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Patterning of Structurally Anisotropic Composite Hydrogel Sheets Elisabeth Prince, Moien Alizadehgiashi, Melissa Campbell, Nancy Khuu, Alexandra Albulescu, Kevin De France, Dimitrije Ratkov, Yunfeng Li, Todd Hoare, and Eugenia Kumacheva Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00100 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Patterning of Structurally Anisotropic Composite Hydrogel Sheets Elisabeth Prince§‡, Moien Alizadehgiashi§‡, Melissa Campbell§, Nancy Khuu§, Alexandra Albulescu§, Kevin De France‡, Dimitrije Ratkov§, Yunfeng Li§, Todd Hoare‡ and Eugenia Kumacheva*§∏£ §

Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, Ontario, Canada, M5S 3H6.

‡

Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada, L8S 4L7. ∏

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, Toronto, Ontario, Canada, M5S 3H6. £

Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College street, Toronto, Ontario, Canada, M5S 3H6.

* E-mail: [email protected]

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Keywords: Hydrogels, cellulose nanocrystals, gelatin, patterning, 3D printing, tissue engineering.

Abstract

Compositional and structural patterns play a crucial role in the function of many biological tissues. In the present work, for nanofibrillar hydrogels formed by chemically crosslinked cellulose nanocrystals (CNC) and gelatin, we report a microextrusion-based 3D printing method to generate structurally anisotropic hydrogel sheets with CNCs aligned in the direction of extrusion. For such hydrogels, we prepared hydrogels with a uniform composition, as well as hydrogels with two different types of compositional gradients. In the first type of patterned hydrogel, the composition of the sheet varied parallel to the direction of CNC alignment. In the second hydrogel type, the composition of the sheet changed orthogonally to the direction of CNC alignment. The hydrogels exhibited gradients in structure, mechanical properties, and permeability, all governed by the compositional patterns, as well as cytocompatibility. These hydrogels have promising applications for both fundamental research and for tissue engineering and regenerative medicine.


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Introduction Mammalian tissues are often spatially heterogeneous, with their compositions and structures varying in high-complexity patterns.1,2 The heterogeneity results in cellular microenvironments with different structures, biochemical cues, and biophysical properties.3 For example, gradients in structure, stiffness, and permeability exist at the boundaries between tissues,4 at the bone/ligament interface, in articular cartilage, and in teeth.5–7 Furthermore, chemical gradients of transcription factors, chemokines, and cytokines play a role in embryogenesis, angiogenesis, and wound healing.7,8 Gradients in the degree of collagen fiber alignment are found across the heart ventricular wall.9 Thus, for both fundamental research and for the applications of hydrogels in tissue engineering and regenerative medicine, it is imperative to fabricate hydrogel scaffolds with compositional and structural gradients. 10 Hydrogel patterning for biological applications should be scalable and compatible with threedimensional cell-culture. The most common approach to hydrogel patterning utilizes photolithography, e.g., photo-initiated radical polymerization of monomers through a photomask.11–14 Alternative methods include acoustic node assembly,15,16 microfluidic patterning,17–19

laser ablation of a photo-labile hydrogel,20 and replica molding.21,22 These

methods are facing a challenge in scalability and compatibility with the encapsulation of cells. Three-dimensional (3D) printing is a powerful tool for hydrogel patterning. In particular, patterned hydrogel sheets have been prepared by continuous extrusion of solutions or dispersions


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of hydrogel precursors through manifold parallel microfluidic (MF) channels, followed by gel formation.19,23–25 Controlled spatial variation in hydrogel composition can be achieved by coextruding several streams of different precursor solutions at varying, yet controllable, relative flow rates.19 This approach is scalable and can be used for the generation of hydrogel sheets with a broad range of patterns by using several distinct hydrogel precursor “inks”. Furthermore, the method is biocompatible and has been used for the generation of cell-laden patterned hydrogel sheets.19,24 Here, we introduce a new composite ink for printing covalently crosslinked hydrogel sheets based on cellulose nanocrystals (CNCs) and gelatin.26 Gelatin, denatured collagen, contains ArgGly-Asp (RGD) sequences that bind to integrin receptors on the cell surface, thereby making the hydrogel bioadhesive.27 On the other hand, CNCs are rod-like, rigid nanoparticles with an average length and diameter of 100-300 nm and 5-20 nm,28,29 respectively, which assemble into a nanofibrillar network30 that resembles the structure of the extracellular matrix proteins.31–34 Furthermore, both gelatin11,35–37 and CNCs38–40 are non-toxic and are cytocompatible to a variety of cell types, both in culture media and in hydrogel constructs. Our rationale was that in the CNC-gelatin hydrogels, the CNCs would act as a filamentous building block (responsible for the nanofibrillar structure), while the gelatin would be a bio-adhesive soft component of the hydrogel. The variation in the composition of this composite hydrogel would provide the ability to tune the structure, stiffness, permeability and adhesive properties of CNC/gelatin hydrogels over a broad range.26 Therefore, the change in hydrogel composition by patterning would lead to the change in its biophysical properties and structure, with potential applications of patterned hydrogels in 3D cell culture and tissue engineering.


