Biomimetic Inks Based on Cellulose Nanofibrils and Cross-Linkable

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Cite This: ACS Appl. Mater. Interfaces 2017, 9, 40878-40886

Biomimetic Inks Based on Cellulose Nanofibrils and Cross-Linkable Xylans for 3D Printing Kajsa Markstedt,†,‡ Alfredo Escalante,§ Guillermo Toriz,†,§ and Paul Gatenholm*,†,‡ †

Wallenberg Wood Science Center, Kemigården 4, 41296 Gothenburg, Sweden Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemigården 4, 41296 Gothenburg, Sweden § Department of Wood, Cellulose and Paper Research, University of Guadalajara, Guadalajara 44100, Mexico

ACS Appl. Mater. Interfaces 2017.9:40878-40886. Downloaded from pubs.acs.org by TULANE UNIV on 01/19/19. For personal use only.

‡

ABSTRACT: This paper presents a sustainable all-wood-based ink which can be used for 3D printing of constructs for a large variety of applications such as clothes, furniture, electronics, and health care products with a customized design and versatile gel properties. The 3D printing technologies where the material is dispensed in the form of liquids, so called inks, have proven suitable for 3D printing dispersions of cellulose nanofibrils (CNFs) because of their unique shear thinning properties. In this study, novel inks were developed with a biomimetic approach where the structural properties of cellulose and the cross-linking function of hemicelluloses that are found in the plant cell wall were utilized. The CNF was mixed with xylan, a hemicellulose extracted from spruce, to introduce crosslinking properties which are essential for the final stability of the printed ink. For xylan to be cross-linkable, it was functionalized with tyramine at different degrees. Evaluation of different ink compositions by rheology measurements and 3D printing tests showed that the degree of tyramine substitution and the ratio of CNFs to xylan−tyramine in the prepared inks influenced the printability and cross-linking density. Both two-layered gridded structures and more complex 3D constructs were printed. Similarly to conventional composites, the interactions between the components and their miscibility are important for the stability of the printed and cross-linked ink. Thus, the influence of tyramine on the adsorption of xylan to cellulose was studied with a quartz crystal microbalance to verify that the functionalization had little influence on xylan’s adsorption to cellulose. Utilizing xylan− tyramine in the CNF dispersions resulted in all-wood-based inks which after 3D printing can be cross-linked to form freestanding gels while at the same time, the excellent printing properties of CNFs remain intact. KEYWORDS: 3D printing, cellulose nanofibrils, cross-linking, hemicellulose, composite

1. INTRODUCTION In the transformation toward a bioeconomy, sustainable materials have an essential role.1 Forests are, therefore, a highly valued resource of which renewable materials are synthesized from carbon dioxide and water. Cellulose, the main component in trees and the most abundant biopolymer on earth, is widely used in the form of wood pulp for use in everyday products such as paper, cardboard, packaging, and textiles. In a bioeconomy, wood-based materials are foreseen to be used in a much more varied way, to an extent replacing plastics but also to be used in high-value products such as electronics,2 lightweight composites,3 and tissue engineering.4,5 Apart from new processing technologies, optimizing the use of wood as a resource for future products requires novel and well-characterized materials. This includes cellulose in a variety of form and modifications, which in turn affect solubility, polydispersity, and ionic charge. Recently, the progress is driven by the development in processing of cellulose nanofibrils (CNFs).6 By introducing pretreatment steps of the pulp (enzymatic, carboxymethylation, TEMPO oxidation), the © 2017 American Chemical Society

energy consumption, and thereby the cost, has been lowered and enabled production of bulk amounts of CNFs.7,8 CNFs have a high surface area which give them suitable properties as a dispersant and rheological modifier.9 It is foreseen that CNFs could be used in a wide range of products such as cosmetics, yoghurts, cement, and paints.10,11 CNFs also show high mechanical strength and can be dried into strong and transparent films and thereby compete with plastic films for packaging applications and electronic devices.12−15 Common for all prospected applications is that all processing of CNFs is in water systems. We have found that the properties of CNFs also are suitable for 3D printing.16,17 3D printing is an emerging technology enabling customized products, minimal material usage, and quick adaptation from idea to product. On the basis of a 3D model, the material is dispensed layer by layer by a printing Received: September 4, 2017 Accepted: October 25, 2017 Published: October 25, 2017 40878

