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Supporting Information

Injectable Anisotropic Nanocomposite Hydrogels

Direct In Situ Growth and Alignment of

Myotubes

Kevin J. De France,† Kevin G. Yager,

‡ Katelyn J.W. Chan,

† Brandon Corbett,

† Emily D.

Cranston,† and Todd Hoare

†,*

† Department of Chemical Engineering, McMaster University, 1280 Main Street West,

Hamilton, ON L8S 4L8, Canada

‡ Center for Functional Nanomaterials, Brookhaven National Laboratory,

Upton, NY, USA

Keywords: cellulose nanocrystals, anisotropic hydrogels, injectable hydrogels, magnetic

alignment, nanocomposites, small angle x-ray scattering

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EXPERIMENTAL

Preparation and characterization of CNC suspensions

CNCs were produced by sulfuric acid hydrolysis of cotton (GE Healthcare Canada, ashless filter aid CAT

No. 1703-050).1 Filter aid (40 g) was blended and suspended in 700 mL of 64 wt% sulfuric acid (Sigma

Aldrich, 95-98%) at 45 °C for 45 min. The suspension was quenched with purified water (Millipore Milli-

Q grade distilled deionized water, minimum 18.2 MΩ cm resistivity) and centrifuged at 6000 rpm for 10

min. The supernatant was decanted, the bottle was replenished with water, and the CNCs were

recentrifuged until a pellet no longer formed. The CNC suspension was subsequently dialyzed (molecular

weight cut-off = 12–14 kDa) against purified water for a minimum of ten 12+ h cycles, sonicated over an

ice bath using a probe sonicator (Sonifier 450, Branson Ultrasonics, Danbury, CT) for three cycles of 15

minutes, and stored as a 1 wt% suspension in its acid form (pH = 3.2). Suspensions were concentrated as

necessary by evaporation of water at ambient conditions. CNC dimensions ranged from 5 – 12 nm in

cross section and 100 – 200 nm in length, as determined by atomic force microscopy (MFP-3D, Asylum

Research and Oxford Instruments Company, Santa Barbara, CA). The sulfate half-ester content on the

surface of the CNCs was determined via conductometric titration (100 mg of CNC in 100 mL of 10 mM

NaCl as the sample and 2 mM NaOH as the titrant),2 yielding a sulfur content of 0.42 wt% (0.30 charges

per nm2). The electrophoretic mobility (measured on 0.25 wt% CNC suspensions in 10 mM NaCl using a

ZetaPlus analyzer, Brookhaven Instruments Corp.) was –1.86 × 10–8 m2 V–1 s–1.

Preparation and characterization of POEGMA copolymers

Aldehyde- and hydrazide-functionalized POEGMA precursor copolymers were synthesized via chain

transfer radical copolymerization method and subsequent post-functionalization hydrolysis or grafting as

described previously.3 For the aldehyde copolymer, 2,2-azobisisobutyric acid dimethyl ester (AIBMe, 100

mg, Wako Chemicals, 98.5%), column-purified oligo(ethylene glycol) methyl ether methacrylate with a

number average molecular weight of 500 g mol–1 (OEGMA500, 8.0 g, Sigma Aldrich, 95%), synthesized

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functional monomer N-(2,2-dimethoxyethyl)-methacrylamide4 (DMAEAm, 1.2 g) and thioglycolic acid

(TGA, 20 µL, Sigma Aldrich, 98%; 10 wt % in dioxane) were added to a 250 mL round bottom flask.

Dioxane (40 mL, Caledon Laboratory Chemicals, reagent grade) was added to the reaction mixture,

which was then purged with nitrogen for at least 20 min. The flask was moved to a pre-heated oil bath at

75 °C under magnetic stirring to polymerize for 4 h, after which the reaction was allowed to cool to room

temperature. After solvent evaporation, 0.33 M HCl (200 mL) was added to the copolymer solution and

left stirring for 24 h to facilitate conversion of acetal groups to aldehyde groups. The polymer was

subsequently dialyzed (MWCO = 3.5 kDa) against purified water for at least six (6+ h) cycles,

lyophilized, and stored as 20 wt % solutions in PBS buffer at 4 °C. A molecular weight of Mn = 27.1 kDa

and dispersity = 2.5 was measured by aqueous size exclusion chromatography (SEC) using a Waters 515

