images taken with an atomic force microscope (AFM) for the ChNC and dChNC samples. We found that even after the alkaline deacetylation, dChNC remained the original nanoscale rod-like structure of ChNC. Table 1 lists their length and width estimated as average ones of 50 nanostructures, from the images. These dimensional data suggest that the chitinous nanostructures have expanded somewhat after the deacetylation treatment.


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Figure 2. AFM topographic images of (a) ChNC, (b) dChNC41.2, and (c) dChNC61.1. Scale bar denotes 1 µm.

Table 1. Dimensional data of ChNC and dChNC, estimated by AFM. These are the average values of fifty non-overlapping nanostructures. sample code

width/nm

length/nm

ChNC

10±4

358±145

dChNC41.2

15±5

557±238

dChNC61.1

17±7

476±183

The reader may have noticed that the DD values determined above from the FT-IR measurement are merely the average ones of the nanostructures as a whole, and the spatial distribution of DD within the respective nanostructure particles is not taken into account. On the other hand, it is well known that chitinous molecules with DD of ~50% and random distribution of the acetylamino substituents are soluble in neutral water. In the deacetylation treatment of ChNC to prepare dChNC, however, since the deacetylation preferentially occurred from the surface of the original ChNC probably, a distribution of DD should be present in the depth direction of the nanostructures.

Therefore, it can be essentially possible to obtain water-

insoluble chitinous nanostructures with an overall average DD of ~50%. Regarding the crystalline forms of the nanostructures, the profiles of wide-angle X-ray diffraction (WAXD) are shown in Figure 3, together with those of as-provided chitin powder and


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chitosan flake for comparison. In ChNC and dChNC41.2, the crystal form was retained as the original α-chitin structure, whereas dChNC61.1 exhibited major diffraction peaks at 2θ = 10, 20, and 22 °. The set of diffractions for the dChNC61.1 was consistent with that of the chitosan flake. The series of data thus supports the above assumption that deacetylation proceeds from the surface: the internal crystal structure of chitin remained in the process of deacetylation of ChNC to obtain dChNC41.2. Moreover, when the deacetylation progressed to a certain extent, the internal crystal changed to that of chitosan. The critical average DD giving rise to the change in the crystal forms seems to be between 41.2 and 61.1 %. Although we here do not deeply pursue such a transition of the crystal structure of ChNC by the alkali treatment, we consider that it is a very interesting research subject.

(e) Intensity (a.u.)

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(d) (c) (b)

(a) 10

15

20 25 2θ/degree

30

35

Figure 3. WAXD profiles of (a) as-provided chitin powder, (b) ChNC, (c) dChNC41.2, (d) dChNC61.1, and (e) as-provided chitosan flake.

Inkjet printing of the chitinous nanostructures. For inkjet printing, 1.25 wt% and 0.20 wt% of ChNC and dChNC aqueous suspensions were used as inks, respectively. According to the manufacturer of our inkjet device, the suitable viscosity and surface tension of inks should be 0.5–40 mPa·s and ≤ 73 mN/m, respectively. These requirements were fulfilled for both the •

ChNC and dChNC aqueous dispersions, since the values were determined to be ~2 mPa·s ( γ =


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1000 s−1) and ~60 mN/m, respectively.

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The driving signal for inkjet was composed of

rectangular pulses as shown in Scheme 1 including the definition of parameters. The pulse sequence was chosen to stabilize the formation of droplets ejected from the nozzle (80-µm diameter), which was monitored by a built-in high-speed video camera. Figure 4 demonstrates the droplet formation of the ChNC dispersion. The time elapsed since the application of the first piezo pulse was taken as t. The parameters of the pulse sequence parameters for the ejection were t1 = 90 µs, tpause = 30 µs, t2nd = 20 µs, and twaiting = 1860 µs. At the initial stage of the discharge, the droplet was connected to the nozzle via a thin filament. Separation of the ChNC dispersion into droplets occurred at t = ~210 µs, and the flight distance of the formed droplet reached 1 mm at t = 380 µs, while satellite droplets can be seen slightly. In this case, the velocity of the droplet was calculated as 5.9 m/s. The ink droplet generally had a diameter of ~100 µm. Eventually, for both the ChNC and dChNC inks, we achieved a constant dropping of ~500-pL droplets at a rate of 5–7 m/s from the nozzle.