DOI: 10.1021/acsami.7b13400 ACS Appl. Mater. Interfaces 2017, 9, 40878−40886

Research Article

ACS Applied Materials & Interfaces

3. METHODS

head controlled by an X, Y, Z-stage. If the dispensed material is liquid, it is referred to as ink. Suitable inks for 3D printing flow easily through the nozzle while solidifying or forming a gel upon dispensing. The speed of the solidification or gelation process will influence both the printing fidelity and the ability to print subsequent layers. The CNF dispersions fulfill the requirements of an ink; because of shear thinning properties, they are easily dispensed and quickly regain a high viscosity once the applied force is removed. For dispensing of CNF dispersions, either an ink-jet or a pneumatic printer head is suitable because they can dispense the material in liquid form. At the same time, CNF also has a high viscosity at zero shear, allowing a whole construct to be printed without collapsing. After printing, different approaches have been applied to crosslink or solidify the printed CNFs to enable handling of the printed construct. For example, CNFs have been printed together with alginate and ionically cross-linked,16 or CNFs have been freeze-dried.17 Also, cellulose nanocrystals which are similar to CNFs have been printed with resin and UV-crosslinked.18 The structure of the secondary cell wall of trees, which is the main building block of wood, is not maintained solely by cellulose. In the cell wall, hemicellulose and lignin serve as a cross-linked matrix, which helps bind and keep the cellulose structure in place. Because of hemicelluloses’ natural affinity to cellulose, several biopolymers have been studied where hemicellulose influences the properties of cellulose19−21 and functions as a plasticizer of cellulose films22 or acts as a crosslinking component in composites.21 The presence of hydroxyl and carboxylic groups on hemicelluloses makes them suitable for chemical modification to introduce new functionalities. To obtain hydrogels from hemicelluloses, several chemical modifications have been studied to introduce cross-linking such as thiol-functionalization,23,24 methacrylate derivatives,25 and grafting of acrylic acid.26 We have previously shown that hemicelluloses extracted from spruce, such as xylan and galactoglucomannan, can be modified with tyramine, a molecule similar to lignin, to allow for enzymatic cross-linking using horseradish peroxidase (HRP).27,28 The enzymatic crosslinking is a fast process and starts within seconds as soon as HRP and H2O2 are present. It is also an irreversible reaction and results in a stable hydrogel. Inspired by the roles cellulose and hemicellulose have in the plant cell wall, we present an allwood-based biomimetic ink for 3D printing consisting of CNFs and cross-linkable xylan.

3.1. Synthesis of XT. The extracted xylan was conjugated with tyramine by a coupling reaction, following the procedure of Kuzmenko et al.27 In brief, xylan was dissolved in deionized (DI) water (25 mg/ mL, 15.3 mol COOH per liter H2O) under stirring and heating at 70 °C for 30 min. For activation of the carboxylic groups, an EDC/NHS mixture was added to the xylan solution, followed by 30 min of magnetic stirring at room temperature. After stirring, tyramine solution in DMF (10.4 mg/mL) was added dropwise to the mixture and stirred under N2 for 3 days at room temperature. The formed XT conjugate was precipitated in cold ethanol and vacuum-filtrated. The amount of reactants was based on the calculated concentration of COOH in xylan: 0.612 mmol per gram xylan. Most batches of XT were prepared with a ratio of 1.8/1.8/1 for EDC/NHS/COOH, and for tyramine/ COOH, the ratio was 1.5/1. The batches were either prepared from 500 mg xylan or 1500 mg xylan. For 500 mg xylan, a batch with less reactants (EDC/NHS/COOH ratio of 1.35/1.35/1) was also prepared. The resulting degree of substitution of tyramine from the different batches was determined by 1H NMR analysis (400 Hz, Varian-Agilent spectrometer). XT samples were dissolved in D2O (16 mg/mL). The ratio between glucuronic acid units conjugated with tyramine and the total amount of acidic units in the initial xylan was used to evaluate the degree of substitution (DS). The DS was measured from the integral of the signals given by the aromatic protons on tyramine (δ 6.75 and 7.05) and anomeric protons of xylan (δ 5.15−5.30). At full substitution, the expected ratio between the aromatic protons (H10 + H11) and anomeric protons (H1) would be 4:1. Setting the integral of H1 to 1, the degree of substitution was thus calculated as DS = 100 ×

H10 + H11 4

(1)