HPLC pump, Waters 717 Plus autosampler, three Ultrahydrogel columns (30 cm × 7.8 mm i.d. with

exclusion limits of 0–3 kDa, 0–50 kDa and 2–300 kDa) and a Waters 2414 refractive index detector. A

mobile phase consisting of 25 mM N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, 500 mM

NaNO3 and 10 mM NaN3 at a flow rate of 0.8 mL min–1 was used, with results calibrated using narrow-

dispersed PEG standards (106 to 584 × 103 g mol–1, Waters). 1H-NMR (Bruker AVANCE 600 MHz

spectrometer, deuterated chloroform solvent) indicated that 28 mol% of monomer repeat units were

functionalized with aldehyde groups, essentially quantitative functionalization based on the 30 mol%

functionalization target.

For the hydrazide copolymer, AIBMe (74 mg), OEGMA500 (8.0 g), acrylic acid (AA, 1050 µL; Sigma

Aldrich, 99%) and TGA (150 µL, 10 wt % in dioxane) were added to a 250 mL round bottom flask. 40

mL of dioxane was added to the reaction mixture, which was then purged with nitrogen at ambient

conditions for at least 20 min. The reaction was allowed to proceed for 4 h at 75 °C under magnetic

stirring, after which the flask was cooled. Following rotary evaporation of the solvent, DIW (200 mL) and

adipic acid dihydrazide (ADH, 8.66 g, Alfa Aesar, 98%) were added to the polymer, and the solution pH

was adjusted to 4.8 ± 0.1 using 1 M HCl. N’-ethyl-N-(3-dimethylaminopropyl)-carbodiimide (EDC, 3.87

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g, Carbosynth, commercial grade) was then added to mediate grafting of hydrazide groups to carboxylic

acid groups from AA residues, during which the pH was maintained at 4.8 ± 0.1 via dropwise addition of

1 M HCl over 4 h. The mixture was left stirring overnight. The polymer was then dialyzed (MWCO = 3.5

kDa) against purified water for at least six (6+ h) cycles, lyophilized, and stored as 20 wt % solutions in

PBS buffer at 4 °C. SEC (using the same conditions described previously) indicated Mn = 30.3 kDa and

dispersity = 2.6. Conductometric titration (0.1 M NaOH titrant, 50 mg polymer in 50 mL of 1 mM NaCl

sample, ManTech) indicated that 34 mol% of the monomer repeat units were functionalized with

hydrazide groups, again close to the stoichiometric 30 mol% target functionalization.

Investigation of internal hydrogel structure and CNC alignment

Hydrogel samples were extruded from a double barrel syringe directly into quartz capillaries (inner

diameter = 1 mm) and centered between a rare earth magnet mounted in a Bruker Nanostar small-angle x-

ray scattering (SAXS) instrument. The magnet was mounted on a track so that the spacing between poles,

and therefore field strength, could be controlled up to 1.2 T at minimum separation. SAXS experiments

were performed with a Cu K-α rotating anode source (8.04 keV x-rays, wavelength λ = 4.0784 Å–1). A

two-pinhole scheme (using scatterless pinholes) was used for beam collimation. The SAXS area detector

(2048 × 2048 pixels) was positioned 1.150 m from the sample; this configuration yields a q-resolution of

≈ 0.001 Å–1. A typical x-ray exposure time was 60 s, yielding ~500,000 total counts. Detector images

were converted to q-space using a silver behenate calibration standard.

When fitting the experimental data (intensity along angular direction) to the predictions of the scattering

model, the best fit is taken to be the theoretical curve with the smallest χ² difference between the

experimental and model data. Note that in some cases the model curve does not exactly reproduce the

experimental data (due to, e.g., imperfect background subtraction or self-shadowing of the scattering

pattern from the sample). Nevertheless, the shape of the model and experimental curves are well-matched,

and the trends in the calculated S values are consistent with theory. To estimate the error in the S values,

we estimated the sensitivity of the fit parameters by computing the variation in S while allowing for a 5%

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variation in the χ² value. The reported errors are the resulting half-width of the χ² vs. fit-parameter (S)

curves.