Figure 4. Images captured by the CCD camera on the ejection behavior of the ChNC aqueous dispersion at different elapsed times t since the application of the first piezo pulse.

Figure 5 illustrates optical microscopic images of dot-like as well as latticed fine moldings by discharging dChNC61.1 aqueous suspension onto cellophane films by the inkjet printer. The parameters of the pulse sequence for the ejection were t1 = 80 µs, tpause = 0 µs, t2nd = 0 µs, and twaiting = 1920 µs. The dot-shaped dChNC61.1 fine molded articles were formed with droplets of


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1–10 shots. According to the definition of the parameters (Scheme 1) for inkjet printing, the m was 1–10 in this case. Dots of 180 and 500 µm in diameter were obtained by one and ten shots, respectively, indicating that the dot diameter can be controlled by the number of droplets. On the other hand, if the dots are continuously ejected while moving the stage unidirectionally at a constant speed, a line-shaped fine molded product can be obtained. For the line-drawing, the dots are ejected by overlapping part of the dots at regular intervals (pitch, p). If the p is too large, dots are not connected as a line, whereas bulges are formed when the p is too small. In our experiments, by setting p = 200 µm and the stage moving velocity was 30 mm/s, lines could successfully be drawn without forming bulges. In that case, the line width was ~200 µm. Figure 5b demonstrates the lines were with a spacing of 600 µm and arranged in a lattice pattern.

Figure 5. Bright-field micrographs of patterned (a) dots and (b) grid-like lines drawn with 0.20wt% dChNC61.1 aqueous dispersion onto a cellophane substrate by means of ink-jet. Numerals beside the dots correspond to the number of shots of the dispersion.

Scale bars

indicate 300 µm.

Figure 6 displays the surface topology and cross-sectional profile imaged with a white light interferometer (WLI) for dChNC61.1 fine molded materials discharged on a cellophane film. The WLI method is a non-contact technique used to obtain three-dimensional images for quantitatively measuring surface texture of materials with a high lateral resolution.29 Figure 6a shows the morphology of a dot formed with one droplet. From the inserted crosssectional view, it was observed that the central portion of the dot was recessed. This dent was probably caused by the cellophane curving due to water absorption. This behavior was seen for


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the dots produced with the condition of m = 2–6 as well. Also, we observed an accumulation of shaped objects at the edge of the dots, which can be attributed to so-called coffee stain effect. While it should be explained by hydrodynamic terms such as the Marangoni flow and the Weber and Reynolds numbers, it phenomenologically is observed that deposits with a concave cross section are formed after droplets are dried on the solid surface.30 Thus, desired shaping by inkjet is often impaired, but it can be prevented by control of drying behavior of ink and patterning procedure.31,32 Figure 6b shows an image of a dot-like molded object formed with 10 droplets. From the cross-sectional view, the fine molded product was convex.

The formation of the convex

structure unlike the case where the m was 1–6 can be ascribable to a difference in the drying process of the ink: (i) Since a large number of droplets were ejected consecutively, drying of them was delayed on the substrate. (ii) Consequently, the motion of the ink fluid from the center to the edge of the dot became not conspicuous, and the shape of the droplet immediately after ejection was maintained to some extent [12].

The dot-shaped dChNC61.1 microstructures

consisting of 7–9 droplets exhibited a convex structure, too. In this way, we have found that the dot-shaped molded articles become concave or convex depending on the number of ejections. This indicates that if we select discharge conditions, desired shaping can be performed with preventing the coffee stain phenomenon. Figure 6c demonstrates a line-shaped object of dChNC61.1. From the cross-sectional view, we can see that the edges were raised, which represents that the coffee stain effect is occurring in this system. This effect arose because this line was created by discharging droplets one by one while shifting positions. When the other two suspensions of ChNC and dChNC41.2 were used as ink, ejection by the inkjet apparatus was successfully performed as well and dots and lines could be drawn adequately.