3.2. Quartz Crystal Microbalance with Dissipation. The XT conjugate was compared to the unmodified xylan with regard to cellulose affinity upon adsorption. Adsorption kinetics of xylan and XT onto CNF model surfaces were monitored with quartz crystal microbalance with dissipation (QCM-D, Q-Sense E4, Biolin Scientific, Sweden). Cellulose-coated quartz crystal sensors (QSX 333, Biolin Scientific, Sweden) were rinsed with Milli-Q water and ethanol and plasma-treated prior to use. In QCM, adsorption is measured as a change in oscillation frequency, Δf, which is proportional to the adsorbed mass per area, m, including water. This relation is expressed by the Sauerbrey equation m=C

Δf n

(2)

where C is the sensitivity constant (−0.177 mg·m−2·Hz−1) and n is the overtone number (3rd, 15 MHz).30 Also, the change in energy dissipation can be monitored to give information on the rigidity of the adsorbed layers. For the QCM-D experiment, the quartz crystal was exposed, in a flow cell (150 μL/min, 25 °C), to either xylan or XT solutions in water (0.1 g/L). Rinsing steps with Milli-Q water were included both before flowing of xylan/XT to stabilize swelling of the cellulose coating on the quartz crystal and after flowing to desorb loosely attached mass. 3.3. Ink Preparation. Inks for 3D printing were prepared from CNFs and xylan with different degrees of tyramine substitution. The synthesized XT was added to CNFs in two different manners: as a dry powder or in solution (300 mg/mL in H2O, stirring at 60 °C, 60 min). In the case of the XT solution, the ink was mixed by hand followed by running the ink in a SpeedMixer (DAC 150 FVZ-K, Synergy Devices Ltd) for 2 × 30 s. For the addition of XT as a dry powder, the ink was mixed by hand at 60 °C for 5 min followed by sonication for 1 min. The ink was stored at 2−8 °C overnight. Before using the ink, speed mixing was conducted for 2 × 30 s. To ensure uniform cross-linking of the inks, HRP (0.6 mg/mL) was added to the final inks and mixed with a spatula before usage in cross-linking measurements or printing of the ink.

2. MATERIALS CNF dispersion (3%) in water was kindly provided by Stora Enso (Karlstad, Sweden). The CNFs were processed from soft wood pulp as described by Päak̈ kö7 et al. by enzymatic pretreatment followed by mechanical shearing and high-pressure homogenization. For ink preparation, CNF dispersions were concentrated to 3.6% by centrifugation at 4000 rpm for 2 × 30 min. Arabinoglucuronoxylan (xylan) was extracted from Norway spruce by alkali extraction following the Wise and Timell methods as previously reported by Escalante et al.29 The extracted xylan had a molecular weight of 12 700 g/mol and contained 10 wt % 4-O-methyl-glucuronic acid. For the conjugation of xylan with tyramine, chemicals were purchased from Sigma-Aldrich: tyramine, N-hydroxysuccinimide (NHS), N,N-dimethylformamide (DMF) (anhydrous, 99.8%), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (≥99.0%). For enzymatic cross-linking of inks prepared from CNF and xylan− tyramine (XT), H2O2 (30 wt %), and HRP (256 U/mg), were purchased from Sigma-Aldrich. 40879


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Friendly by Tetralite, Thingiverse31) was printed. The printing speed was set to 40 mm/min, and the layer height was set to 400 μm. After printing, the printed constructs were cross-linked by submerging them in a bath of 1% H2O2 solution in water. On the basis of the results from the rheology study, 10 min was chosen as a sufficient time for cross-linking.

3.4. Atomic Force Microscopy. The diameter of the CNFs in the CNF dispersion and in inks with XT was measured by atomic force microscopy (AFM). The samples were prepared by diluting the CNF dispersion and inks with DI water to obtain a CNF concentration of 10 ppm followed by centrifugation for 15 min at 1000 rpm to remove larger aggregates. For each sample, the supernatant was dropped onto freshly cleaved mica surfaces and left to dry at room temperature for 24 h. The dried samples were examined by AFM using a Digital Instrument Dimension 3000 with a type G scanner. The measurement was performed in a tapping mode with a standard silicon tip to determine the width of the CNFs. 3.5. Rheology. Oscillation time measurements were conducted to study the cross-linking time and the difference in gel strength. The inks were measured with an 8 mm plate−plate at a gap distance of 500 μm. After starting the measurement, 0.5% H2O2 was added to the rim, and the change in storage modulus was registered. Storage modulus of the inks was given by amplitude sweeps at a frequency of 1 Hz and controlled stress from 1 to 5000 Pa. The yield stress of the inks was defined as applied stress at the crossover point of the storage modulus, G′, and the loss modulus, G″ (G′ = G″). 3.6. Compression. The mechanical properties of the cross-linked inks were evaluated by using a universal testing machine (Instron model 5565A, UK) equipped with a 10 N load cell. Measurements were conducted at room temperature in unconfined compression, with a strain rate of 1% s−1 until 50% compressive strain was reached. Bluehill software (Instron) was used to calculate the compressive stress and strain, compressive stiffness (tangent modulus) at 30% strain, and compressive stress at 30% strain for all samples (n = 5 per group). Measurements were performed on discs prepared from Ink19a and Ink19c (Table 1). The discs were casted by dispensing droplets of