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We report 3D printed hydrogel sheets with lateral or longitudinal gradient patterns in their composition, structure, and properties. We used aldehyde-modified cellulose nanocrystals (aCNCs), which reacted with gelatin to form imine crosslinks. In order to form the hydrogel sheets, the gelatin solution and a suspension of a-CNCs were mixed and extruded from a manifold MF device. Covalent crosslinking started immediately after mixing a-CNCs and gelatin, while complete gelation took place after the a-CNC/gelatin suspension exited the MF device. The variation in the hydrogel composition along or across the hydrogel sheet was achieved by varying the relative flow rates of the streams of the suspension of a-CNCs and gelatin solution that were supplied to the MF device. In addition to the gradual variation in composition, mechanical properties, and permeability throughout the hydrogel sheet, extrusion rendered these sheets anisotropic due to the flowinduced alignment of a-CNCs in the mixed precursor suspension, which was preserved in the resulting hydrogel due to the chemical crosslinking of a-CNCs and gelatin. Structural anisotropy is an essential property of many biological tissues, and is important for cell guidance and proliferation41 as well as mass transport of nutrients and growth factors.42 Importantly, we patterned gradients in hydrogel composition that were either parallel or perpendicular to the direction of a-CNC alignment. In other words, we independently controlled the alignment of aCNCs and the patterns in the hydrogel’s composition, thus enabling further studies of the role of anisotropy and composition on cell growth, migration, and differentiation. Experimental Section Materials. Type A gelatin (300 g bloom), sodium periodate, butyl acrylate, 9-vinyl anthracene, potassium persulphate, sodium dodecyl sulphate, and acetic acid were purchased from Sigma Aldrich, Canada and used without further purification, unless otherwise specified. An aqueous


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12.2 wt. % suspension of CNCs was purchased from the University of Maine Process Development Center and dialyzed for 7 days against Milli-Q grade distilled deionized water (DI, 18.2 MΩ cm resistivity) before use. Surface modification of CNCs with aldehyde groups. Oxidation of surface hydroxyl groups of CNCs to yield aldehyde-functionalized CNCs (a-CNCs) was performed as described elsewhere.43 Briefly, sodium periodate (NaIO4) was added to a 1 wt. % suspension of CNCs at a NaIO4/CNC weight ratio of 4:1. The pH was adjusted to 3.5 with acetic acid. The flask was covered with aluminum foil to prevent photodecomposition of NaIO4. The suspension was stirred at 25 °C for 2 h and subsequently quenched by adding ethylene glycol. The suspension of a-CNCs was dialyzed against deionized water for seven days, with replacing of twice a day, and then concentrated by rotary evaporation. The presence of aldehyde groups on the CNC surface was confirmed with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) using a Bruker Vertex 70 spectrometer with a 1.85 mm diameter diamond crystal. The aldehyde group content of the CNC surface was determined by first, converting these groups to carboxylic acid groups in 0.1 M NaOH using an intra-molecular Cannizzaro reaction,43 and subsequently, by titrating with sulfuric acid to determine the consumption of hydroxide ions.43 Quantification of amine groups on gelatin. The TNBS (trinitrobenzene sulfonic acid) assay was used to quantify the number of primary amine groups in gelatin, as described elsewhere.35,44 Briefly, gelatin was dissolved in 0.1 M sodium carbonate buffer (pH = 9) to a final concentration of 0.5 wt. %. Trinitrobenzene sulfonic acid (TNBS) was added to the solution to a final concentration of 0.1 w/v %, and the solution was equilibrated for 4 h at 37 °C. The absorbance of the solution was measured at ߣ = 500 nm. A calibration curve was prepared by measuring the