For electron microscopy, hydrogel samples were prepared in cylindrical molds as described above and

submerged in 10 mM PBS to swell for at least 24 h. A solvent exchange to ethanol was performed to

minimize the collapse of pore structure, using ethanol solutions of 0, 10, 20, 30, 40, 50, 75, 95 and 100

vol %. Pieces of hydrogel were quick frozen in liquid nitrogen and placed into a pre-cooled (–145°C) FC

4E cryochamber attached to an Ultracut E ultramicrotome (Reichert-Jung Wien, Austria). Thin sections

(unstained) were cut with a diamond knife, placed onto Formvar-coated Cu grids, and allowed to warm to

room temperature prior to imaging. Images were collected using a JEOL JEM 1200 EX TEMSCAN

transmission electron microscope (JEOL, Peabody, MA) operating at an accelerating voltage of 80 kV.

Hydrogel preparation, swelling, and rheology

Hydrogels were prepared by coextruding aldehyde and hydrazide copolymer solutions at 16 wt% in PBS

buffer from a double barrel syringe equipped with a static mixer (MedMix, L-System). CNCs were

incorporated at different loadings (0.2 – 1.65 wt % total mass) in equal amounts in both barrels. Mixtures

were extruded into cylindrical silicone molds (3.5 mm in height x 12.7 mm in diameter) and allowed to

gel both as is and centered between a 0.56 T rare earth magnet (both on the benchtop).

For gravimetric swelling measurements, hydrogel discs were placed in a 12-well cell culture plate and

completely submerged in 10 mM PBS (pH 7.4, 5 mL) at time t = 0. Samples were incubated at room

temperature (22 °C), removed at specified time intervals, gently wicked to remove non-adsorbed PBS,

and weighed. Discs were then re-submerged in 5 mL PBS and weighed at subsequent time intervals until

equilibrium was reached (generally 24 h).

Mechanical tests were conducted at 22°C using a Mach-1 mechanical tester (Biomomentum Inc., Laval,

QC, Canada) operating under parallel-plate geometry. Cylindrical hydrogel discs with a diameter of 12.7

mm and a height of 3.5 mm were used for analysis. Unconstrained compression testing was performed to

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25% of the sample height at a rate of 3% per second to determine the compressive modulus. Shear testing

was also performed whereby samples were pre-compressed by 25% and then subjected to strain sweeps

with amplitudes ranging from 0.1 to 2.2 degrees at a frequency of 0.5 Hz to determine the linear

viscoelastic region (LVE). Dynamic frequency sweeps were subsequently performed within the hydrogel

LVE from 0.1 to 2.2 Hz to determine the shear storage modulus (G’) of the samples. All samples were

tested in at least triplicate; results are presented as an average value, with error bars representing one

standard deviation.

C2C12 mouse myoblast culture, growth, differentiation, and staining

C2C12 Mus musculus mouse myoblast cells were obtained from ATCC: Cedarlane Laboratories

(Burlington, ON) and cultured in growth media (Dulbecco’s modified Eagle medium (DMEM),

supplemented with 10 % fetal bovine serum (FBS) and 1% penicillin streptomycin (PS)) according to

manufacturer recommended protocols. Cells were passaged when 80 – 90 % confluency was reached, and

cells were not used beyond passage 30. For cell growth experiments, hydrogels were extruded into 96

well tissue culture polystyrene plates, allowed to gel in the presence or absence of a 0.56 T magnetic

field, and subsequently seeded with myoblasts at a density of 10,000 cells per well. Following complete

gelation (~ 1 hr), cell-seeded hydrogel samples were supplemented with growth media and incubated at

37 °C for 48 hours. The seeded hydrogel constructs were then stained with CFSE (ThermoFisher

CellTrace Kit) for total protein and DAPI (Sigma Aldrich) as a nuclear counterstain according to

manufacturer recommended protocols and imaged using an Olympus inverted microscope.

