Biomimetic inks based on cellulose nanofibrils and crosslinkable

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Biomimetic inks based on cellulose nanofibrils and crosslinkable xylans for 3D printing Kajsa Markstedt, Alfredo Escalante, Guillermo Toriz, and Paul Gatenholm ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13400 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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ACS Applied Materials & Interfaces

Biomimetic inks based on cellulose nanofibrils and crosslinkable xylans for 3D printing Kajsa Markstedtab, Alfredo Escalantec, Guillermo Torizac, Paul Gatenholmab* AUTHOR ADDRESS a

Wallenberg Wood Science Center, Kemigården 4, 41296 Gothenburg, Sweden

b

Department of Chemistry and Chemical Engineering, Chalmers University of Technology,

Kemigården 4, 41296 Gothenburg, Sweden c

Department of Wood, Cellulose and Paper Research, University of Guadalajara, Guadalajara

44100, Mexico *Corresponding author

KEYWORDS 3D printing, Cellulose nanofibrils, Crosslinking, Hemicellulose, Composite

ACS Paragon Plus Environment

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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 customized design and versatile gel properties. 3D printing technologies where material is dispensed in the form of liquids, so called inks, have proven suitable for 3D printing dispersions of cellulose nanofibrils (CNF) due to their unique shear thinning properties. In this study, novel inks were developed with a biomimetic approach where the structural properties of cellulose and the crosslinking function of hemicelluloses that are found in the plant cell wall were utilized. 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 crosslinkable, 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 CNF versus xylan-tyramine in the prepared inks influenced the printability and crosslinking 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 crosslinked ink. Thus, the influence of tyramine on the adsorption of xylan to cellulose was studied with quartz crystal microbalance to verify that the functionalization had little influence on xylans adsorption to cellulose. Utilizing xylan-tyramine in the CNF dispersions resulted in all wood based inks which after 3D printing can be crosslinked to form free standing gels while at the same time the excellent printing properties of CNF remain intact.

1. INTRODUCTION


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In the transformation towards 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 being used in high value products such as electronics2, light weight composites3 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 (CNF) 6

. By introducing pretreatment steps of the pulp (enzymatic, carboxymethylation, TEMPO

oxidation) the energy consumption, and thereby the cost, has been lowered and enabled production of bulk amounts of CNF

7-8

. CNF have a high surface area which give them suitable

properties as a dispersant and rheological modifier 9. It is foreseen that CNF could be used in a wide range of products such as cosmetics, yoghurts, cement and paints 10-11. CNF 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 the

prospected applications is that all the processing of CNF is in water systems. We have found that the properties of CNF 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. Based on a 3D model, material is dispensed layer by layer by a printing head controlled by an X, Y, Z - stage. If the dispensed material is liquid, it is referred to


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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. CNF fulfills the requirements of an ink; due to shear thinning properties it is easily dispensed and quickly regains a high viscosity once the applied force is removed. For dispensing of CNF dispersions, either an ink-jet or a pneumatic printer head are suitable since they can dispense 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 CNF to enable handling of the printed construct. For example, CNF has been printed together with alginate and ionically crosslinked 16, or CNF has been freeze-dried 17. Also, cellulose nanocrystals which are similar to CNF, have been printed with resin and UVcrosslinked 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 crosslinked matrix, which helps bind and keep the cellulose structure in place. Due to hemicelluloses natural affinity to cellulose, several biopolymers have been studied where hemicellulose influences the properties of cellulose

19-21

and functions as a plasticizer of

cellulose films22 or acts as a crosslinking component in composites21. The presence of hydroxyl and carboxylic groups on hemicelluloses make them suitable for chemical modification to introduce new functionalities. To obtain hydrogels from hemicelluloses, several chemical modifications have been studied to introduce crosslinking such as thiol-functionalization methacrylate derivatives

25

and grafting of acrylic acid

26

23-24

,

. 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 crosslinking using horse


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radish peroxidase 27-28. The enzymatic crosslinking is a fast process and starts within seconds as soon as horse radish peroxidase and H2O2 are present. It is also an irreversible reaction and results in a stable hydrogel. Inspired by the role of which cellulose and hemicellulose have in the plant cell wall, we present an all wood based biomimetic ink for 3D printing consisting of CNF and crosslinkable xylan.

