3D Printing of a Bio-Based Ink Made of Cross-Linked Cellulose Nanofibrils With Various Metal Cations

3D Printing of a Bio-Based Ink Made of Cross-Linked Cellulose Nanofibrils With Various Metal Cations | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 3D Printing of a Bio-Based Ink Made of Cross-Linked Cellulose Nanofibrils With Various Metal Cations Jakob Mietner, Xuehe Jiang, Ulrica Edlund, Bodo Saake, Julien Navarro This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-122100/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Mar, 2021 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract In this work, we present an approach to cross-link cellulose nanofibrils (CNFs) with various metallic cations (Fe 3+ , Al 3+ , Ca 2+ , and Mg 2+ ) to produce inks suitable for three-dimensional (3D) printing application. The printability of each hydrogel ink was evaluated, and several parameters such as the optimal ratio of M n+ :TOCNF:H 2 O were discussed. CNF suspensions were produced by mechanical disintegration of cellulose pulp with a microfluidizer and then oxidized with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). Finally, metal cations were introduced to the deprotonated TEMPO-oxidized CNF (TOCNF) suspension to cross-link the nanofibrils and form the corresponding hydrogels. The performances of each gel-ink were evaluated by rheological measurements and 3D printing. Only the gels incorporated with divalent cations Ca 2+ and Mg 2+ were suitable for 3D printing. The 3D printed structures were freeze-dried and characterized with Fourier transform infrared spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM). The better interaction of the TOCNFs with the divalent metallic cations in terms of printability, the viscoelastic properties of the inks, and the variation trends owing to various metal cations and ratios are discussed. Biomaterials Materials Chemistry Cellulose nanofibrils TEMPO mediated oxidation Metal-ion crosslinking Hydrogel 3D printing Nanocellulose Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The need to replace petroleum-based products with biodegradable and renewable resources, to produce high-performance functional materials is one of the greatest challenges for a future sustainable society. For this purpose, bio-based polymers have attracted considerable attention over the past decades. Cellulose, and its derivatives, fulfill those needs by offering many advantages such as renewability, biodegradability, and to some extent recyclability. 1 – 3 Cellulose nanofibrils (CNF) is a highly promising candidate for a wide panel of applications, ranging from composites, water purification to drug delivery. 4 – 7 CNF can be extracted from numerous lignocellulosic source materials through mechanical disintegration. 8 Typical CNF dimensions comprise widths between 5–20 nm and a wide range of lengths, typically several micrometers. 9 Recently, several groups demonstrated that CNF could be structured through 3D printing processes. 10 – 16 The demonstrated 3D printed CNF objects showed great potential as 3D printed tablets for controlled drug release and as 3D printed bioactive composites in tissue engineering and wound dressing applications. Markstedt et al. 17 produced a biobased ink made of cross-linked CNF, alginate, and CaCl 2 . The CNF-alginate cross-linked structure proved to be a viable scaffold for hosting human nasoseptal chondrocyte cells. Leppiniemi et al. 18 developed a 3D ink based on alginate, avidin protein-modified CNF and glycerin. In this study Leppiniemi et al. were using CNF as a strengthening additive and CaCl 2 as a cross-linker, what leads to a significantly more stable shape fidelity after 3D printing. Moreover, the 3D printed object showed good tissue compatibility and great potential in biomedical applications such as in wound dressings. Li et al. 19 produced a 3D printed structure made of nanocellulose and carbon nanotubes. The object was first 3D printed and cross-linked with CaCl 2 later on. Several drying protocols were applied to the printed hydrogel and freeze-drying was found to be the most efficient strategy. CNF has an abundance of hydroxyl groups on the fibril surfaces which lead to strong hydrogen interaction i) between fibrils (inter-fibrils interaction that leads to fibril agglomeration) and ii) with water molecules, endowing viscoelasticity and shear thinning properties that are advantageous for 3D printing. 20 The viscoelastic properties help to maintain the structural shape integrity of the CNF structure after complete removal of water from a CNF hydrogel upon freeze-drying, under appropriate conditions. 14 To enhance and enlarge the application range of those nanofibrils and increase the compatibility and adhesion to other matrices (such as hydrophobic thermoplastics), several surface modification chemistries were explored. 21 , 22 Among them, the catalytic oxidation with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) proved to be an efficient method for the modification of CNF hydroxyl groups. 23 One advantage of the TEMPO-mediated oxidation is that the reaction can be carried out in water and under mild conditions. The resulting TEMPO-mediated oxidized CNF (TOCNF) has a high anionic charge density on the fibril surfaces. TOCNF suspensions behave like gels under moderate concentrations, however, they cannot withstand a high shear rate as the gel is easily disrupted. 24 As previously reported, stable TOCNF gels can be obtained through the cross-linking of the CNF carboxylate groups with various divalent and trivalent metal cations. 