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absorbance of the solution at ߣ = 500 nm for standard solutions of beta alanine in 0.1 M sodium carbonate buffer, containing 0.1 w/v % TNBS. The calibration curve is shown in Figure S1a. Mechanical Characterization. The Young’s modulus of the hydrogels was determined in cyclic compression experiments using a Mach-1 Mechanical tester (Biomomentum Inc., QC) operating in parallel plate geometry. The hydrogel disks for mechanical testing were 3.25 mm in height and 14 mm in diameter. The disks were compressed by applying 20 % strain in the zdirection at a rate of 0.03 mm/s. The Young’s modulus of the hydrogels was determined by fitting the linear portion of the resulting stress-strain curve. All hydrogels were equilibrated for 24 h before the measurements. Determination of Gelation Time. The gelation time for a-CNC/gelatin hydrogels was determined by the inversion test. A 1 mL vial containing 500 µL of the mixed suspension of aldehyde-functionalized CNCs and gelatin was inverted every 5 min at room temperature. Gelation time was determined as the time when no flow was observed upon inversion. Rheology experiments. The rheological properties of the a-CNC/gelatin hydrogels were characterized using a rheometer (AR-1000 TA Instruments) with a cone and plate geometry, with a cone angle and diameter of 0.9675o and 40 mm, respectively. An integrated Peltier plate was used to control the temperature, and a solvent trap was utilized to minimize solvent evaporation. Frequency sweep experiments were conducted at 37 oC with an amplitude of 1 to 100 Hz and a strain of 1 %. Scanning Electron Microscopy. Hydrogel samples for scanning electron microscopy (SEM) were prepared by first, freezing the hydrogel samples by immersing them in liquid propane and then lyophilizing them to remove water. Liquid propane was used to suppress the formation of ice crystals and preserve the hydrogel structure during freezing.44 The dried hydrogels were


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freeze-fractured and gold-coated using an SC7640 High Resolution Sputter Coater (Quorum Technologies). The samples were imaged with the Quanta FEI Scanning Electron Microscope. Characterization of Hydrogel Permeability. To determine Darcy permeability of the aCNC/gelatin hydrogels, a hydrogel sample with the dimensions 3 mm x 3 mm x 13.7 mm (width x height x length) was formed in a chamber fabricated in poly(dimethyl siloxane) (PDMS). Perfluoroalkoxyalkane tubing (IDEX Health & Science) was used to connect the ends of the chamber to inlet and outlet reservoirs containing 1X HBSS solution. A pressure difference was applied across the hydrogel by varying the height of the inlet reservoir relative to that of the outlet reservoir. The HBSS solution was under the influence of the pressure drop. The value of the volumetric flow rate (Qp) of the HBSS solution perfused through the hydrogel sample was determined by measuring the change in the mass of the outlet reservoir over a particular time interval. The Darcy permeability was determined as ‫ܭ‬௦ =

ఎ௅ொ೛ ஺∆௉

, where A is the hydrogel’s cross-

sectional area (9 mm2), L is the hydrogel length (= 13.7 mm), ∆ܲ is the pressure drop across the hydrogel, and η is the viscosity of HBSS solution (taken as 1.002 cP, the viscosity of water at room temperature). Printing Hydrogel Sheets. To print hydrogel sheets, we used a MF extrusion device19,23–25 with a modified design. A schematic of the experimental setup for printing hydrogel sheets is shown in Figure S8. The precursor liquids, a suspension of a-CNCs and a solution of gelatin, both in HBSS, were supplied to the MF device via two separate inlets (positions 1 and 2 in Figure 6a), mixed in the meandering channel (position 3 in Fig. 5a), split into 32 microchannels, and subsequently, extruded as a sheet from the MF device. The flow rates of the precursor liquids were controlled using two independently controlled syringe pumps (Harvard Apparatus PHD 2000 Syringe Pump, USA). The device was connected to a stage that moved in the direction


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opposite to the direction of extrusion. The linear velocity of the substrate was controlled by using the software Pronterface (http://www.pronterface.com). Synthesis of anthracene-labeled poly(butyl acrylate) latex nanoparticles. Fluorescent latex nanoparticles were synthesized by emulsion polymerization of butyl acrylate (BuA) that was labeled with vinyl anthracene, as described elsewhere.45–47 The average hydrodynamic diameter of the nanoparticles of 50 nm was determined by dynamic light scattering (Figure S6, Supporting Information), and the electrokinetic potential of the nanoparticles was -40 mV. Cell culture. Prior to the encapsulation in the a-CNC/gelatin hydrogel, human breast cancer MCF-7 cells were cultured in 250 mL polystyrene tissue culture flasks. To each flask, 10 mL of Dulbecco’s Modified Eagle Medium with 4.5 g/L glucose, L-glutamine, and sodium pyruvate (DEME, FIBCO), supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen) and 1% (v/v) penicillin/streptomycin, was added. The flasks were incubated at 37 oC with a constant 5% CO2 supply in the incubator. For cell passage, a Trypsin-EDTA solution (0.25 wt. %, GIBO) was used to detach cells from the basement support. After detachment, 5 mL of fresh media was added and the suspension was centrifuged at 184 × g and 20 oC for 3 min. The supernatant was removed, and the pellet was re-suspended in 1 mL fresh media. 300 µL of the cell suspension was then transferred to fresh media in a new flask. Cells were passaged every 5 days. Cell Encapsulation. Prior to cell encapsulation into a-CNC/gelatin hydrogels, a 3 wt.% a-CNC suspension and a 10 wt. % gelatin solution were prepared in Hank’s balanced salt solution (HBSS). Both solutions were sterilized by exposure to ultraviolet light (Sterilaire Lamp, 254 nm, 345 µW/cm2) for 5 min. The gelatin solution and a-CNC suspension were then mixed with a cell suspension to give a final hydrogel composition of Ctotal = 3 wt.%, Cgelatin = 2 wt.% and CaCNC