4. RESULTS Cross-linkable xylans, XT, were designed and synthesized to be used as a component together with CNFs in a biomimetic ink. To evaluate the influence of tyramine on the cross-linking ability of XT in the inks, xylan with three different degrees of substitution (DS) was synthesized. The DS was determined by 1 H NMR. Figure 1 shows 1H NMR spectra of XT dissolved in

Table 1. Ink Composition for Inks Prepared from CNF and XT with Different Degrees of Substitutiona

Figure 1. 1H NMR spectra of XT dissolved in D2O. (A) Anomeric protons. (B) Aromatic protons.

final composition ink

DS (%)

ϕCNF

water (wt %)

CNF (wt %)

XT (wt %)

Ink0b Ink15 Ink32 Ink19c Ink19b Ink19a

0 15 32 19 19 19

33 33 33 20 27 34

92.3 92.3 88.4 86.8 92.2 91.2

2.6 2.6 3.3 2.7 2.0 3.0

5.11 5.11 6.6 10.6 5.8 5.8

D2O where the peaks for the anomeric protons (δ 5.15−5.30) and the aromatic protons (δ 6.75 and 7.05) were identified. From the integral of these peaks, the degree of substitution was calculated by eq 1. The DS varied for different samples of XT, as a result of varying batch sizes. The synthesis was performed on xylan samples of 2000, 1500, and 500 mg which resulted in DS values of 15, 19, and 32%, respectively. Thus, smaller batches resulted in higher DS. Moreover, the smallest batch was synthesized with lower amounts of EDC and NHS. This indicates that the synthesis can be further optimized to minimize the amount of reagents, especially to increase the DS for larger batches. Xylan with four different degrees of substitution (DSs of 0, 15, 19, and 32%) was mixed with CNFs to prepare inks. Additionally, to prepare inks from XT with different DSs, XT with a DS of 19% was used in inks where the ratio of CNF to XT was varied. The ratio of CNF, ϕCNF, was given by mCNF ϕCNF = 100· mCNF ·mXT (3)

The final concentration of water, CNF, and XT is noted as the weight percentage with regard to the total weight of the ink. All inks also contained 2 μL HRP/mg XT. bInk0 was prepared from CNF and xylan which has not been modified with tyramine. Thus, 5.11 is the wt % of pure xylan. a

ink in casting units (Q-Gel bio, Lausanne, Switzerland) followed by cross-linking in 1 wt % H2O2 for 10 min. Each disc was then punched to a diameter of 6 mm. The measured thickness of the discs was 1.78 and 1.88 mm for Ink19a and Ink19c, respectively. The results are presented as mean ± SD and statistically analyzed with a one-way analysis of variance with a posthoc Scheffé test comparing each set of data. 3.7. Printability. 3D printing of the XT/CNF inks was evaluated with the 3D bioprinter 3D Discovery from RegenHU, Switzerland. The inks were compared primarily with regard to their line resolution by printing circular grid structures consisting of two layers. The grids (10 mm diameter, 1.5 mm line width, 0.4 mm layer height) were printed with a conical needle tip (420 μm outlet diameter) connected to a pneumatic printer head. The printing speed was set to 10 mm/min for all inks. The printing pressure was adjusted for each ink so that a continuous flow was achieved and so that the flow was sufficient to form continuous dispensed lines at the given printing speed. The ink which showed best printability and cross-linking at the given printing parameters was used for printing a 3D structure. For the 3D structure, a 3D model of a chess piece, either queen or rook (Chess SetPrint

where all masses, m, are given as dry weight. A total of 6 different inks were evaluated and are summarized in Table 1. The inks were named after the DS of the XT used in each ink sample. A critical parameter of 3D printing with liquids, so called inks, is printability. There is a lack of standards to define printability, and the evaluation method of printability depends on the application. The printed ink must at least be able to keep the shape given by dispensing, for example, printed lines should be continuous, not flow out, and have a diameter similar to the 40880