24 The cross-linking process can strengthen the network structures of the oxidized CNF-based hydrogels. Our primary aim was to develop 3D printable CNF hydrogel inks by cross-linking TEMPO-oxidized CNF with divalent and trivalent metal cations. The properties and 3D printing performance of the cross-linked TOCNF-based hydrogels were studied and evaluated and the ability of different metal ions to serve as stabilizing cross-linkers was assessed. Methods Materials The dry cellulose source, elemental chlorine free (ECF) bleached softwood kraft pulp, was obtained from MERCER Stendal GmbH, Germany. The Northern bleached softwood kraft pulp was made from pine (30 - 60 %) and spruce (40 - 70 %), PFI-milled at 23 °C and 50 % relative humidity. CNF was produced by passing the softwood kraft pulp through an M-110EH-30 Microfluidizer from Microfluidics. The grinding degree was analyzed with a Schopper-Riegler analyzer (KARL SCHRÖDER KG, Germany). 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, 98 %), hydrochloric acid (37 %, HCl), ethanol (96 %), sodium hydroxide solution (0.5 M, NaOH), sodium bromide (99 %, NaBr), iron(III) chloride (98 %, FeCl 3 ), aluminum nitrate nonahydrate (98 %, Al(NO 3 ) 3 ), calcium chloride dehydrate (99 %, CaCl 2 ), and magnesium nitrate hexahydrate (99 %, Mg(NO 3 ) 2 ) were purchased from Sigma-Aldrich and used as received. Sodium hypochlorite pentahydrate (available chlorine min. 40.0 %) was purchased from TCI EUROPE N.V. and used as received. All syntheses were performed using MilliQ water. MilliQ water was purified via a PURELAB® Option-Q System, 0.055 µS cm -1 . Characterization The morphology of the different CNF gels was observed via ultra-high-resolution field emission scanning electron microscopy (FE-SEM) using a Hitachi S-4800. The dried CNF samples were mounted on sample supports using carbon tape and coated with a 5 nm layer of Pd/Pt with a Cressington 208HR under an inert atmosphere. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) was performed using a Bruker Vector 33 spectrometer. Measurements were performed by accumulating 256 scans in the spectral region of 4000–550 cm -1 with a spectral resolution of 2 cm -1 . Rheological tests were carried out with a TA Instrument AR 2000ex and the Advantage Software v5.8.2. Rheological tests were carried out with a 40 mm parallel-plate configuration and 1000 μm gap distance. The cation-cross-linked hydrogel was distributed onto the bottom plate. The frequency sweep was set up between 0.1 and 100 rad/s and a strain sweep was performed at an angular frequency of 6.28 rad/s to ensure the measurements were made in the linear viscoelastic region. The conductivity titration was performed with a 721 NET Titrino from Metrohm. For purification and concentration, a centrifuge (Sorvall LYNX 6000) from Thermo SCIENTIFIC was used. Production of Cellulose Nanofibrils CNF was produced via microfluidic treatment similar to previously described processes. 8 In a typical procedure 10 g dry cellulose pulp was suspended in 200 mL water and grinded until a degree of grinding of 75­–80 °SR [1] was reached. A Microfluidizer (M-110EH-30 Microfluidics) was used to disintegrate cellulose fibers into CNFs. The fiber suspension firstly passed through two z-shaped channels of 400 μm and 200 μm diameter under high pressure (15,000 Psi). This operation was repeated two times. Then, the fiber suspension passed through two thinner chambers with orifice widths of 200 μm and 100 μm successively under the pressure of 25,000 Psi. This operation was repeated four times. The CNF suspension was then concentrated by centrifugation, resulting in a 2.0 wt% CNF aqueous gel. TEMPO-mediated Oxidation of the CNF TEMPO-mediated oxidized CNF (TOCNF) was obtained by TEMPO-mediated oxidation in water at pH 10 as described previously. 28 In a typical synthesis, 500 mL CNF suspension (0.58 wt%) was added to a 100 mL solution of TEMPO (0.05 g, 0.32 mmol) and NaBr (0.3 g, 2.9 mmol). NaClO·5H 2 O (4.9 g, 66 mmol) was then added to initiate the reaction. The mixture was kept at room temperature and the pH was maintained to a value of 10 through the addition of 0.5 M NaOH solution over a period of 5 h. After 5 h, no further pH variation was observed, indicating the end of the reaction. The reaction was quenched by adding 15 mL ethanol. HCl solution (37 wt%) was then added to adjust the pH to 4. The suspension was concentrated by centrifugation (20.000 g for 45 min) yielding a solid content of 2.64 wt%. A conductivity titration was performed to determine the carboxylate content 29 of the TOCNF by titration with 0.05 M NaOH standard solution. A carboxylate group content of 1.94 mmol/g was measured. If compared to other already published articles, the carboxylate group contents of our TOCNF is higher, as other carboxylate group contents are more in the range of 1.0 – 1.5 mmol/g. 24,26,30–33 This high carboxylate group content has two mayor reasons. First of all, the cellulose was already fibrillated prior the oxidation process. This leads to a better accessibility for the oxidizing agent to the cellulose fibril compared to a procedure were the oxidation is part of the fibrillating process. More important is the fact, that a freshly made NaClO solution from a solid NaClO·5H 2 O source was used. In most other published procedures an already solved solution of NaClO in water is used and this solution will degrade over time and depending on the age of this solution the concentration will be lower. Preparation of Cation-Cross-linked TOCNF Hydrogels CNF hydrogels were produced through the addition of various metal cation solutions to cross-link the TOCNF in suspension. Before the addition of metal cation solutions, the pH of the TOCNF suspension was adjusted to 6 with a 0.5 M NaOH solution. The corresponding amount of the metal cation solution (50 mM, FeCl 3 , Al(NO 3 ) 3 , CaCl 2 , or Mg(NO 3 ) 2 ) was added dropwise into the TOCNF suspensions. After 12 h, the hydrogels were collected through centrifugation for 20 min at defined g-force values of 4430 g, 8860 g or 13290 g, which were redefined in this study as 1×G, 2×G, 3×G respectively. The impact of centrifugation at different g-force values on hydrogel performance for 3D printing was also investigated. The hydrogels were characterized through ATR-FTIR spectroscopy and rheology measurements. 