2. MATERIALS 3 % cellulose nanofibril dispersion (CNF) in water was kindly provided by Stora Enso (Karlstad, Sweden). CNF was processed from soft wood pulp as described by Pääkö

7

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 x 30 minutes. 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 12700 g/mol and contained 10 wt% 4-O-methylglucuronic 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 crosslinking of inks prepared from CNF and xylan-tyramine, H2O2 (30 wt %), and peroxidase from horseradish, HRP (256 U/mg), were purchased from Sigma Aldrich.

3. METHODS 3.1. Synthesis of xylan-tyramine


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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 DI-water (25 mg/ml, 15.3 mol COOH per liter H2O) under stirring and heating at 70o 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 xylan-tyramine conjugate (XT) was precipitated in cold ethanol and vacuum filtrated. The amount of reactants were based on the calculated concentration of COOH in xylan: 0.612mmol 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 500mg 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 (400Hz, VarianAgilent 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 tyramine substitution (DS). 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 tyramine substitution was thus calculated as ‫ = ܵܦ‬100 ∗

ுଵ଴ାுଵଵ ସ

(1)

3.2. Quartz Crystal Microbalance with Dissipation


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Xylan-tyramine conjugate was compared to the unmodified xylan with regard to cellulose affinity upon adsorption. Adsorption kinetics of xylan and XT onto cellulose nanofibril model surfaces were monitored with 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, ∆f, which is proportional to the adsorbed mass per area, m, including water. This relation is expressed by the Sauerbrey equation ݉=‫ܥ‬

∆௙ ௡

(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 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, 25o 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 CNF and xylan with different degrees of tyramine substitution. The synthesized XT was added to CNF in two different manners: as a dry powder or in solution (300 mg/ml in H2O, stirring at 60o 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 x 30 seconds. For addition of XT as dry powder, the ink was mixed by hand at 60o C for 5 minutes followed by sonication for 1 minute. The ink was stored at 2-8o C overnight. Before using the ink, speed mixing was conducted for 2 x 30 s. To ensure uniform crosslinking


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of the inks, horse radish peroxidase (0.6mg/ml) was added to the final inks and mixed with a spatula before usage in crosslinking measurements or printing of the ink. 3.4. Atomic Force Microscopy The diameter of the cellulose nanofibrils in the CNF dispersion and in inks with xylantyramine were measured by 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 let 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 tapping mode with a standard silicon tip to determine the width of the cellulose nanofibrils. 3.5. Rheology Oscillation time measurements were conducted to study the crosslinking 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 were 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 crosslinked 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%


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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 ink in casting units (Q-Gel bio, Lausanne, Switzerland) followed by crosslinking in 1 wt% H2O2 for 10 minutes. Each disc was then punched to a diameter of 6 mm. The measured thickness of the discs was 1.78 mm and 1.88 mm for Ink19a and Ink19c respectively. The results are presented as mean ± standard deviation and statistically analyzed with a one-way analysis of variance (ANOVA) with a post-hoc 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 the flow was sufficient to form continuous dispensed lines at the given printing speed. The ink which showed best printability and crosslinking 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 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


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crosslinked by submerging them in a bath of 1 % H2O2 solution in water. Based on the results from the rheology study, 10 minutes was chosen as a sufficient time for crosslinking.

4. RESULTS Crosslinkable xylans, xylan-tyramine (XT), were designed and synthesized to be used as a component together with CNF in a biomimetic ink. To evaluate the influence of tyramine on the crosslinking ability of XT in the inks, xylan with three different degrees of tyramine substitution (DS) were synthesized. The DS was determined by 1H-NMR. Figure 1 shows a 1H-NMR spectra of XT dissolved in 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 equation 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 mg, 1500 mg 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 amount of reagents, especially in order to increase the DS for larger batches.