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Figure 6. Three dimensional WLI images of patterned dots with (a) 1 and (b) 10 droplets and (c) line drawn with the 0.20-wt% dChNC61.1 aqueous dispersion onto a cellophane substrate via inkjet printing. The figures on the right display the cross-sectional profiles of the part where a white dashed line is inserted in each 3D image.

Cell adhesion on the chitinous microfabricated objects.

In order to investigate the

availability of ChNC and dChNC micro-molding materials for cell micropatterning, mouse L929 fibroblasts were seeded on those discharged onto cellophane substrates by inkjet. Indeed, PET films and polystyrene plates were preliminarily evaluated as substrates. Even though the printing was successfully performed, these substrates themselves exhibited a considerable cell adhesion


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ability. Because the cellophane films are inert to the cell adhesion, they are appropriate as substrates for monitoring the cell adhesiveness of the respective chitinous fine molded articles. The optical micrograph of Figure 7 illustrates the cells being cultured for 48 h on the fine dots modeled with ChNC, dChNC41.2, and dChNC61.1 inks. These dots were formed by ejecting five shots of droplets ten times at 20-s intervals. The figure includes bright field images as well as fluorescent ones obtained by staining cytoplasm of living cells (green) and nucleic acid of dead cells (red).

Since dead cells were not observed markedly, the cytotoxicity of these

chitinous nanostructures was commonly low. Regarding the individual chitinous ink, first, no cells adhered to ChNC. This result is consistent with the report that the cell adhesion is strongly influenced by the DD and that cells less adhere to chitinous substance with low DD.21,26 On the other hand, for dChNC-based objects, cells adhered preferentially onto the dots. However, although we can see the adhesion of the cells to the dot of dChNC41.2, their spreading was not significant. In contrast, for dChNC61.1 with relatively higher DD, we observed not only better adhesion of cells but also their stretching with pseudopod extension.

Accordingly, this

deacetylated chitin nanocrystal has well-balanced hydrophilicity and charge, suitable for adhesion and extension of the fibroblasts. As has been revealed by the WAXD data (Figure 3), the crystal form of this dChNC of DD = 61.1 % was that of chitosan unlike the other two chitinous nanostructures, which may affect the cell adhesiveness.

This is currently under

investigation in detail and we would like to report in the near future.


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Figure 7. Bright-field and fluorescent images of L929 fibroblast cell growth on the patterned dots with five droplets of the NC inks discharged ten times onto a cellophane film by the inkjet printer. Culture time was 48 h. Fluorescence micrographs are displayed for living (green) and dead (red) cells. Scale bars denote 100 µm.

Using the cytocompatible dChNC61.1 ink, various fine molded products were fabricated by inkjet and evaluated as cell micropatterning culture scaffolds. As the sample for micropatterning of cells, the patterns (dots and grid lines) shown in Figure 5 were used. The results are illustrated in Figure 8a and 8b, respectively. In general, the cells are selectively adhered onto any of the shaped objects.


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Figure 8. Examples of the mouse fibroblast L929 preferentially adhered on (a) dots and (b) lattice, each printed with the dChNC61.1 ink by the inkjet device. These are the images obtained by performing the cell adhesion test on the objects shown in Figure 5. Numerals beside the dots (a) correspond to the number of dispersions. Culture time was 48 h in each case. The images are illustrated as fluorescent ones via staining cytoplasm of living cells.