3D Printing of Hydrogels A cube model of 10×10×5 mm was designed and 3D printed by pneumatic extrusion. 3D printing was performed with an INKREDIBLE 3D printer from CELLINK. The cubes were 3D printed using two different conical nozzles diameters (0.84 mm or 0.58 mm). The weights of the 3D printed cubes were measured before and after the drying process to determine the solid content of the hydrogels. The 3D printed cubes were according to their performances dried either in an oven overnight at 60 °C or through freeze-drying. The 3D printed structures were characterized through visual inspection, ATR-FTIR spectroscopy and SEM. [1] °SR: Schopper-Riegler degrees, determined using SCHOPPER-RIEGLER method (DIN EN ISO 5267-1). Results And Discussion Cross-linked hydrogels were prepared from TEMPO-oxidized CNF with various metallic cations (Fe 3+ , Al 3+ , Ca 2+ , and Mg 2+ ). Gelation of the TOCNF suspension occurred immediately upon the addition of the metal cation solution, through diffusion of the metal cations into the deprotonated TOCNF dispersion followed by electrostatic interactions between the metal cations and the negative charge of the TOCNF carboxylate groups. All the hydrogels (TOCNF-M n+ ) were left undisturbed overnight to enable thorough diffusion of cations into the preformed gels. All gels prepared through this method were macroscopically homogeneous and were slightly less transparent than the TOCNF starting dispersions. The Fe 3+ cross-linked TOCNF gels (TOCNF-Fe 3+ ) were yellow (which is typical for this ion complex) while the TOCNF gels cross-linked with Al 3+ (TOCNF-Al 3+ ), Ca 2+ (TOCNF-Ca 2+ ) and Mg 2+ (TOCNF-Mg 2+ ) remained colorless. The cross-linked gels were characterized with ATR-FTIR spectroscopy (Figure 1). Table 1. IR Wavenumbers for CNF, TOCNF, and TOCNF-M n+ hydrogels. Samples ʋ OH (H-bonded) / cm -1 ʋ C=O / cm -1 ʋ as, OCO / cm -1 ʋ s, OCO / cm -1 CNF 3335 - - - TOCNF 3334 1725 - - CNF-Fe 3+ 3326 1725 1601 1412 CNF-Al 3+ 3330 1735 1617 1419 CNF-Ca 2+ 3330 1744 1590 1418 CNF-Mg 2+ 3330 1744 1593 1418 As shown in Figure 1, the spectrum of the initial and unmodified CNF exhibits the characteristic bands of the nanocellulose with bands localized at 3335 cm ‑1 (ʋ OH ), 2905 cm -1 and 2860 cm -1 (ʋ C-H ), 1637 cm -1 (δ OH ), 1429 cm -1 (δ CH2 ), 1369 cm -1 (δ C‑H ) and 1335 cm -1 (δ O-H ). In addition to those characteristic bands, the TOCNF spectrum shows a strong additional absorption band localized at 1725 cm ‑1 which is attributed to the vibration of the carbonyl bond (ʋ C=O ) in the carboxylic group. The presence of this new band confirms the successful chemical conversion of CNF into TOCNF. After cross-linking of the TOCNF with a metal cation, new bands appear in the region 1650–1400 cm -1 . Vibration assignments for the most relevant bands are listed in Table 1. With or without cross-linking, the broad bands localized between 3297-3335 cm -1 (ʋOH stretching vibrations) remain unchanged. As mentioned earlier, the TOCNF spectrum exhibits a strong additional absorption band localized at 1725 cm ‑1 (ʋ C=O ). A shift of this carbonyl band is observed after metal ion cross-linking of the TOCNF. The bands for these vibrations in TOCNF-M n+ spectra are attributed to un-complexed carboxylate groups that still exist in the carboxylic acid form. Thus, the divalent cations Ca 2+ and Mg 2+ incorporated better with deprotonated TOCNF than the trivalent cations Fe 3+ and Al 3+ , due to the relatively stronger ʋ s, OCO stretching vibration of TOCNF-Ca 2+ and TOCNF-Mg 2+ , whereas the hydrogels with trivalent cations Fe 3+ and Al 3+ had less incorporation and more un-complexed C=O groups that exist as carboxylic acid form. 24 In the TOCNF-M n+ spectra, the symmetric and asymmetric bands (ʋ as/s, OCO ) are also shifted towards lower wavenumbers, which is due to the formation of ionic bonds between the cations and the carboxylate groups of the surface-modified cellulose. 25 Various cations and various M 2+ :TOCNF:H 2 O ratios were investigated (Table 2). The gelation process was faster with the addition of trivalent cations (Fe 3+ , Al 3+ ) than with divalent cations (Ca 2+ , Mg 2+ ). Lower yields were observed when divalent ions were used for the cross-linking. The amounts of TOCNF-Fe 3+ and TOCNF-Al 3+ gels were similar and about twice as high as the yields of TOCNF-Ca 2+ and TOCNF-Mg 2+ . Hydrogels synthesized with a M n+ :TOCNF ratio of 1:1 and without water dilution did not exhibit any fluidity and were mechanically too robust and rigid to pass through the 3D printer nozzle and could therefore not be 3D printed. The same was observed for M n+ :TOCNF gels with a M n+ :TOCNF:H 2 O ratio of 1:1:1, regardless of the valency of the cation. To obtain 3D printable gels, the hydrogels were swollen through the addition of water. The addition of water during gel preparation (M n+ :TOCNF:H 2 O ratios ranging from 1:1:10 to 1:1:25) influenced the rheological behavior of the gels and the rigidity decreased in the order TOCNF-Fe 3+ > TOCNF-Al 3+ > TOCNF-Ca 2+ > TOCNF-Mg 2+ (Figure 4). The synthesized hydrogels obtained with trivalent cations were unprintable regardless of the M n+ :TOCNF:H 2 O ratio, probably due to the high gel density that does not meet the specific rheological requirements (for example shear thinning) and therefore could not be pneumatically extruded since they blocked the 