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Figure 1. 1H-NMR spectra of xylan-tyramine dissolved in D2O. (A) Anomeric protons. (B) Aromatic protons. Xylan with four different degrees of substitution (DS of 0%, 15%, 19%, 32%) were mixed with CNF to prepare inks. Additionally to preparing inks from XT with different DS, 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: ߶஼ேி = 100 ∙ ௠

௠಴ಿಷ

(3)

಴ಿಷ ∙௠೉೅

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.

Table 1. Ink composition for inks prepared from CNF and XT with different degree of substitution (DS). 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. Final composition Water (wt%)

CNF (wt%)

XT (wt%)

33

92.3

2.6

5.11

15

33

92.3

2.6

5.11

Ink32

32

33

88.4

3.3

6.6

Ink19c

19

20

86.8

2.7

10.6

Ink19b

19

27

92.2

2.0

5.8

Ink19a

19

34

91.2

3.0

5.8

Ink

DS (%)

Ink0*

0

Ink15

߶஼ேி


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* Ink0 was prepared from CNF and xylan which has not been modified with tyramine. Thus, 5.11 is the wt% of pure xylan.

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, e.g. printed lines should be continuous, not flow out, and have similar diameter to the printing nozzle. If crosslinking 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 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 CNF can be used to build up a 3D construct. Unfortunately, the queen could not be crosslinked and therefor easily fell apart. Solutions of XT show the opposite behavior (fig. 2C), they are easily crosslinked27 but were not printable. In the case of mixing CNF and XT, the resulting ink may either be printable or crosslinkable depending on the ink composition. Figure 2A illustrates how the concentrations of CNF and XT were predicted to influence the ink properties.


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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 crosslinking ability. Blue section: Inks are printable but not crosslinkable. Green section: Inks are printable and crosslinkable. White section: Inks are neither printable nor crosslinkable. Yellow section: Inks are crosslinkable but not printable. (B) A queen chess piece printed with pure CNF which cannot crosslink. (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 crosslinking ability. As shown in previous studies by Markstedt et al. the concentration of CNF 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 printing with hydrogels32, 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 crosslinking, it is instead the concentration of XT which is critical. The minimum concentration required for gel formation upon addition of the crosslinking agents, HRP and H2O2, will depend on the degree of substitution of tyramine. Crosslinking 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 & D, which helped determine the required ratio of CNF versus 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 crosslinkable where the cellulose nanofibrils reinforced the crosslinked matrix of


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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 therefor expected to be both printable and crosslinkable. Ink0 however lies outside of the composition window since it is not expected to crosslink 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. Since 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 CNF 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 crosslinking and the printability. Also the miscibility of XT and CNF was crucial for obtaining a homogenous ink. Since there is no crosslinking taking place between CNF and XT it is beneficial that xylan has a natural affinity 19, 33 to cellulose which aids the mixing. The affinity to cellulose is due to hydrogen bonding and strong attractive forces as a result of similar chemical structure and van der Waals forces. Since the crosslinked ink consists of fibrils (CNF) in a polymer matrix (XT), it could be classified as a composite. In successful composites the fibers and matrix are well dispersed and have strong interactions at the interface to avoid mechanical failure. Likewise, for a strong gel to form after crosslinking 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 while


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the adsorption of XT was 0.7 mg/m2. The calculated masses are estimations, since equation 1 is valid for thin, rigid and uniform films and thereby underestimates the mass for viscoelastic films when ∆D < 1

34-35

. The difference in adsorption is governed by several factors. Adsorption is

highly associated to the solubility where low solubility leads to larger aggregates which tend to adsorb more to surfaces. In xylan-tyramine, 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 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.