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Figure 2. (A) Ink composition graph with varying CNF concentration and XT concentration divided into four parts with respect to predicted printability and cross-linking ability. Blue section: inks are printable but not cross-linkable. Green section: inks are printable and cross-linkable. White section: inks are neither printable nor cross-linkable. Yellow section: inks are cross-linkable but not printable. (B) A queen chess piece printed with pure CNFs which cannot cross-link. (C) An unsuccessful grid printed with XT. (D) Ink composition graph where the stars indicate the placement of the prepared inks with respect to predicted printability and cross-linking ability.

Figure 3. (A) Change in frequency (top) and dissipation (bottom) as a function of time for adsorption of xylan (black) and XT (red) on cellulose model surfaces. The rinsing step with Milli-Q water is seen at 22 min. Measurements were conducted at 296 K. Results are shown for the third overtone of quartz crystal resonance frequency. (B) AFM images showing CNFs and XT of Ink19c (diluted to 10 ppm CNF) on mica after drying. Left: topographical image and right: phase-contrast image.

printing with hydrogels,32 gives bad printing fidelity and inhibits the possibility of printing several layers. Viscosity is also the reason why an XT solution cannot be printed on its own. Even at high concentrations (300 mg/mL), the viscosity is just slightly higher than for water. Considering cross-linking, it is instead the concentration of XT which is critical. The minimum concentration required for gel formation upon the addition of the cross-linking agents, HRP and H2O2, will depend on the degree of substitution of tyramine. Cross-linking tests of XT solutions showed that concentrations down to 50 mg/mL were able to form gels. The concentration limitations of CNF and XT gave basis for a predicted composition window, shown as the green box in Figure 2A which helped determine the required ratio of CNF to XT for a successful ink. Likewise to a composite, the properties of two different materials, CNF and XT, were used to form a third material with their combined properties: an ink which was printable and cross-linkable where the CNFs reinforced the cross-linked matrix of XT. In Figure 2D, the prepared inks, listed in Table 1, have been placed in the composition diagram. Four of the inks were within the predicted composition window (green box in Figure 2D) and were therefore expected to be both printable and cross-linkable. Ink0, however, lies outside of the composition window because

printing nozzle. If cross-linking is not applied immediately while printing, it must also be possible to print several layers of the ink without the printed structure collapsing. The crosslinking of an ink is important for further handling of the final printed structure. If printing with a shear thinning ink, such as the CNF, the printed shape will collapse if subjected to mechanical force when handling the printed material. Crosslinking locks the material structure and helps the printed material keep its shape intact upon handling. Figure 2B of a printed queen chess piece exemplifies how CNFs can be used to build up a 3D construct. Unfortunately, the queen could not be cross-linked and therefore easily fell apart. Solutions of XT show the opposite behavior (Figure 2C); they are easily crosslinked27 but are not printable. In the case of mixing CNF and XT, the resulting ink may either be printable or cross-linkable depending on the ink composition. Figure 2A illustrates how the concentrations of CNF and XT were predicted to influence the ink properties. As shown in previous studies by Markstedt et al., the concentration of CNFs should preferably be above 2.5% to be printable.16 Below this concentration, the viscosity at low shear becomes too low for the ink to print nicely and not flow out upon dispensing. Too low viscosity, a common issue when 40881


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Figure 4. (A) Storage modulus measured by oscillation time sweep for six different inks: +Ink0, ●Ink15, ■Ink19a, ▲Ink19b, ◆Ink19c, and ▼Ink32. Cross-linking started at t = 60 s upon the addition of H2O2. (B) Storage modulus, G′ (closed symbols), and loss modulus, G″ (open symbols), vs the oscillation stress. Yield stress marked by a red cross for the five different inks: ●Ink15, ■Ink19a, ▲Ink19b, ◆Ink19c, and ▼Ink32.