3D printer nozzle. On the contrary, hydrogels cross-linked with divalent cations could be 3D printed, however, the printed objects became inhomogeneous (heavy structural defects) at M n+ :TOCNF:H 2 O ratios ranging from 1:1:10 - 1:1:20. When the ratio was 1:1:25, the gels of TOCNF-Ca 2+ offer the best printing performance while the TOCNF-Mg 2+ hydrogel was not firm enough for shape retention when printed in the form of a cube. When the ratio was kept at 1.5:1:25, the TOCNF-Ca 2+ -gel was inhomogeneously printed and the TOCNF-Mg 2+ -gel was still too fluid, although this would be the perfect ratio according to the Derjaguin–Landau–Verwey–Overbeek (DLVO [1] ) theory, based on calculations from Fukuzumi et al. 26 in a study on the dispersion stability and aggregation behavior of TEMPO-oxidized cellulose nanofibrils in water as a function of salt addition. The initial TOCNF suspension is opaque, nearly transparent and very fluidic. The direct 3D printing of the initial TOCNF suspension in a bath what contains the metal cation solution for post-printing cross-linking was investigated, but was unfortunately not successful. Centrifugation with higher rotation speed (Table 3) had no significant impact on the performance of TOCNF-Ca 2+ (solid content remained unchanged: 1.39 wt%) but affected the TOCNF-Mg 2+ hydrogel with an increase of the solid content to 1.39 wt%. With this centrifugation step, TOCNF-Mg 2+ hydrogels could be 3D printed as a cube with a good shape fidelity. After the freeze-drying process, the 3D printed TOCNF-Mg 2+ hydrogels maintained good structural integrity (Figure 2). The CNF, TOCNF, and the 3D printed and freeze-dried TOCNF-M 2+ samples were also analyzed by Scanning Electron Microscopy (SEM) and the SEM images are shown in Figure 3. The SEM images of CNF and TOCNF in Figure 3 show an agglomerated network of isolated fibrils. The images of the 3D printed and freeze-dried cubes of the cross-linked samples TOCNF-Ca 2+ and TOCNF-Mg 2+ show a highly porous structure with dense pore walls made from the cross-linked TOCNF. Cross-linking in combination with freeze-drying leads to a very high degree of interfibril interaction and the formation of dense sheets as observed in the TOCNF-Ca 2+ sample (Figure 3, middle right). Table 2. Solid contents of 3D printed TOCNF-Ca 2+ and TOCNF-Mg 2+ hydrogels at various ratios of M 2+ :TOCNF:H 2 O. M 2+ :TOCNF:H 2 O TOCNF-Ca 2+ wt% TOCNF-Mg 2+ wt% 1:1:1 1.67 1.17 1:1:10 1.03 0.79 1:1:20 0.52 0.95 1:1:25 1.39 0.92 1.5:1:25 1.19 1.00 Table 3. Solid contents of 3D printed TOCNF-Ca 2+ and TOCNF-Mg 2+ hydrogels at the ratio of 1:1:25 with various centrifugation intensities. Centrifugation Intensities TOCNF-Ca 2+ wt% TOCNF-Mg 2+ wt% 1×G 1.39 0.92 2×G 1.40 1.33 3×G ––– [2] 1.39 Table 2 and Table 3 list the solid contents of the 3D printed TOCNF-Ca 2+ and TOCNF-Mg 2+ hydrogels after freeze-drying. The solid content of pristine TOCNF (2.64 wt%) decreases after the cross-linking process (TOCNF-M 2+ ) probably due to the insufficient interaction between the metal cations and deprotonated TOCNF dispersions. Higher water contents within the TOCNF-Mg 2+ (ratio 1:1:25) were decreased by more intensive centrifugation (Table 3), thus leading to a higher solid content and a better 3D printing performance of the resulting TOCNF-Mg 2+ hydrogels. ­ Viscoelastic properties of the hydrogels, storage modulus (G') and loss modulus (G''), are shown in Figure 4. Moduli were measured as a function of a dynamic frequency sweep between 0.1 and 100 rad/s. The G' values of the hydrogels are consistently larger than the G'' values in the entire angular frequency range. Moreover, both G' and G'' values show similar small variations with frequency in the defined range, which indicate a stable gel state of TOCNF-M n+ . The dynamic moduli of TOCNF-Fe 3+ and TOCNF-Al 3+ are clearly higher than for TOCNF-Ca 2+ and TOCNF-Mg 2+ , and they present a significant declining trend after dilution with water during the gelation process. The highest storage modulus of the hydrogels with the ratio of M n+ :TOCNF at 1:1 (up to Gʹ = 40 kPa for Fe 3+ :TOCNF, 1:1) demonstrated the high rigidity and unprintability of those gels. Interestingly, increasing the proportion of metal cations to a M n+ :TOCNF:H 2 O ratio of 1.5:1:25 decreased the dynamic modulus, if compared with the ratio of 1:1:25, at which TOCNF-Ca 2+ had the best 3D printing performance. It is possibly due to more substantial intra-fibril interactions rather than an inter-fibril cross-linking, at higher amounts of metal cations and wider dispersed TOCNFs. 27 Additionally, the rheological measurements of hydrogels with M n+ :TOCNF ratios of 1:1 and the TOCNF-Al 3+ hydrogel (M n+ :TOCNF:H 2 O ratio 1:1:25) present some deviations, and the hydrogel performance in the viscoelastic area under the strain sweep at a frequency of 6.28 rad/s should be further ensured. The G' value of TOCNF-Ca 2+ , at an M n+ :TOCNF:H 2 O ratio of 1:1:25, is one order of magnitude higher than G’ for the original deprotonated TOCNF dispersion, indicating a better elasticity of the hydrogels due to the incorporation of the metal cations (cross-linking). The impact of centrifuging intensity on hydrogels was further studied (Figure 5). An increase of the centrifugation force increases the viscoelasticity of the hydrogels to a certain extent. The centrifugation effect on the TOCNF-Mg 2+ hydrogel (0.92 to 1.39 % solid content) is more significant than on the TOCNF-Ca 2+ hydrogel (solid content remains constant). The viscoelastic properties of the TOCNF-Mg 2+ after intensive centrifuging, were in the similar range as of the optimal TOCNF-Ca 2+ and also had a comparable good 3D printing performance as of the TOCNF-Ca 2+ . [1] This theory explains that chemical factors, such