Figure 3. (A) Change in frequency (top) and dissipation (bottom) as a function of time for adsorption of xylan (black) and xylan-tyramine (red) on cellulose model surface. The rinsing step with Milli-Q water is seen at 22 min. Measurements were conducted at 296 K. Results are shown for the 3rd overtone of quartz crystal resonance frequency. (B) AFM images showing CNF and


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XT of Ink19c (diluted to 10 ppm CNF) on mica after drying. Left: topographical image, right: phase-contrast image. The adsorption of XT on CNF after mixing the inks could influence the diameter of the fibrils which in turn could have an effect on the inks viscosity. Figure 3B shows an AFM image of CNF after mixing with XT where both fibrils and aggregates of XT can be seen. The fibril diameter measured by AFM (n=30) for pure CNF, Ink15 and Ink19c averaged 8.0±2.6 nm, 8.7±2.1 nm and 8.9±2.3 nm respectively. An increase in thickness due to addition of xylan in the samples could not be concluded since the difference between the samples is within the measurement standard deviation. Several hydrogel systems used for 3D printing are based on ionic crosslinking which is a reversible process and depends on the concentration of crosslinking ions. Other types of CNF, which are negatively charged, form free-standing gels by ionic interactions caused by decreased pH or addition of salts. The crosslinking in this system was enabled by the enzyme horse radish peroxidase 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 crosslinked network of XT. For the crosslinking reaction to start, both horse radish peroxidase (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 crosslinking 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 crosslinks were forming between tyramine groups within the ink (fig. 4A). Both the DS of the XT used in the ink and the ratio of CNF to xylan, ߶஼ேி , 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 addition of H2O2. Neither inks which contained XT with a low


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DS (DS=15), nor inks with a low concentration of XT were able to form enough crosslinks for an increase in storage modulus to be measured. In figure 4A, a clear increase in storage modulus is seen for Ink19a, Ink19c and Ink32. After a certain time the storage modulus leveled out which corresponded to the crosslinking time. Similarly to pure XT solutions reported by Kuzmenko et. al.27, the prepared inks crosslinked within 10 minutes. This crosslinking time is sufficient for the printing process used in the current study where the crosslinking is conducted post printing by submerging the whole construct in H2O2 solution. It is beneficial that the crosslinking reaction is not instantaneous to leave time for the reactants to diffuse into the construct to obtain a homogenously crosslinked structure without the formation of a skin with a non-gelled core. The inks which showed the largest increase in storage modulus, also showed the longest crosslinking time. The higher G’ was due to more bonds formed as a result of the crosslinking reaction which created a denser network. The formed network slowed down the diffusion of H2O2 which in turn slowed down the crosslinking 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 respectively and correlated well with the amount of xylan and 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 crosslinking time and maximum storage modulus. Additionally, even though Ink19b and DS19a had the same XT concentration (5.8 mg/ml), they showed a large difference in crosslinking indicating that also the concentration of CNF influenced the storage modulus of the crosslinked ink. At a higher concentration of CNF, the fibrils act as a reinforcement of the crosslinked structure. The largest increase in storage modulus (23 kPa) was obtained with a DS of 19 and load of xylan equal to


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10.6 wt% which is roughly ten times the value of pure xylan-tyramine gels obtained by Kuzmenko et al. 2014.

Figure 4: (A) Storage modulus measured by oscillation time sweep for six different inks: +Ink0, Ink15, Ink19a, Ink19b, Ink19c, Ink32. Crosslinking started at t=60s upon addition of H2O2. (B) Storage modulus, G’ (closed symbols), and Loss modulus, G’’ (open symbols), versus the oscillation stress. Yield stress marked by a red cross for the five different inks: Ink15, Ink19a, Ink19b, Ink19c, Ink32. Compression testing (fig. 5) confirmed that the storage modulus reflects the mechanical properties of the crosslinked inks. The stress-strain curve from compression testing (fig. 5C) shows that both the maximum stress and the compressive stiffness was higher for Ink19c than Ink19a and corresponds well with the measured storage modulus in figure 4A. The measured compressive stress and stiffness in figure 5A and 5B are higher in relation to values previously reported for pure hemicellulose-tyramine gels27-28. 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


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was resistant to crack formation as a result of a stronger crosslinked network due to the higher concentration of XT.

Figure 5: Results from compression testing at 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) Stressstrain plot representative for samples of Ink19c and Ink19a.

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 higher CNF 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


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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 kPa, 60 kPa and 75 kPa respectively.