Differentiation experiments were carried out both in 2D (seeding the cells on top of the pre-formed

hydrogels) and 3D (encapsulating the cells within the hydrogel precursor solutions and extruding) using

96-well plates. For 2D experiments, hydrogels were extruded into wells in the presence/absence of a

magnetic field as described for the growth experiments, allowed to gel for one hour, removed from the

magnetic field (for aligned samples only), seeded with cells (10,000/well), and moved into an incubator

(37 °C, 5% CO2). For 3D experiments, cells at a density of 500,000/mL were mixed with both hydrogel

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precursor solutions (in PBS), loaded into a double barrel syringe, extruded into wells in the

presence/absence of a magnetic field, and allowed to gel for one hour, after which 200 µL of media was

added and the scaffolds were moved to an incubator. The seeded constructs in both 2D and 3D were

allowed to grow for 48 hours, at which point myoblast differentiation was induced by switching to

differentiation media (DMEM supplemented with 5% horse serum and 1% PS) and culturing for an

additional 8 days. Media was replaced every 2 – 3 days. The seeded constructs were then stained for F-

actin using phalloidin (Abcam CytoPainter Phalloidin-iFluor 488 Reagent, diluted 1:1000 in 2% BSA-

PBS) and DAPI according to manufacturer recommended protocols. Cells were fixed and permeabilized

using 3% paraformaldehyde (PFA) and 0.5% Triton X-100 respectively prior to imaging.

Image analysis

For each dataset, at least 4 fluorescent images with different fields of view were used in order to quantify

overall myotube alignment. Alignment was quantitatively determined in Matlab through an image

intensity gradient algorithm adapted from Karlon et al.5–7 Briefly, for a given microscopy image (contrast

normalized green channel only, using Matlab’s built-in ‘histeq’ function) local F-actin gradient

orientation directions were calculated for each pixel using Matlab’s ‘imgradient’ function. Subsequently,

an accumulator was used to aggregate the dominant local angles in each 35 µm × 35 µm subregion.

Finally, the most commonly occurring overall direction was determined by binning sub-region dominant

angles into 2 degree intervals and selecting the most common. This overall direction was subtracted from

each subregion’s dominant angle to calculate angle deviations for each subregion; the smaller the

deviation angle, the more perfect the alignment. The resulting distributions were visualized as histogram

plots for each image. Histograms from each single image collected were subsequently summed with

those from other images collected (minimum n = 4) under the same experimental condition to remove

bias related to the location of the specific pictures taken.

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ADDITIONAL RESULTS

Small angle x-ray scattering analysis of CNC-POEGMA nanocomposite hydrogels

SAXS data shows how the presence of the hydrogel phase significantly disrupts the long-range ordering

of CNCs in suspension. SAXS data for the pure CNC suspension in the absence of a magnetic field (1.65

wt%, Figure S1, blue curve) are consistent with literature reports,8–10 whereby the relatively broad low-q

scattering peak arises due to the preferred CNC-CNC packing distance between nematic pseudo-planes.

Alternately, the pure POEGMA hydrogel (16 wt%, Figure S1, purple curve) exhibits a broad scattering

feature at intermediate q and a high-q upturn indicative of polymer clustering, a profile well-described by

a model that includes scattering contributions from polymer clusters and polymer chains.11–13 In

comparison, the scattering curve for the CNC-POEGMA nanocomposite hydrogel outside of a magnetic

field (16 wt% POEGMA, 1.65 wt% CNC, Figure S1, black curve) is distinct from the two component

materials, suggesting that the composite yields a new phase with distinct ordering. In particular, the high-

q scattering of the POEGMA hydrogel (associated with the solvation properties of the polymer chains) is

significantly suppressed in the CNC-POEGMA nanocomposite hydrogel, suggesting that polymer

solvation has been modified by introduction of CNCs. This observation is likely due to the strong

interactions between the polymer chains and CNCs, which we have previously observed through both

quartz crystal microbalance and isothermal titration calorimetry experiments.3 Simultaneously, the

suppression of the CNC low-q peak suggests that their mixture with the hydrogel has disrupted the

conventional liquid crystal packing of CNCs, further confirming the presence of strong interactions

between the two components.