= 1 wt. % (weight ratio of a-CNC to gelatin (R) is 0.5). The cell density was


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5.0 x 104 cells/well. Cells were cultured in a 96-well plate and incubated at 37 oC at a constant 5% CO2 supply for 24 h before adding an additional 100 µl of fresh media to each well. Live/Dead Assay and Fluorescence Staining. On day 0, 8 and 15 of cell culture, the cells were stained with calcein-AM (Invitrogen, Carlsbad; green fluorescence) and ethidium homodimer-1 (Invitrogen, Carlsbad; red fluorescence) to identify live vs. dead cells. To each well, 100 µL of the assay solution was added and incubated for 45 min at 37 oC. The cells were then imaged by fluorescence microscopy (Nikon, Eclipse Ti). Results and Discussion Figure 1 shows the schematic of the preparation of a-CNC/gelatin hydrogel. Upon mixing of an aqueous suspension of a-CNCs and an aqueous gelatin solution, the aldehyde groups on the aCNC surface react with the primary amino groups on gelatin’s lysine residues to form imine crosslinks.26 In the present work, we explored the properties of a-CNC/gelatin hydrogel formed at varying weight concentration ratios, R, of a-CNC-to-gelatin and the total solids concentration (a-CNC plus gelatin), Ctotal.

Figure 1. Schematic of the formation of a-CNC/gelatin hydrogels. The reaction of aldehyde groups on a-CNC surface with lysine residues of gelatin yields imine crosslinks.


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The a-CNCs were prepared by oxidizing the CNC surface using sodium periodate.43 Transmission electron microscopy (TEM) was used to confirm that CNC modification did not affect their filamentous structure. Figure 2a shows TEM images of CNCs prior to and after modification with aldehyde groups. Both the a-CNCs and CNCs had a characteristic rod-shaped structure. The CNCs were 194 ± 52 and 180 ± 46 nm-long before and after modification, respectively. Thus, there was no significant change in the CNC length after modification (student’s t-test, p > 0.1). The ATR-FTIR spectra of CNCs and a-CNCs are shown in Figure 2b. A peak at 1731 cm-1 in the a-CNC spectrum corresponds to the aldehyde carbonyl stretch, thereby confirming that aldehyde groups are introduced to the a-CNC surface. The degree of aldehyde modification was 8050 µmol of aldehyde groups per gram CNC, determined by titration. Gelation of the mixed suspension of a-CNCs and gelatin was confirmed in oscillatory shear rheology experiments at 37 oC (the temperature of cell culture). While gelatin solutions do not gel at 37 oC, covalent crosslinking with a-CNCs led to gel formation. In rheology experiments, a frequency sweep conducted at 37 oC for an a-CNC/gelatin hydrogel at Ctotal = 3 wt. % and R = 0.5, the storage modulus (G’) was greater than the loss modulus (G’’) over the full frequency range screened (Figure S2, Supporting Information,).48,49


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Figure 2. Surface functionalization of CNCs with aldehyde groups. (a) TEM image of a-CNCs and pristine CNCs (inset). The scale bar is 500 nm. (b) ATR-FTIR spectra of pristine CNCs (blue) and a-CNCs (green). Inset shows a zoomed in part of the spectra corresponding to the aldehyde carbonyl stretch. Prior to the patterning, we examined the composition-dependent properties of the hydrogels with varying Ctotal and R. First, we examined the gelation time of the mixed suspension, which is an important characteristic for extruded patterned hydrogels. Following extrusion from the MF device, slow gelation of the mixed a-CNC/gelatin suspension diminishes control over compositional and structural patterns, while fast gelation causes viscosity buildup and possible blocking of the microchannels. Figure 3a shows the variation of the gelation time of the aCNC/gelatin suspension with composition. The gelation time reduced from 55 to 10 min when Ctotal increased from 3 to 6 wt. %. At higher Ctotal, the concentration of complimentary crosslinkable aldehyde groups on the a-CNCs and lysine groups on gelatin, increased, and the crosslinking rate and the rate of gelation increased. At constant Ctotal and increasing R, no noticeable change in gelation time was observed. This effect can be explained by the ratio