Figure 6. Printed grids of two crossing layers with 5 different inks: Ink15 (A, F); Ink19b (B, G); Ink19a (C, H); Ink19c (D, I); Ink32 (E, J). Top row: Images taken directly after printing. Bottom row: Images taken after crosslinking 10 min in 1% H2O2. 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 (fig. 6A & 6B). 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 crosslinking 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 crosslinks for the printed grids to be


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handled and lifted from the printing plate after immersion in H2O2. Also the crosslinking of Ink19a was not sufficient for the grid to hold together. However, when crosslinked as a disk the crosslinking was sufficient to keep the structure intact as seen in figures 7A-C. These images also show the difference in mechanical strength between the inks with high and low crosslinking density. The crosslinked disk of Ink19c, shown on the left in figure 7A-C, kept its shape when lifted on a spatula (fig. 7A) and kept a very well defined shape both after crosslinking (fig. 7B), and after swelling in water (fig. 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 (fig. 4A and fig. 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 (fig. 7G) and could be deformed without breaking in comparison to Ink19b (fig. 7D & 7E). Also Ink32 could be crosslinked 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 crosslinked and could thereby be handled without falling apart.


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Figure 7. Handling of printed and crosslinked inks, all scale bars indicate 10mm.

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(A-C)

Comparison of crosslinked discs prepared from Ink19c (left) and Ink19a (right). The images show the (A) crosslinked discs from the side, (B) the top and (C) the crosslinked discs after three days in water. Printed and crosslinked grids of Ink19b (D) and Ink19c (E) after handling with spatula. (F) Freestanding printed and crosslinked cylinder with Ink32. (G) Printed and crosslinked grid with Ink19c handled and bent in air. (H) Printed and crosslinked rook chess piece, held upside down. 5. CONCLUSIONS By presenting an all wood based biomimetic ink, we have demonstrated how the inherent properties of natures’ own building blocks may be utilized for 3D printing. The resulting composite ink consisted of a crosslinked hemicellulose matrix reinforced by cellulose nanofibrils. As for trees which cannot grow tall and strong without cellulose, 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 crosslinking density by using different degrees of substitution. Since the printed constructs were crosslinked 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. 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 CNC18, CNF at concentrations below 1 wt%36 , and for diluted CNF dispersions in microfluidic devices37 it is of interest to further investigate the possibility to align CNF also at higher concentrations. Since the printed ink forms a gel with high water content it shows potential in


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being used as a bioink for tissue engineering or printing of wound dressings. Apart from fulfilling the requirements of printability and crosslinking, 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.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] Department of Chemistry and Chemical Engineering Chalmers University of Technology Kemigården 4, 41296 Gothenburg, Sweden

Author Contributions Experiments were designed by 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 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. ACKNOWLEDGMENT 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 CNF, Cellulose Nanofibrils; XT, Xylan-Tyramine; HRP, Horse Radish Peroxidase; DS, Degree of Substitution; QCM-D, Quartz Crystal Microbalance with Dissipation