While the extent of magnetically induced CNC alignment observed here is lower than that observed for

pure CNC suspensions at high concentrations (where perfect alignment, S ≈ –0.5, is possible), it is notable

that pure CNC suspensions at this dilute concentration (1.65 wt%) show no signs of alignment (S = –0.02

± 0.02) at magnetic field strengths up to 1.2 T.10 We attribute the observed CNC alignment within the

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viscous hydrogel matrix to the reduced Brownian motion (and thus slower relaxation to the isotropic

state) of the CNCs coupled with the smaller free volume available for CNCs to move. Above a critical

concentration in suspension, CNCs are sufficiently crowded that they spontaneously order into liquid

crystalline phases to maximize their translational entropy; we expect that the confined environment

created by the hydrogel similarly encourages CNC alignment. As such, the hydrogel environment and

polymer–CNC interactions together appear to enhance the cooperative behavior of CNCs to effectively

amplify the orienting effect of a weak magnetic field. Our group has previously noted that adsorbing

polymers can induce CNC ordering by effectively shifting the onset of liquid crystal phase formation to

lower CNC concentrations, whereby dilute suspensions of CNCs (0.5 – 3.0 wt%) mixed with adsorbing

nonionic polysaccharide solutions (below the polymer overlap concentration) resulted in gels with regions

of high nematic order.14 We hypothesize that POEGMA adsorption to CNCs similarly acts to increase the

effective CNC volume fraction, leading to increased nematic structuring and therefore increased

susceptibility to magnetic alignment.

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SUPPORTING FIGURES

Figure S1. One-dimensional, circularly-averaged small angle x-ray scattering curves for a 1.65 wt% CNC suspension (blue, no hydrogel), a POEGMA hydrogel (purple, no CNCs), and a CNC-POEGMA nanocomposite hydrogel containing 1.65 wt% CNCs (black).

Figure S2. Kinetics of CNC alignment during gelation for a 1.65 wt% CNC-POEGMA nanocomposite hydrogel exposed to a 1.2 T magnetic field as measured by intensity of the SAXS signal over time during gelation; as the POEGMA network fully crosslinks, aligned CNCs are ‘pulled apart’ slightly, disrupting their otherwise close-packed behavior

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Figure S3. Small angle x-ray scattering (SAXS) angular linecuts taken after 1 hour after initial exposure to a 0 T (A-C), 0.56 T (D-F), or 1.2 T (G-I) magnetic field for a CNC-POEGMA hydrogel containing 0.2 (A, D, G), 0.96 (B, E, H), or 1.65 (C, F, I) wt% CNC. Linecuts were obtained in a representative region of the low-q scattering regime (0.05 Å–1).

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Figure S4. Effects of CNC alignment on directing 2D myoblast differentiation over a larger field of view relative to Fig. 4 (10x magnification): (A) Differentiated myotubes on TCPS, unaligned, and aligned hydrogels after 10 days total of culture. Cells were plated at a concentration of 10,000 cells/well. (B) Image analysis on myotube F-actin filaments from the images in panel A.

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Figure S5. Effects of CNC alignment on directing 3D myoblast differentiation over a larger field of view relative to Fig. 5 (10x magnification): (A) Differentiated myotubes on TCPS, and within unaligned, and aligned hydrogels after 10 days total of culture. Cells were encapsulated within hydrogel precursor solutions at a concentration of 500,000 cells/mL. (B) Image analysis on myotube F-actin filaments from the images in panel A.

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Figure S6. Effects of exposure to a 0.56 T magnetic field on C2C12 myoblast differentiation: (A) Differentiated myotubes on TCPS, having been either unexposed to the magnetic field or exposed for 1 hr directly following cell plating (10,000 cells/well). Images were obtained after 10 days total of culture. (B) Image analysis on myotube F-actin filaments from the images in panel A, and (C) histograms of their orientational order. Histograms are compiled from at least four images for both conditions.

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