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between the concentration of aldehyde groups and the amine groups: as shown in Figure S1b, aldehyde groups were present in excess relative to amine groups, and thus increasing the amount of a-CNCs did not significantly affect the gelation time. The mechanical properties of the a-CNC/gelatin hydrogels were characterized via cyclic compression at 20 % compressive strain (Figure S3). Figure 3b shows the variation in the Young’s modulus, E, with varying R for hydrogels prepared with Ctotal of 2.5 or 6 wt. %. At both values of Ctotal, the hydrogels became softer as R increased from 0.25 to 4. For 0.25≤R≤4.0, the value of E changed from 14 to 4.3 kPa at Ctotal = 6.0 wt. %, and from 3 to 1.4 kPa for Ctotal = 2.5 wt. %. Both results indicated the capability of tuning hydrogel mechanical properties over a broad range by varying its composition. Since individual CNCs possess high strength,50 the reduction in E with an increasing content of a-CNCs suggested that the change of hydrogel structure, that is, its increasing porosity with increasing R (see Figure 4 below) dominated hydrogel mechanical properties. Since hydrogel permeability governs nutrient supply and waste removal from encapsulated cells,51,52 next, we examined the variation in hydrogel permeability. To ensure that the pressure drop, ∆P, applied to the hydrogel does not lead to the change in hydrogel structure, we ensured that a linear relationship exists between ∆P and the volumetric flow rate of the HBSS solution in the range 1500≤∆P≤3490 Pa for hydrogels (Figure S4). This result also suggested that the hydrogel structure did not change during the perfusion of the HBSS and that a-CNCs and gelatin were chemically crosslinked.


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Figure 3. Composition-dependent properties of a-CNC/gelatin hydrogels. (a) Dependence of gelation time on Ctotal at R = 0.5 ( ), R = 1.0 ( ), and R = 2.0 ( ). Inset: picture of a-CNC/gelatin before (left) and after gelation (right). (b) Effect of varying R on the hydrogel’s Young’s modulus at Ctotal of 2.5 and 6.0 wt. %. (c) Variation in hydrogel permeability with varying R, and Ctotal of 2.5 and 6 wt. %. In (a-c), at least three hydrogels were characterized for each composition.

Figure 3c shows the variation in permeability, Ks, as R changed from 0.25 to 4.0 at Ctotal of 2.5 and 6 wt. %. Changing R and Ctotal allowed hydrogel permeability to be varied over four orders of magnitude. For 0.25≤R≤4.0, the value of Ks was in the range from 9.0 ×10-13 to 1.78×10-10 cm2 at Ctotal = 2.5 wt. % and in the range from 8.26×10-14 to 2.35×10-11 cm2 for Ctotal = 6.0 wt. %. Increasing Ctotal at a particular R resulted in decreasing hydrogel permeability. Both results indicated the capability of tuning transport properties of the hydrogel over a broad range by varying its composition. The relationship between Ks and R was supported by examining composition-dependent hydrogel structure for R of 0.1 and 10, a broader R range than shown in Figure 3.

Figures 4a and b show scanning electron microscopy (SEM) images of the a-

CNC/gelatin hydrogels with R of 0.1 and 10.0, respectively, at Ctotal = 5.5 wt. %; Figures 4c and


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d show hydrogels with R of 0.1 and 10.0, respectively, at Ctotal = 3.0 wt. %. At a higher R, the hydrogel structure exhibited a more pronounced nanofibrillar nature with thicker fibers organized in an open network with larger pores. These trends correlated with increasing permeability of hydrogels with higher a-CNC content. At lower R, the hydrogel’s structure was dominated by the gelatin component, as the hydrogel had small pores, consistent with the low measured permeability of these gels. Hydrogel structure was also influenced by Ctotal: at the same value of R, increase in Ctotal resulted in smaller pores, thus correlating with the observed decrease in hydrogel permeability.

Figure 4. Scanning electron microscopy images of the hydrogels with varying composition. (a) R = 0.1, Ctota = 2.5 wt. % (b) R = 10, Ctotal = 2.5 wt. %. (c) R = 0.1, Ctotal = 5.5 wt. % (d) R = 10, Ctotal = 5.5 wt. %. Scale bars are 1 µm.