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REFERENCES 1. Scarlat, N.; Dallemand, J.-F.; Monforti-Ferrario, F.; Nita, V., The Role of Biomass and Bioenergy in a Future Bioeconomy: Policies and Facts. Environmental Development 2015, 15, 334. 2. Hoeng, F.; Denneulin, A.; Bras, J., Use of Nanocellulose in Printed Electronics: A Review. Nanoscale 2016, 8 (27), 13131-13154. 3. Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.; Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; Gindl, W.; Veigel, S.; Keckes, J.; Yano, H.; Abe, K.; Nogi, M.; Nakagaito, A. N.; Mangalam, A.; Simonsen, J.; Benight, A. S.; Bismarck, A.; Berglund, L. A.; Peijs, T., Review: Current International Research into Cellulose Nanofibres and Nanocomposites. J Mater Sci 2010, 45 (1), 1-33. 4. Hua, K.; Carlsson, D. O.; Ålander, E.; Lindström, T.; Strømme, M.; Mihranyan, A.; Ferraz, N., Translational Study between Structure and Biological Response of Nanocellulose from Wood and Green Algae. RSC Adv. 2014, 4 (6), 2892-2903. 5. Syverud, K.; Pettersen, S. R.; Draget, K.; Chinga-Carrasco, G., Controlling the Elastic Modulus of Cellulose Nanofibril Hydrogels—Scaffolds with Potential in Tissue Engineering. Cellulose 2014, 22 (1), 473-481. 6. Nechyporchuk, O.; Belgacem, M. N.; Bras, J., Production of Cellulose Nanofibrils: A Review of Recent Advances. Ind. Crops Prod. 2016, 93, 2-25. 7. Paakko, M.; Ankerfors, M.; Kosonen, H.; Nykanen, A.; Ahola, S.; Osterberg, M.; Ruokolainen, J.; Laine, J.; Larsson, P. T.; Ikkala, O.; Lindstrom, T., Enzymatic Hydrolysis Combined with Mechanical Shearing and High-Pressure Homogenization for Nanoscale Cellulose Fibrils and Strong Gels. Biomacromolecules 2007, 8 (6), 1934-1941. 8. Saito, T.; Nishiyama, Y.; Putaux, J.-L.; Vignon, M.; Isogai, A., Homogeneous Suspensions of Individualized Microfibrils from Tempo-Catalyzed Oxidation of Native Cellulose. Biomacromolecules 2006, 7 (6), 1687-1691. 9. Klemm, D.; Kramer, F.; Moritz, S.; Lindstrom, T.; Ankerfors, M.; Gray, D.; Dorris, A., Nanocelluloses: A New Family of Nature-Based Materials. Angew. Chem. Int. Ed. Engl. 2011, 50 (24), 5438-5466. 10. Sun, X.; Wu, Q.; Lee, S.; Qing, Y.; Wu, Y., Cellulose Nanofibers as a Modifier for Rheology, Curing and Mechanical Performance of Oil Well Cement. Sci Rep 2016, 6, 31654. 11. Göran Ström, C. Ö. a. M. A. Nanocellulose as an Additive in Foodstuff; 403; Innventia: April 2013, 2013. 12. Galland, S.; Berthold, F.; Prakobna, K.; Berglund, L. A., Holocellulose Nanofibers of High Molar Mass and Small Diameter for High-Strength Nanopaper. Biomacromolecules 2015, 16 (8), 2427-2435. 13. Österberg, M.; Vartiainen, J.; Lucenius, J.; Hippi, U.; Seppälä, J.; Serimaa, R.; Laine, J., A Fast Method to Produce Strong Nfc Films as a Platform for Barrier and Functional Materials. ACS Appl. Mater. Interfaces 2013, 5 (11), 4640-4647. 14. Henriksson, M.; Berglund, L. A.; Isaksson, P.; Lindström, T.; Nishino, T., Cellulose Nanopaper Structures of High Toughness. Biomacromolecules 2008, 9 (6), 1579-1585. 15. Nogi, M.; Iwamoto, S.; Nakagaito, A. N.; Yano, H., Optically Transparent Nanofiber Paper. Adv. Mater. 2009, 21 (16), 1595-1598.