as pH and electrolyte concentration, can reduce the thickness of the electrical bilayers of colloids and cause an aggregation of colloids through Brownian motion. [2] Not measured, because no need for 3×G at TOCNF-Ca 2+ . Conclusion And Outlook In this study, CNF-based hydrogel inks for 3D printing were prepared from TEMPO-oxidized CNF (TOCNF) with a solid content of 2.64 wt% and a carboxylate content 1.94 mmol/g. Divalent and trivalent metal cations were introduced to cross-link the deprotonated TOCNFs to form the corresponding hydrogels. The chemical functional groups of the original CNF suspension, the TOCNFs and TOCNF-M n+ hydrogels were analyzed with FT-IR, which demonstrated a better interaction between carboxylate anions and the divalent cations Ca 2+ and Mg 2+ than with the trivalent cations Fe 3+ and Al 3+ . The storage modulus (G') and loss modulus (G'') of hydrogels incorporating with trivalent cations Fe 3+ and Al 3+ were significantly higher than thoughts with divalent cations Ca 2+ and Mg 2+ . Hydrogel 3D printing performance was evaluated and showed that gel cross-linked with the divalent cations Ca 2+ and Mg 2+ had good printability and that the TOCNF-Ca 2+ prepared with an M n+ :TOCNF:H 2 O ratio of 1:1:25 under 1×G centrifugation was the best. This gel had a solid content of 1.39 wt% and a storage modulus of Gʹ = 2 kPa. A comparable performance was achieved with TOCNF-Mg 2+ at the same ratio by 2×G centrifugation. Declarations Acknowledgements B. Saake and B. Mietner thank the Fachagentur Nachwachsende Rohstoffe e.V. for financial support (FNR Project number BMEL-22004518). J. Navarro thank the Fachagentur Nachwachsende Rohstoffe e.V. for financial support (FNR Project number BMEL-2200HV024X). 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The Effect of Oxidation Conditions on Chemical and Crystal Structures of the Water-Insoluble Fractions. Biomacromolecules 5 , 1983–1989 (2004). Shinoda, R. et al. Relationship of Distribution of Carboxy Groups to Molar Mass Distribution of TEMPO-Oxidized Algal, Cotton, and Wood Cellulose Nanofibrils. Biomacromolecules 13 , 1–3 (2019). Saito, T., Kimura, S., Nishiyama, Y. & Isogai, A. Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose. Biomacromolecules 8 , 2485–2491 (2007). Okita, Y., Saito, T. & Isogai, A. Entire surface oxidation of various cellulose microfibrils by TEMPO-mediated oxidation. Biomacromolecules 11 , 1696–1700 (2010). Shinoda, R., Saito, T., Okita, Y. & Isogai, A. Relationship between Length and Degree of Polymerization of TEMPO- Oxidized Cellulose Nanofibrils ; Supporting Information. Biomacromolecules 13 , 1–3 (2012). Cite Share Download PDF Status: Published Journal Publication published 19 Mar, 2021 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Major revision 01 Feb, 2021 Reviewers agreed at journal 09 Jan, 2021 Reviews received at journal 08 Jan, 2021 Reviewers agreed at journal 28 Dec, 2020 Reviewers invited by journal 07 Dec, 2020 Editor assigned by journal 07 Dec, 2020 Editor invited by journal 07 Dec, 2020 Submission checks completed at journal 07 Dec, 2020 First submitted to journal 04 Dec, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-122100","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":5970310,"identity":"f1ee7861-8702-47b2-bef2-616e68750954","order_by":0,"name":"Jakob Mietner","email":"","orcid":"https://orcid.org/0000-0001-5954-1774","institution":"Universität Hamburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jakob","middleName":"","lastName":"Mietner","suffix":""},{"id":5970311,"identity":"578b6ba1-885e-4bb1-b210-6c3af40e04cc","order_by":1,"name":"Xuehe Jiang","email":"","orcid":"","institution":"Universität Hamburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuehe","middleName":"","lastName":"Jiang","suffix":""},{"id":5970312,"identity":"671eea10-351a-421c-9b04-cb2d1df2fca9","order_by":2,"name":"Ulrica Edlund","email":"","orcid":"https://orcid.org/0000-0002-1631-1781","institution":"Royal Institute of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ulrica","middleName":"","lastName":"Edlund","suffix":""},{"id":5970313,"identity":"1034dbb2-4709-452d-8439-9ed31fdf48ff","order_by":3,"name":"Bodo Saake","email":"","orcid":"https://orcid.org/0000-0003-4389-9506","institution":"Universität Hamburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bodo","middleName":"","lastName":"Saake","suffix":""},{"id":5970314,"identity":"107fd63a-9f54-48b1-9dfe-a50fc0a79a5d","order_by":4,"name":"Julien Navarro","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8791-6190","institution":"Universität Hamburg","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Julien","middleName":"","lastName":"Navarro","suffix":""}],"badges":[],"createdAt":"2020-12-04 19:59:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-122100/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-122100/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-021-85865-4","type":"published","date":"2021-03-19T19:04:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":4131004,"identity":"1d06335a-56f0-455f-a6a6-ea52af204a57","added_by":"auto","created_at":"2020-12-09 17:01:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":75966,"visible":true,"origin":"","legend":"ATR-FTIR spectra of the dried TOCNF suspension and TOCNF-Mn+ hydrogels: (a) pristine CNF (b) TOCNF, (c) TOCNF-Fe3+, (d) TOCNF-Al3+, (e) TOCNF-Ca2+, (f) TOCNF-Mg2+. Left: full spectra, right: region of interest.","description":"","filename":"OnlineFig1.Png","url":"https://assets-eu.researchsquare.com/files/rs-122100/v1/d610c2ef97c9fe8e773255b5.Png"},{"id":4131005,"identity":"b7600fe2-f502-4d74-aefb-78256fc21271","added_by":"auto","created_at":"2020-12-09 17:01:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97693,"visible":true,"origin":"","legend":"Representative