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Since tissue engineering and three-dimensional cell culture are key potential applications of patterned a-CNC/gelatin hydrogel sheets, we explored the ability of this hydrogel to support cell culture by examining its cytotoxicity with respect to breast cancer MCF-7 cells. Prior to these experiments, to ensure that a-CNCs are not cytotoxic, we cultured MCF-7 cell in a 1 wt. % aCNC suspension (Figure S5). Figure 5 shows fluorescence microscopy images of live/death staining of MCF-7 cells after 1, 8, and 15 days of culture within a-CNC/gelatin hydrogels with R = 0.5 and Ctotal = 6 wt. %. Live cells were stained by calcein-AM (green), while dead cells were stained with ethidium homodimer (red). The MCF-7 cells show good viability, indicating that the hydrogel is not cytotoxic. Furthermore, on Day 15, the MCF-7 cells grew to form colonies (Figure 5c). Thus, we conclude that a-CNC/gelatin hydrogels can support threedimensional culture.

Figure 5. Culture of MCF-7 cells in a-CNC/gelatin hydrogel sheet (Ctotal = 3 wt. %, R = 0.5). Cells were stained with calcein-AM (green) and ethidium homodimer (red) on (a) day 1, (b) day 8, and (c) day 15. Scale bars are 200 µm.

Next, we proceeded with 3D printing of a-CNC/gelatin hydrogel sheets using a MF extrusion device. The design of the device (reported elsewhere19,23–25 and shown in Figure S7a) included


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two inlets (one for the a-CNC suspension and the other for the gelatin solution), a mixing zone with posts to promote mixing of the a-CNC suspension and gelatin solution, an array of hierarchically branched parallel microchannels, the outlet for sheet extrusion, and a movable stage. We extruded hydrogel sheets with a uniform composition at R = 1 and Ctotal = 6 wt. % (the properties of these hydrogels are shown in Figure 3). A 6 wt. % a-CNC suspension and a 6 wt. % solution of gelatin in HBSS were supplied to inlets 1 and 2 (Figure 6a), respectively, at a flow rate of 3 mL/min, each. Green dye was added to both the a-CNC suspension and the gelatin solution. After mixing in the meandering microchannel and splitting between the parallel microchannels, a high viscosity suspension of a-CNC and gelatin was extruded from the MF device onto a glass slide that was placed on a stage moving at a velocity of 150 mm/min. Under these conditions, the thickness of the extruded gel was 1.58 ± 0.23 mm. The sheet gelled within 10 min is shown in Figure 6b.


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Figure 6. Extrusion of uniform hydrogel sheets. (a) Photograph of the MF sheet-extruding device. The microchannels are filled with a solution of the green food dye. Positions 1 and 2 are the inlets for the supply of the a-CNC suspension and gelatin solution. The precursor solutions are mixed in region 3 and extruded from the outlet 4. Scale bar is 25 mm. (b) A photograph of the uniform a-CNC/gelatin hydrogel sheet (R = 1, Ctotal = 6 wt.%). Green food dye was added to both the precursor a-CNC suspension and gelatin solutions. Scale bar is 20 mm. (c) Schematic illustration of flow-induced a-CNC orientation in the direction of extrusion (indicated by the black arrow). (d) Polarized optical microscopy (POM) image of the extruded a-CNC/gelatin hydrogel as in (b). Inset shows a POM image of the cast a-CNC/gelatin hydrogel with the composition as in (b). Scale bars are 200 µm. Arrows indicate the direction of the polarizer and analyzer relative to the direction of extrusion.


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Figure 6c shows a schematic of the expected flow-induced alignment of a-CNCs in the extruded hydrogel sheets, as indicated by the arrow.53 Indeed, the hydrogel sheets exhibited birefringence when imaged using polarized optical microscopy (Figure 6d), in contrast with a cast a-CNC/gelatin hydrogel sheet with the same composition (Figure 6d, inset). The birefringence was caused by the shear-induced alignment of the a-CNCs during extrusion. While the relaxation time of a-CNCs is on the order of several miliseconds,54 the crosslinking of aCNCs and gelatin and the resulting increase in viscosity preserved the orientation of a-CNCs in the hydrogel. Next, we extruded a-CNC/gelatin hydrogel sheets with two types of gradients in composition (and thus structure and properties). In the first case, the hydrogel had a longitudinal gradient in R that was parallel to the long axis of the sheet (L axis); in the second case, the hydrogel had a transverse gradient in Ctotal, which was parallel to the short axis of the sheet (W axis) (Figure 7a).