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16. Markstedt, K.; Mantas, A.; Tournier, I.; Martinez Avila, H.; Hagg, D.; Gatenholm, P., 3d Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications. Biomacromolecules 2015, 16 (5), 1489-1496. 17. Håkansson, K. M. O.; Henriksson, I. C.; de la Peña Vázquez, C.; Kuzmenko, V.; Markstedt, K.; Enoksson, P.; Gatenholm, P., Solidification of 3d Printed Nanofibril Hydrogels into Functional 3d Cellulose Structures. Adv. Mater. Technol. 2016, 1 (7), 1600096. 18. Siqueira, G.; Kokkinis, D.; Libanori, R.; Hausmann, M. K.; Gladman, A. S.; Neels, A.; Tingaut, P.; Zimmermann, T.; Lewis, J. A.; Studart, A. R., Cellulose Nanocrystal Inks for 3d Printing of Textured Cellular Architectures. Adv. Funct. Mater. 2017, 27 (12), 1604619. 19. Eronen, P.; Österberg, M.; Heikkinen, S.; Tenkanen, M.; Laine, J., Interactions of Structurally Different Hemicelluloses with Nanofibrillar Cellulose. Carbohydr. Polym. 2011, 86 (3), 1281-1290. 20. Prakobna, K.; Kisonen, V.; Xu, C.; Berglund, L., Strong Reinforcing Effects from Galactoglucomannan Hemicellulose on Mechanical Behavior of Wet Cellulose Nanofiber Gels. J Mater Sci 2015, 50 (22), 7413-7423. 21. Oinonen, P.; Krawczyk, H.; Ek, M.; Henriksson, G.; Moriana, R., Bioinspired Composites from Cross-Linked Galactoglucomannan and Microfibrillated Cellulose: Thermal, Mechanical and Oxygen Barrier Properties. Carbohydr. Polym. 2016, 136, 146-153. 22. Kisonen, V.; Eklund, P.; Auer, M.; Sjöholm, R.; Pranovich, A.; Hemming, J.; Sundberg, A.; Aseyev, V.; Willför, S., Hydrophobication and Characterisation of O-AcetylGalactoglucomannan for Papermaking and Barrier Applications. Carbohydr. Res. 2012, 352, 151-158. 23. Pahimanolis, N.; Kilpeläinen, P.; Master, E.; Ilvesniemi, H.; Seppälä, J., Novel ThiolAmine- and Amino Acid Functional Xylan Derivatives Synthesized by Thiol–Ene Reaction. Carbohydr. Polym. 2015, 131 (Supplement C), 392-398. 24. Maleki, L.; Edlund, U.; Albertsson, A. C., Thiolated Hemicellulose as a Versatile Platform for One-Pot Click-Type Hydrogel Synthesis. Biomacromolecules 2015, 16 (2), 667674. 25. Dax, D.; Chavez, M. S.; Xu, C.; Willfor, S.; Mendonca, R. T.; Sanchez, J., Cationic Hemicellulose-Based Hydrogels for Arsenic and Chromium Removal from Aqueous Solutions. Carbohydr. Polym. 2014, 111, 797-805. 26. Sun, X.-F.; Wang, H.-h.; Jing, Z.-x.; Mohanathas, R., Hemicellulose-Based Ph-Sensitive and Biodegradable Hydrogel for Controlled Drug Delivery. Carbohydr. Polym. 2013, 92 (2), 1357-1366. 27. Kuzmenko, V.; Hägg, D.; Toriz, G.; Gatenholm, P., In Situ Forming Spruce Xylan-Based Hydrogel for Cell Immobilization. Carbohydr. Polym. 2014, 102, 862-868. 28. Markstedt, K.; Xu, W.; Liu, J.; Xu, C.; Gatenholm, P., Synthesis of Tunable Hydrogels Based on O-Acetyl-Galactoglucomannans from Spruce. Carbohydr. Polym. 2017, 157, 13491357. 29. Escalante, A.; Gonçalves, A.; Bodin, A.; Stepan, A.; Sandström, C.; Toriz, G.; Gatenholm, P., Flexible Oxygen Barrier Films from Spruce Xylan. Carbohydr. Polym. 2012, 87 (4), 2381-2387. 30. Höök, F.; Rodahl, M.; Brzezinski, P.; Kasemo, B., Energy Dissipation Kinetics for Protein and Antibody−Antigen Adsorption under Shear Oscillation on a Quartz Crystal Microbalance. Langmuir 1998, 14 (4), 729-734. 31. Chess Set - Print Friendly by Tetralite. Thingiverse: June 29, 2014.