images of printed TOCNF-Mg2+-gel (1:1:25, 2×G). Gel cube a-) (10×10×5 mm) after printing in wet state, and b,c) after freeze-drying.","description":"","filename":"OnlineFig2.Png","url":"https://assets-eu.researchsquare.com/files/rs-122100/v1/c349e92deb450c8f027585e9.Png"},{"id":4131006,"identity":"ab52aa9f-395e-46b1-857c-408d2f0f6ca6","added_by":"auto","created_at":"2020-12-09 17:01:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":721745,"visible":true,"origin":"","legend":"Representative SEM images of CNF, TOCNF, TOCNF-Ca2+ and TOCNF-Mg2+ at different magnifications.","description":"","filename":"OnlineFig3.Png","url":"https://assets-eu.researchsquare.com/files/rs-122100/v1/2f75cf784f4f569b50715652.Png"},{"id":4131007,"identity":"d6a2ad03-4dac-44fb-b034-de8d4ec423cb","added_by":"auto","created_at":"2020-12-09 17:01:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45271,"visible":true,"origin":"","legend":"Viscoelastic properties of the TOCNF-Mn+ hydrogels with various cations:Fe3+, Al3+, Ca2+, and Mg2+ are presented in red, blue, green, and orange, respectively. Mn+:TOCNF(:H2O) ratios: a) 1:1, b) 1:1:25, and c) 1.5:1:25. Storage modulus (G') and loss modulus (G'') are symbolized with filled and open symbols, respectively.","description":"","filename":"OnlineFig4.Png","url":"https://assets-eu.researchsquare.com/files/rs-122100/v1/57d28cba1fee7a00191f3b6d.Png"},{"id":4131008,"identity":"78c8c45f-93ef-49ca-b2c6-1a2a5c5d5129","added_by":"auto","created_at":"2020-12-09 17:01:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":44888,"visible":true,"origin":"","legend":"Viscoelastic properties of the deprotonated TOCNF dispersion and the hydrogels prepared from various divalent cations concentrations and centrifuging intensity: (a) hydrogels with defined ratio of M2+:TOCNF:H2O = 1:1:25 centrifuged with 1×G and 2×G, (b) TOCNF-Mg2+ with various Mg2+ concentrations and centrifuging intensities. Storage modulus (G') and loss modulus (G'') are symbolized with filled and open symbols, respectively.","description":"","filename":"OnlineFig5.Png","url":"https://assets-eu.researchsquare.com/files/rs-122100/v1/b62a4ebd02fe7e415b7b9c08.Png"},{"id":13630116,"identity":"ba29fc98-b6fd-4aaf-8137-fc93b392f78a","added_by":"auto","created_at":"2021-09-17 08:10:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3000312,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-122100/v1/3bf321a3-47fc-4590-acdd-94fc0ce75bca.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e3D Printing of a Bio-Based Ink Made of Cross-Linked Cellulose Nanofibrils With Various Metal Cations\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eThe need to replace petroleum-based products with biodegradable and renewable resources, to produce high-performance functional materials is one of the greatest challenges for a future sustainable society. For this purpose, bio-based polymers have attracted considerable attention over the past decades. Cellulose, and its derivatives, fulfill those needs by offering many advantages such as renewability, biodegradability, and to some extent recyclability.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Cellulose nanofibrils (CNF) is a highly promising candidate for a wide panel of applications, ranging from composites, water purification to drug delivery.\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e CNF can be extracted from numerous lignocellulosic source materials through mechanical disintegration.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Typical CNF dimensions comprise widths between 5\u0026ndash;20\u0026nbsp;nm and a wide range of lengths, typically several micrometers.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRecently, several groups demonstrated that CNF could be structured through 3D printing processes.\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e The demonstrated 3D printed CNF objects showed great potential as 3D printed tablets for controlled drug release and as 3D printed bioactive composites in tissue engineering and wound dressing applications. Markstedt \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e produced a biobased ink made of cross-linked CNF, alginate, and CaCl\u003csub\u003e2\u003c/sub\u003e. The CNF-alginate cross-linked structure proved to be a viable scaffold for hosting human nasoseptal chondrocyte cells. Leppiniemi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e developed a 3D ink based on alginate, avidin protein-modified CNF and glycerin. In this study Leppiniemi \u003cem\u003eet al.\u003c/em\u003e were using CNF as a strengthening additive and CaCl\u003csub\u003e2\u003c/sub\u003e as a cross-linker, what leads to a significantly more stable shape fidelity after 3D printing. Moreover, the 3D printed object showed good tissue compatibility and great potential in biomedical applications such as in wound dressings. Li et \u003cem\u003eal.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e produced a 3D printed structure made of nanocellulose and carbon nanotubes. The object was first 3D printed and cross-linked with CaCl\u003csub\u003e2\u003c/sub\u003e later on. Several drying protocols were applied to the printed hydrogel and freeze-drying was found to be the most efficient strategy.\u003c/p\u003e \u003cp\u003eCNF has an abundance of hydroxyl groups on the fibril surfaces which lead to strong hydrogen interaction i) between fibrils (inter-fibrils interaction that leads to fibril agglomeration) and ii) with water molecules, endowing viscoelasticity and shear thinning properties that are advantageous for 3D printing.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e The viscoelastic properties help to maintain the structural shape integrity of the CNF structure after complete removal of water from a CNF hydrogel upon freeze-drying, under appropriate conditions.