Figure 7. Longitudinal hydrogel patterning. a) Photograph of the longitudinal gradient hydrogel. Red arrow indicates the direction of increase in R; the blue arrow indicates the direction of aCNC alignment. (b) Variation in PL intensity (λexc = 405 nm) across the hydrogel sheet. Inset shows the positions at which PL intensity was measured (W = 5 mm ( ), 12 mm ( ), 20 mm ( ). Inset shows schematically the positions of the measurements of PL intensity along the hydrogel. In the longitudinal direction, PL intensity was measured at 20 values of L, as determined by the


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software of the microscope. (c) POM image of the hydrogel taken at L = 43 mm and W = 17 mm. Scale bar is 300 µm. To achieve longitudinal patterning, the ratio of flow rates of the individual liquid streams carrying the a-CNC suspension and the gelatin solution was continuously changed during the course of extrusion. More specifically, the flow rates of the streams of the a-CNC suspension and gelatin solution varied from 6-to-1.5 mL/min to 1.5-to-6 mL/min, over 14 sec (the linear velocity of the substrate was 200 mm/min). The a-CNC suspension and gelatin solution had concentrations of 6 wt. %, each, and a green food dye and anthracene-labeled 50 nm latex nanoparticles were added to the stream of the gelatin solution). Extrusion yielded a hydrogel sheet with Ctotal = 6 wt. % and R varying along the sheet from 4 to 0.25. Figure 7a shows an image of a 1.65 ± 0.14 mm-thick hydrogel sheet with L = 45 mm and W = 25 mm, in which the value of R increased from the back side of the sheet (L = 0) to the front of the sheet (L = 45 mm). To characterize the gradient in composition in this hydrogel, following extrusion and gelation, we measured the variation in photoluminescence (PL) intensity in the longitudinal direction in the patterned sheet (Supporting Information, Section S10). To examine pattern consistency, the PL intensity was measured at several positions (W) along the L-axis. Figure 7b (inset) shows schematically the positions of the measurements of PL intensity along the hydrogel for 1≤W≤15 mm at L of 5, 15, 25, and 30 mm for each value of W. Figure 7b shows a gradual decrease in PL intensity from L = 0 to L = 30 mm, indicating that there is a gradual reduction in the concentration of anthracene-labeled latex markers and thus a gradual decrease in gelatin concentration at the expense of increasing a-CNC content (and thus increase in R). The variation in PL intensity with L was consistent at all the values of W, indicating that the printed pattern was consistent across the hydrogel width. As follows from Figure 3, the existence of the gradient


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in R resulted in a gradient in the hydrogel physical properties: from R ≈ 0.25 to R ≈ 4 along the hydrogel the Young’s modulus decreased from 14 to 4.3 kPa and the permeability increased from 8.63×1014 to 2.35×10-11 cm2. Polarized optical microscopy (POM) in Figure 7c revealed that the a-CNCs in the longitudinally patterned hydrogel sheet were aligned, and therefore the hydrogel had an anisotropic structure. Notably, the alignment of a-CNCs and the direction of the variation in R were both parallel to the direction of extrusion, that is, in the L direction. The direction of gradient and a-CNC alignment are indicated by the red and blue arrows in Figure 7a. Next, we conducted transverse hydrogel patterning, that is, the hydrogel composition was changed along the W direction (perpendicular to the direction of extrusion). To prepare these hydrogels, two pre-mixed a-CNC/gelatin suspensions in HBSS (Hank’s Balanced Salt Solution) were introduced into inlets 1 and 2 of the MF device (as shown in Figure 5a): one at Ctotal = 6 wt.%, and the other at Ctotal = 2.5 wt. % (each at R = 1). Anthracene-labeled latex nanoparticles and green food dye were added to the liquid stream with Ctotal = 6 wt. %. Both liquids were supplied to the MF device at a flow rate of 3 mL/min. The linear velocity of the stage was 200 mm/min. The device used to prepare the transverse gradient had a modified design, such that there was less mixing between the two components (design shown in Figure S7b). Due to the difference in the viscosities of the liquids with different Ctotal, and their laminar flow in the meandering mixing zone, mixing of the two suspensions was incomplete,55 thus giving rise to a gradient in Ctotal across the hydrogel sheet. The experimental details of extrusion are provided in Supporting Information (Figure S8 and S9). Figure 8a shows an image of the 1.3±0.2 mm-thick a-CNC/gelatin hydrogel sheet with R = 1 and transverse gradient in Ctotal. The red arrow in Figure 8a indicates the direction of the increase