has an adsorption to cellulose. The amount of adsorption of XT to CNF within the prepared inks cannot be concluded from these measurements, but it showed that there was an interaction between XT and CNF even after conjugation of tyramine to xylan. The adsorption of XT on CNFs after mixing the inks could influence the diameter of the fibrils which in turn could have an effect on the ink viscosity. Figure 3B shows an AFM image of CNFs after mixing with XT, where both fibrils and aggregates of XT can be seen. The fibril diameters measured by AFM (n = 30) for pure CNFs, Ink15, and Ink19c averaged 8.0 ± 2.6, 8.7 ± 2.1, and 8.9 ± 2.3 nm, respectively. An increase in thickness due to the addition of xylan in the samples could not be concluded because the difference between the samples is within the measured SD. Several hydrogel systems used for 3D printing are based on ionic cross-linking which is a reversible process and depends on the concentration of cross-linking ions. Other types of CNF, which are negatively charged, form freestanding gels by ionic interactions caused by decreased pH or addition of salts. The cross-linking in this system was enabled by the enzyme HRP which together with H2O2 catalyzed the formation of radicals on the phenol group of tyramine. The covalent bonds formed by the radicals created an irreversible cross-linked network of XT. For the cross-linking reaction to start, both HRP and H2O2 had to be present in the ink. HRP was mixed in the prepared inks so that only H2O2 had to be added to initiate the crosslinking. The cross-linking reaction was monitored for the different inks by an oscillation time sweep experiment, where an increase in storage modulus, upon addition of H2O2, confirmed if cross-links were forming between tyramine groups within the ink (Figure 4A). Both the DS of the XT used in the ink and the ratio of CNF to xylan, ϕCNF, influenced the crosslinking and the strength of the final gel. The storage modulus for an ink prepared from unmodified xylan (DS = 0) showed no increase upon the addition of H2O2. Neither inks which contained XT with a low DS (DS = 15) nor inks with a low concentration of XT were able to form enough cross-links for an increase in the storage modulus to be measured. In Figure 4A, a clear increase in the storage modulus is seen for Ink19a, Ink19c, and Ink32. After a certain time, the storage modulus leveled out which corresponded to the cross-linking time. Similarly, to pure XT solutions reported by Kuzmenko et al.,27 the prepared inks cross-linked within 10 min. This cross-linking

it is not expected to cross-link at all because it consists of unmodified xylan and not xylan functionalized with tyramine. Apart from an ink with a balance of printability and crosslinking, successful printability requires homogenous inks to enable a consistent flow without clogging while printing. Because the CNF used in the inks was produced by enzymatic pretreatment, the CNF has a low charge, thus ionic repulsion was avoided, which could otherwise lead to phase separation. When mixing the inks, XT was added to CNFs in two ways, either as solid or as solution. Both methods gave a homogenous ink with no macroscopic aggregates or phase separation. However, adding XT as solution diluted the ink, leading to a change in rheological properties which may both influence the cross-linking and the printability. Also, the miscibility of XT and CNFs was crucial for obtaining a homogenous ink. Because there is no cross-linking taking place between CNFs and XT, it is beneficial that xylan has a natural affinity19,33 to cellulose which aids the mixing. The affinity to cellulose is due to hydrogen bonding and strong attractive forces as a result of a similar chemical structure and van der Waals forces. Because the cross-linked ink consists of fibrils (CNF) in a polymer matrix (XT), it could be classified as a composite. In successful composites, the fibers and the matrix are well-dispersed and have strong interactions at the interface to avoid a mechanical failure. Likewise, for a strong gel to form after cross-linking the printed ink, it is important that xylan, after substitution of tyramine, was still able to interact with cellulose. Figure 3A shows the adsorption of xylan compared to XT (DS = 32) on cellulose model surfaces measured by QCM. From the Sauerbrey equation, the adsorption of xylan was 0.9 mg/m2, whereas the adsorption of XT was 0.7 mg/m2. The calculated masses are estimations because eq 1 is valid for thin, rigid, and uniform films, and thereby underestimates the mass for viscoelastic films when ΔD < 1.34,35 The difference in adsorption is governed by several factors. Adsorption is highly associated with the solubility where low solubility leads to larger aggregates which tend to adsorb more to surfaces. In XT, carboxylic groups of xylan are replaced by tyramine. When deprotonated, carboxylic acids are negatively charged compared to tyramine which would increase the solubility and thereby decrease the adsorption. Tyramine is also a more bulky group and could give more steric hindrance which would also hinder aggregation and adsorption. QCM measurements showed that even though tyramine is conjugated to xylan, the modified xylan 40882


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Figure 5. Results from compression testing at a strain rate of 1% s−1. (A) Compressive stress at 30% strain, n = 6. (B) Compressive stiffness (tangent modulus) at 30% strain, n = 5. (C) Stress−strain plot representative of samples of Ink19c and Ink19a.