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e To enhance and enlarge the application range of those nanofibrils and increase the compatibility and adhesion to other matrices (such as hydrophobic thermoplastics), several surface modification chemistries were explored.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Among them, the catalytic oxidation with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) proved to be an efficient method for the modification of CNF hydroxyl groups.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e One advantage of the TEMPO-mediated oxidation is that the reaction can be carried out in water and under mild conditions. The resulting TEMPO-mediated oxidized CNF (TOCNF) has a high anionic charge density on the fibril surfaces. TOCNF suspensions behave like gels under moderate concentrations, however, they cannot withstand a high shear rate as the gel is easily disrupted.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e As previously reported, stable TOCNF gels can be obtained through the cross-linking of the CNF carboxylate groups with various divalent and trivalent metal cations.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The cross-linking process can strengthen the network structures of the oxidized CNF-based hydrogels.\u003c/p\u003e \u003cp\u003eOur primary aim was to develop 3D printable CNF hydrogel inks by cross-linking TEMPO-oxidized CNF with divalent and trivalent metal cations. The properties and 3D printing performance of the cross-linked TOCNF-based hydrogels were studied and evaluated and the ability of different metal ions to serve as stabilizing cross-linkers was assessed.\u003c/p\u003e "},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dry cellulose source, elemental chlorine free (ECF) bleached softwood kraft pulp, was obtained from MERCER Stendal GmbH, Germany. The Northern bleached softwood kraft pulp was made from pine (30 - 60\u0026nbsp;%) and spruce (40 - 70\u0026nbsp;%), PFI-milled at 23\u0026nbsp;\u0026deg;C and 50\u0026nbsp;% relative humidity. CNF was produced by passing the softwood kraft pulp through an M-110EH-30 Microfluidizer from Microfluidics. The grinding degree was analyzed with a Schopper-Riegler analyzer (KARL SCHR\u0026Ouml;DER KG, Germany).\u003c/p\u003e\n\u003cp\u003e2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO, 98\u0026nbsp;%), hydrochloric acid (37\u0026nbsp;%, HCl), ethanol (96\u0026nbsp;%), sodium hydroxide solution (0.5\u0026nbsp;M, NaOH), sodium bromide (99\u0026nbsp;%, NaBr), iron(III) chloride (98\u0026nbsp;%, FeCl\u003csub\u003e3\u003c/sub\u003e), aluminum nitrate nonahydrate (98\u0026nbsp;%, Al(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e), calcium chloride dehydrate (99\u0026nbsp;%, CaCl\u003csub\u003e2\u003c/sub\u003e), and magnesium nitrate hexahydrate (99\u0026nbsp;%, Mg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e) were purchased from Sigma-Aldrich and used as received. Sodium hypochlorite pentahydrate (available chlorine min. 40.0\u0026nbsp;%) was purchased from TCI EUROPE N.V. and used as received. All syntheses were performed using MilliQ water. MilliQ water was purified via a PURELAB\u0026reg; Option-Q System, 0.055 \u0026micro;S cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe morphology of the different CNF gels was observed via ultra-high-resolution field emission scanning electron microscopy (FE-SEM) using a Hitachi S-4800. The dried CNF samples were mounted on sample supports using carbon tape and coated with a 5\u0026nbsp;nm layer of Pd/Pt with a Cressington 208HR under an inert atmosphere.\u003c/p\u003e\n\u003cp\u003eAttenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) was performed using a Bruker Vector 33 spectrometer. Measurements were performed by accumulating 256 scans in the spectral region of 4000\u0026ndash;550\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e with a spectral resolution of 2\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eRheological tests were carried out with a TA Instrument AR 2000ex and the Advantage Software v5.8.2. Rheological tests were carried out with a 40\u0026nbsp;mm parallel-plate configuration and 1000\u0026nbsp;\u0026mu;m gap distance. The cation-cross-linked hydrogel was distributed onto the bottom plate. The frequency sweep was set up between 0.1 and 100\u0026nbsp;rad/s and a strain sweep was performed at an angular frequency of 6.28\u0026nbsp;rad/s to ensure the measurements were made in the linear viscoelastic region.\u003c/p\u003e\n\u003cp\u003eThe conductivity titration was performed with a 721 NET Titrino from Metrohm. For purification and concentration, a centrifuge (Sorvall LYNX 6000) from Thermo SCIENTIFIC was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of Cellulose Nanofibrils\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCNF was produced via microfluidic treatment similar to previously described processes.\u003csup\u003e8\u003c/sup\u003e In a typical procedure 10\u0026nbsp;g dry cellulose pulp was suspended in 200\u0026nbsp;mL water and grinded until a degree of grinding of 75\u0026shy;\u0026ndash;80\u0026nbsp;\u0026deg;SR\u003ca href=\"#_ftn1\" name=\"_ftnref1\"\u003e\u003csup\u003e\u003csup\u003e[1]\u003c/sup\u003e\u003c/sup\u003e\u003c/a\u003e was reached. A Microfluidizer (M-110EH-30 Microfluidics) was used to disintegrate cellulose fibers into CNFs. The fiber suspension firstly passed through two z-shaped channels of 400\u0026nbsp;\u0026mu;m and 200\u0026nbsp;\u0026mu;m diameter under high pressure (15,000\u0026nbsp;Psi). This operation was repeated two times. Then, the fiber suspension passed through two thinner chambers with orifice widths of 200\u0026nbsp;\u0026mu;m and 100\u0026nbsp;\u0026mu;m successively under the pressure of 25,000\u0026nbsp;Psi. This operation was repeated four times. The CNF suspension was then concentrated by centrifugation, resulting in a 2.0\u0026nbsp;wt% CNF aqueous gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTEMPO-mediated Oxidation of the CNF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEMPO-mediated oxidized CNF (TOCNF) was obtained by TEMPO-mediated oxidation in water at pH\u0026nbsp;10 as described previously.