Non-proteolytic cell detachment. In in vitro cell culture systems, an efficient recovery of cells from the culture substrate is an essential process for their further utility such as cell sheet engineering as a platform technology for regenerative medicine. In general, the recovery of cultured cells from the solid substrates requires treatment with a proteolytic enzyme such as trypsin. The treatment can damage cell membranes by hydrolyzing various cell membranebound and extracellular proteins, which can give rise to an impairment of cell function.33–35 In this context, Okano et al. have achieved a non-proteolytic removal of cell sheets formed on thermo-responsive substrates of poly(N-isopropylacrylamide), by a temperature control.27 Alternatively, considering that the dChNC61.1 ink is of biological origin, we supposed that the present micropatterning substrates could be decomposed with enzymes and eventually, the cells adhered and grown on the scaffolds could be recovered nonproteolytically. We thus performed the experiments of cell exfoliation. For this purpose, we used two commercial lytic enzymes which hydrolyze the polysaccharides constituting bacterial and fungal cell walls: lysozyme (human, expressed in plants) for eubacteria and YatalaseTM (digestive enzyme of cell walls of filamentous fungi; origin, Corynebacterium sp. OZ-21) for filamentous fungi.

These have


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chitinase and chitosanase activities.36 As a result, however, the lysozyme did not detach the cells and chitinous substrates. On the other hand, when Yatalase was used, it was possible to remove the dChNC61.1 micropatterned articles considerably from the cellophane bottom layer. However, the detachment behavior of the cells depended on the concentration of Yatalase; the removal rate of cells was not much at 0.25 w/v% of Yatalase, and it was substantial for the case with a 1.0 w/v% solution. As shown in Figure 9, by culturing the detached cells again, it was found that the cells proliferated almost at the same number and speed as those detached with the commonly used trypsin. It is widely known that not only proteins but also sugar chains are present in cell membranes and extracellular matrices. Therefore, we are afraid that such sugar chains can be degraded by the glycolytic enzyme treatment. Nonetheless, we may apply the combination of “cell culture on the chitinous micropatterning substrate” and “cell detachment with glycolytic enzyme” for the experimental systems that analyze the function of proteins and that deal with the cells weak against trypsin, such as hepatocyte.27

In the current situation, the present chitinous

micropatterning substrates can be used for applications such as drug screening in which a fixed number of cells should be attached and their responsiveness is assayed.

5 Living cells × 10-4/cells/well

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Trypsin Yatalase

4 3 2 1 0

0

2

4 6 Culture time/days

8

10

Figure 9. Time-course of the numbers of living cells of mouse fibroblast L929 cultured after detaching from the dChNC61.1 micro-patterned scaffolds with Yatalase and trypsin. The cell


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detachment treatment was conducted with 0.25 w/v% solutions of Yatalase and trypsin for 5 min. The cells suspended by this treatment were cultured in 96-well plates and their numbers were quantified with a Cell Counting Kit-8. The amount of the formazan dye is directly proportional to the number of living cells (Mean ± SD, n = 5).

CONCLUSIONS In order to open up the application of NCs with a simple and reasonable processing method, we established a concept of inkjet technology for micropatterning of animal cells, where aqueous suspensions of chitinous NC were used as an ink for the printing. Since the cell adhesion onto chitinous materials is generally affected by DD, we prepared chitin NC and its deacetylated ones (dChNC) under alkaline conditions. When deacetylated intensively, the original α-chitin crystal form could be changed to that of chitosan, but the nanocrystalline morphology was remained. The series of aqueous suspensions of NCs was successfully discharged by a research inkjet printer, and the deposit form of microstructures could be controlled by printing conditions. The dChNC fine moldings printed on a cellophane substrate were good scaffolds for mouse fibroblasts and the cell micropatterning was accomplished. The present concept can be used for drug screening applications. There are still fundamental and important research elements such as detailed examination of the relationship between DD and crystal system of the chitinous NCs, expansion of cell type, and recognition of the nano- and microstructures by cells.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +81 58 293 2917.

ACKNOWLEDGMENT


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The authors wish to acknowledge Mr. Akira Kagami of Kyowa Interface Science Co., Ltd. for their help with measuring surface tension. This work was financially supported by Grant-in-Aids for Young Scientists (A) (No. 26252025 to YT), Challenging Exploratory Research (No. 26620178 to YT), and Scientific Research (A) (No. 17H01480) from the Japan Society for the Promotion of Science and by the Environment Research and Technology Development Fund (3K153010) of the Ministry of the Environment, Japan.

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Simple Inkjet Process To Fabricate Microstructures of Chitinous

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