Figure 6. Printed grids of two crossing layers with 5 different inks: Ink15 (A,F); Ink19b (B,G); Ink19a (C,H); Ink19c (D,I); and Ink32 (E,J). Top row: images taken directly after printing. Bottom row: images taken after cross-linking 10 min in 1% H2O2.

time is sufficient for the printing process used in the current study, where the cross-linking is conducted after printing by submerging the whole construct in H2O2 solution. It is beneficial that the cross-linking reaction is not instantaneous to leave time for the reactants to diffuse into the construct to obtain a homogenously cross-linked structure without the formation of a skin with a nongelled core. The inks which showed the largest increase in the storage modulus, also showed the longest cross-linking time. The higher G′ was due to more bonds formed as a result of the cross-linking reaction which created a denser network. The formed network slowed down the diffusion of H2O2 which in turn slowed down the cross-linking and network formation. This was evident for Ink32 and Ink19c which had the highest increase in G′. The slope became steeper for Ink19a, Ink32, and Ink19c and correlated well with the amount of xylan and the degree of substitution. The oscillation measurements also showed differences when comparing inks prepared from XT with the same DS but with different ink composition (ratio of XT/CNF and water content). Ink19a, Ink19b, and Ink19c all have a DS of 19 but exhibit differences in cross-linking time and maximum storage modulus. Additionally, even though Ink19b and Ink19a had the same XT concentration (5.8 mg/mL), they showed a large difference in cross-linking, indicating that the concentration of CNFs also influenced the storage modulus of the cross-linked ink. At a higher concentration of CNFs, the fibrils act as a reinforcement of the cross-linked structure. The largest increase in the storage modulus (23 kPa) was obtained with a DS of 19 and a load of xylan equal to 10.6 wt % which is roughly ten

times the value of pure XT gels obtained by Kuzmenko et al. in 2014. Compression testing (Figure 5) confirmed that the storage modulus reflects the mechanical properties of the cross-linked inks. The stress−strain curve from compression testing (Figure 5C) shows that both the maximum stress and the compressive stiffness were higher for Ink19c than Ink19a and correspond well with the measured storage modulus in Figure 4A. The measured compressive stress and stiffness in Figure 5A,B are higher in relation to values previously reported for pure hemicellulose−tyramine gels.27,28 This shows that the cellulose also has a reinforcing effect and contributes with strength and stiffness to the hydrogels. As demonstrated by the stress−strain curve in Figure 5C, cracks were formed in Ink19a above 30% strain. Ink19c was resistant to crack formation as a result of a stronger cross-linked network because of the higher concentration of XT. When 3D printing, it is the shear thinning properties and the yield stress of the ink that will determine the printability of the ink. The yield stress is the applied stress at which the storage modulus is equal to the loss modulus. The ink goes from a gel state to a liquid state and can therefore start flowing. The yield stress of the prepared inks was determined from the amplitude sweep measurement shown in Figure 4B. The yield stress and storage modulus were influenced by the ink composition and in general increased with increasing solid content and CNF concentration. All inks were shear thinning and had appropriate viscosity for printing. However, the ink with a CNF concentration around or below 2.5% could not be printed with as high line resolution as the inks with a higher CNF 40883


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Figure 7. Handling of printed and cross-linked inks, all scale bars indicate 10 mm. (A−C) Comparison of cross-linked discs prepared from Ink19c (left) and Ink19a (right). The images show the (A) cross-linked discs from the side, (B) the top, and (C) the cross-linked discs after three days in water. Printed and cross-linked grids of Ink19b (D) and Ink19c (E) after handling with spatula. (F) Freestanding printed and cross-linked cylinder with Ink32. (G) Printed and cross-linked grid with Ink19c handled and bent in air. (H) Printed and cross-linked rook chess piece, held upside down.