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32. Murphy, S. V.; Skardal, A.; Atala, A., Evaluation of Hydrogels for Bio-Printing Applications. J. Biomed. Mater. Res., Part A 2013, 101 (1), 272-284. 33. Linder, Å.; Bergman, R.; Bodin, A.; Gatenholm, P., Mechanism of Assembly of Xylan onto Cellulose Surfaces. Langmuir 2003, 19 (12), 5072-5077. 34. Lozhechnikova, A.; Dax, D.; Vartiainen, J.; Willför, S.; Xu, C.; Österberg, M., Modification of Nanofibrillated Cellulose Using Amphiphilic Block-Structured Galactoglucomannans. Carbohydr. Polym. 2014, 110, 163-172. 35. Höök, F.; Kasemo, B.; Nylander, T.; Fant, C.; Sott, K.; Elwing, H., Variations in Coupled Water, Viscoelastic Properties, and Film Thickness of a Mefp-1 Protein Film During Adsorption and Cross-Linking: A Quartz Crystal Microbalance with Dissipation Monitoring, Ellipsometry, and Surface Plasmon Resonance Study. Anal. Chem. 2001, 73 (24), 5796-5804. 36. Sydney Gladman, A.; Matsumoto, E. A.; Nuzzo, R. G.; Mahadevan, L.; Lewis, J. A., Biomimetic 4d Printing. Nat Mater 2016, 15 (4), 413-418. 37. Hakansson, K. M. O.; Lundell, F.; Prahl-Wittberg, L.; Soderberg, L. D., Nanofibril Alignment in Flow Focusing: Measurements and Calculations. J. Phys. Chem. B 2016, 120 (27), 6674-6686.


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Figure 1. 1H-NMR spectra of xylan-tyramine dissolved in D2O. (A) Anomeric protons. (B) Aromatic protons. 85x53mm (300 x 300 DPI)



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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 crosslinking ability. Blue section: Inks are printable but not crosslinkable. Green section: Inks are printable and crosslinkable. White section: Inks are neither printable nor crosslinkable. Yellow section: Inks are crosslinkable but not printable. (B) A queen chess piece printed with pure CNF which cannot crosslink. (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 crosslinking ability. 177x56mm (300 x 300 DPI)


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Figure 3. (A) Change in frequency (top) and dissipation (bottom) as a function of time for adsorption of xylan (black) and xylan-tyramine (red) on cellulose model surface. The rinsing step with Milli-Q water is seen at 22 min. Measurements were conducted at 296 K. Results are shown for the 3rd overtone of quartz crystal resonance frequency. (B) AFM images showing CNF and XT of Ink19c (diluted to 10 ppm CNF) on mica after drying. Left: topographical image, right: phase-contrast image. 177x63mm (300 x 300 DPI)



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Figure 4: (A) Storage modulus measured by oscillation time sweep for six different inks: +Ink0, []Ink15, []Ink19a, []Ink19b, []Ink19c, []Ink32. Crosslinking started at t=60s upon addition of H2O2. (B) Storage modulus, G’ (closed symbols), and Loss modulus, G’’ (open symbols), versus the oscillation stress. Yield stress marked by a red cross for the five different inks: []Ink15, []Ink19a, []Ink19b, []Ink19c, []Ink32. 147x68mm (300 x 300 DPI)


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Figure 5. Results from compression testing at 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 for samples of Ink19c and Ink19a. 59x19mm (600 x 600 DPI)



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Figure 6. Printed grids of two crossing layers with 5 different inks: Ink15 (A,F); Ink19b (B,G); Ink19a (C,H); Ink19c (D,I); Ink32 (E,J). Top row: Images taken directly after printing. Bottom row: Images taken after crosslinking 10 min in 1% H2O2. 177x89mm (300 x 300 DPI)


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Figure 7. Handling of printed and crosslinked inks, all scale bars indicate 10mm. (A-C) Comparison of crosslinked discs prepared from Ink19c (left) and Ink19a (right). The images show the (A) crosslinked discs from the side, (B) the top and (C) the crosslinked discs after three days in water. Printed and crosslinked grids of Ink19b (D) and Ink19c (E) after handling with spatula. (F) Freestanding printed and crosslinked cylinder with Ink32. (G) Printed and crosslinked grid with Ink19c handled and bent in air. (H) Printed and crosslinked rook chess piece, held upside down. 177x69mm (300 x 300 DPI)



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Biomimetic inks based on cellulose nanofibrils and crosslinkable

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