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in Ctotal along the short axis of the sheet. The consistency of transverse patterning was characterized by measuring the variation in photoluminescence (PL) intensity across the hydrogel sheet (parallel to the W-axis) at several values of L. Figure 8b, inset shows schematically the positions of the measurements of PL intensity across the hydrogel (for W from 1 to 15 mm), at L of 5, 10, 15, and 20 mm. Figure 8b shows a gradual decrease in PL intensity from W = 1 (Ctotal ≈ 6 wt.%) to W = 25 mm (Ctotal ≈ 2.5 wt. %), consistent for all the values of L. A gradual decrease in PL intensity with W indicated a gradual reduction in Ctotal across the hydrogel sheet. The variation in PL intensity with W was consistent at each value of L, indicating that the pattern was reproducible across the hydrogel sheet. The existence of the transverse gradient in Ctotal resulted in a gradient in the hydrogel properties; from W = 0 (Ctotal ≈ 6 wt.%, R = 1.0) to W = 25 mm (Ctotal ≈ 2.5 wt. %, R = 1.0) the Young’s modulus changed from 8.7 to 2.1 kPa and the permeability increased from 9.95×10-13 to 2.57×10-12 cm2. Figures 8c shows a POM image of the hydrogel sheet with a transverse gradient in Ctotal. The hydrogel was birefringent, indicating the shear-induced alignment of a-CNCs. The direction of the gradient in Ctotal was orthogonal to the direction of a-CNC alignment, as shown with red and blue arrows in Figure 8a, in contrast to the longitudinal gradient hydrogel.


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Figure 8. Transverse hydrogel patterning. (a) Photograph of the gradient hydrogel. The red arrow indicates the direction of the increase in Ctotal changes; the blue arrow indicates the direction of a-CNC alignment. The W and L axes are indicated. (b) Variation in PL intensity (λexc = 405 nm) across the hydrogel sheet. Inset shows the positions at which PL intensity was measured (L = 5 mm ( ), 15 mm ( ), 25 mm ( ), and 35 mm ( )). In the transverse direction, PL intensity was measured at 15 values of W, as determined by the software of the microscope. Lines and shaded region are added for eye guidance. (c) POM image of the hydrogel taken at L = 23 mm and W = 2 mm. Scale bar is 150 µm.

Conclusion We report the preparation of composite nanofibrillar a-CNC/gelatin hydrogels with tunable gelation time, permeability, mechanical properties, and structure. These properties were changed in two ways: by changing R (the ratio of the concentrations of a-CNC and gelatin) and Ctotal (the total concentration of a-CNCs and gelatin in the precursor mixed suspension). With increasing R, the pore size increased, which resulted in higher hydrogel permeability and lower stiffness. Stiffer and less permeable hydrogels were prepared as Ctotal increased. The hydrogels were used for cell culture and for the growth of colonies of MCF-7 cells. A microextrusion 3D-printing method was used to prepare a-CNCs/gelatin hydrogel sheets with a uniform composition, as well as with two different types of compositional gradients. In the first type of patterned hydrogel, the concentration of a-CNC-to-gelatin (R) was varied along the long axis of the hydrogel sheet, that is, parallel to the direction of flow-induced a-CNC alignment. In the second type of patterned hydrogel, the total concentration of a-CNCs and gelatin (Ctotal) was varied across the hydrogel, that is, orthogonal to the direction of a-CNC


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alignment. In this pattern, the a-CNCs were aligned in the direction of flow during the extrusion, which rendered an anisotropic structure to the hydrogel sheets. As such, the printing strategy enables the production of hydrogels with well-defined compositional gradients in the desired direction, and thus the variation in structure, permeability, and mechanical properties. Owing to the cytocompatibility of both the hydrogel components and the microextrusion method,19,24 the scalability of the patterning method, the ability to control the composition and anisotropic properties of the hydrogel, and the low cost of the hydrogel constituents, the microextruded patterned a-CNC/gelatin hydrogel sheets have promising applications for fundamental studies of the role of anisotropy in cell behavior and as a scaffolds for cell culture and tissue engineering. ASSOCIATED CONTENT Supporting Information The supporting information includes a description of the fabrication of the microfluidic devices, the methods and results for characterizing the molar ratio between amine groups and aldehyde groups in the hydrogel, rheology data, stress-strain experiments for a-CNC/gelatin, a description of the methods for measuring hydrogel permeability, live-death staining images of cell culture in a-CNC suspensions, size distribution of anthracene labeled latex nanoparticles, the design of the MF devices, the description of the experimental set up for microextrusion, and the method used to measure PL intensity in the hydrogel sheets. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was supported by NSERC Canada (Discovery and Strategic grants). E.K. is grateful to the Canada Research Chair program (NSERC Canada). E.P acknowledges NSERC Canada Graduate Scholarship. M.A. thanks NSERC Vanier Canada Graduate Scholarship.

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