concentration. The XT also helped bind the water of the CNF dispersion, and more XT in the ink resulted in more homogenous-looking inks. The printability of the inks was evaluated by comparing the line resolution in printed grids. All grids were printed at a speed of 10 mm/min. The applied pressure was adapted to each ink so that a continuous flow was reached and so that the grids could be printed with continuously dispensed lines. When printing with a conical needle (outlet diameter 420 μm), a pressure of 25 kPa was applied for Ink15 and Ink19b. Ink19a, Ink19c, and Ink32 were printed at a pressure of 47, 60, and 75 kPa, respectively. All inks could be dispensed by the printer with the pneumatic printer head using the conical needle tip. However, the printed lines of Ink15 and Ink19b were very broad with line widths above 750 μm, and the difference between the two printed layers was only slightly visible (Figure 6A,B). The bad resolution when printing was due to their low viscosity as a consequence of the too low solid content of 7.7 wt %. The printed grids before and after cross-linking are shown in Figure 6. Ink19c was printed with the best resolution and had a line width of 560 μm. The inks with the worst printability did not form strong enough cross-links for the printed grids to be handled and lifted from the printing plate after immersion in H2O2. Also, the cross-linking of Ink19a was not sufficient for the grid to hold together. However, when cross-linked as a disk, the cross-linking was sufficient to keep the structure intact as seen in Figure 7A−C. These images also show the difference in mechanical strength between the inks with high and low crosslinking density. The cross-linked disk of Ink19c, shown on the left in Figure 7A−C, kept its shape when lifted on a spatula (Figure 7A) and kept a very well-defined shape both after crosslinking (Figure 7B) and after swelling in water (Figure 7C). In comparison, Ink19a formed a much softer hydrogel which hangs from the spatula and also deforms slightly when crosslinked and swelled in water. This observation corresponds well with the studied mechanical properties of the gels after crosslinking (Figures 4A and 5). The amount of xylan and the degree of substitution dictate the final mechanical performance of the 3D-printed objects in wet conditions. Ink19c was easy to handle even as a grid structure (Figure 7G) and could be deformed without breaking in comparison to Ink19b (Figure 7D,E). Also Ink32 could be cross-linked as a grid and in Figure 7F, a cylinder printed with Ink32 is shown which could withhold its shape even when lying on its side. Finally, a rook

chess piece was printed with Ink19c which was successfully cross-linked and could thereby be handled without falling apart.

5. CONCLUSIONS By presenting an all-wood-based biomimetic ink, we have demonstrated how the inherent properties of nature’s own building blocks may be utilized for 3D printing. The resulting composite ink consisted of a cross-linked hemicellulose matrix reinforced by CNFs. As for trees which cannot grow tall and strong without cellulose, the CNF was essential in the prepared inks to enable 3D printing of complex structures built up of several layers. Using xylan substituted with tyramine as the matrix enabled tuning of the cross-linking density by using different degrees of substitution. Because the printed constructs were cross-linked irreversibly, they could easily be swelled and deswelled in water where CNF inks that are ionically crosslinked would have disrupted. The difference in swelling could also be utilized for applications in 4D printing where movement may be induced by change in humidity. The 4D printing is pronounced for anisotropic materials, and it would therefore be beneficial if CNF could be aligned along the fiber direction to enhance the mechanical properties. As orientation has been shown for 3D printing of cellulose nanocrystals,18 CNFs at concentrations below 1 wt %,36 and for diluted CNF dispersions in microfluidic devices,37 it is of interest to further investigate the possibility to align CNFs also at higher concentrations. Because the printed ink forms a gel with high water content, it shows potential in being used as a bioink for tissue engineering or printing of wound dressings. Apart from fulfilling the requirements of printability and cross-linking, the all-wood-based ink is a sustainable choice for future materials to be used for 3D printing of clothes, packaging, health care products and furniture.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kajsa Markstedt: 0000-0001-6737-6887 Paul Gatenholm: 0000-0002-5107-0389 Author Contributions

Experiments were conceived and designed by K.M., P.G., and G.T. and were conducted by K.M. Extraction of xylan was carried out by G.T. and A.E. and xylan−tyramine was 40884


Research Article


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synthesized by G.T., A.E., and K.M. The main paper was written by K.M. All authors have revised the paper and given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The Knut and Alice Wallenberg Foundation is kindly acknowledged for financial support of this research in the framework of the Wallenberg Wood Science Center (WWSC). Saina Kishani is acknowledged for assistance with QCM measurements at the department of fiber and polymer technology, KTH.

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ABBREVIATIONS

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REFERENCES

CNF, cellulose nanofibrils; XT, xylan−tyramine; HRP, horseradish peroxidase; DS, degree of substitution; QCM-D, quartz crystal microbalance with dissipation

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Biomimetic Inks Based on Cellulose Nanofibrils and Cross-Linkable

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