\u003csup\u003e28\u003c/sup\u003e In a typical synthesis, 500\u0026nbsp;mL CNF suspension (0.58\u0026nbsp;wt%) was added to a 100\u0026nbsp;mL solution of TEMPO (0.05\u0026nbsp;g, 0.32\u0026nbsp;mmol) and NaBr (0.3\u0026nbsp;g, 2.9\u0026nbsp;mmol). NaClO\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO (4.9\u0026nbsp;g, 66\u0026nbsp;mmol) was then added to initiate the reaction. The mixture was kept at room temperature and the pH was maintained to a value of 10 through the addition of 0.5 M NaOH solution over a period of 5\u0026nbsp;h. After 5\u0026nbsp;h, no further pH variation was observed, indicating the end of the reaction. The reaction was quenched by adding 15\u0026nbsp;mL ethanol. HCl solution (37\u0026nbsp;wt%) was then added to adjust the pH to 4. The suspension was concentrated by centrifugation (20.000 g for 45 min) yielding a solid content of 2.64 wt%.\u003c/p\u003e\n\u003cp\u003eA conductivity titration was performed to determine the carboxylate content\u003csup\u003e29\u003c/sup\u003e of the TOCNF by titration with 0.05\u0026nbsp;M NaOH standard solution.\u0026nbsp; A carboxylate group content of 1.94\u0026nbsp;mmol/g was measured. If compared to other already published articles, the carboxylate group contents of our TOCNF is higher, as other carboxylate group contents are more in the range of 1.0 \u0026ndash; 1.5 mmol/g.\u003csup\u003e24,26,30\u0026ndash;33\u003c/sup\u003e This high carboxylate group content has two mayor reasons. First of all, the cellulose was already fibrillated prior the oxidation process. This leads to a better accessibility for the oxidizing agent to the cellulose fibril compared to a procedure were the oxidation is part of the fibrillating process. More important is the fact, that a freshly made NaClO solution from a solid NaClO\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO source was used. In most other published procedures an already solved solution of NaClO in water is used and this solution will degrade over time and depending on the age of this solution the concentration will be lower.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Cation-Cross-linked TOCNF Hydrogels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCNF hydrogels were produced through the addition of various metal cation solutions to cross-link the TOCNF in suspension. Before the addition of metal cation solutions, the pH of the TOCNF suspension was adjusted to 6 with a 0.5\u0026nbsp;M NaOH solution. The corresponding amount of the metal cation solution (50\u0026nbsp;mM, FeCl\u003csub\u003e3\u003c/sub\u003e, Al(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e, CaCl\u003csub\u003e2\u003c/sub\u003e, or Mg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e) was added dropwise into the TOCNF suspensions. After 12\u0026nbsp;h, the hydrogels were collected through centrifugation for 20\u0026nbsp;min at defined g-force values of 4430\u0026nbsp;g, 8860\u0026nbsp;g or 13290\u0026nbsp;g, which were redefined in this study as 1\u0026times;G, 2\u0026times;G, 3\u0026times;G respectively. The impact of centrifugation at different g-force values on hydrogel performance for 3D printing was also investigated. The hydrogels were characterized through ATR-FTIR spectroscopy and rheology measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3D Printing of Hydrogels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA cube model of 10\u0026times;10\u0026times;5\u0026nbsp;mm was designed and 3D printed by pneumatic extrusion. 3D printing was performed with an INKREDIBLE 3D printer from CELLINK. The cubes were 3D printed using two different conical nozzles diameters (0.84\u0026nbsp;mm or 0.58\u0026nbsp;mm). The weights of the 3D printed cubes were measured before and after the drying process to determine the solid content of the hydrogels. The 3D printed cubes were according to their performances dried either in an oven overnight at 60\u0026nbsp;\u0026deg;C or through freeze-drying.\u003c/p\u003e\n\u003cp\u003eThe 3D printed structures were characterized through visual inspection, ATR-FTIR spectroscopy and SEM.\u003c/p\u003e\n\u003cp\u003e\u003ca href=\"#_ftnref1\" name=\"_ftn1\"\u003e[1]\u003c/a\u003e \u0026deg;SR: Schopper-Riegler degrees, determined using SCHOPPER-RIEGLER method (DIN EN ISO 5267-1).\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003eCross-linked hydrogels were prepared from TEMPO-oxidized CNF with various metallic cations (Fe\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, and Mg\u003csup\u003e2+\u003c/sup\u003e). Gelation of the TOCNF suspension occurred immediately upon the addition of the metal cation solution, through diffusion of the metal cations into the deprotonated TOCNF dispersion followed by electrostatic interactions between the metal cations and the negative charge of the TOCNF carboxylate groups. All the hydrogels (TOCNF-M\u003csup\u003en+\u003c/sup\u003e) were left undisturbed overnight to enable thorough diffusion of cations into the preformed gels. All gels prepared through this method were macroscopically homogeneous and were slightly less transparent than the TOCNF starting dispersions. The Fe\u003csup\u003e3+\u003c/sup\u003e cross-linked TOCNF gels (TOCNF-Fe\u003csup\u003e3+\u003c/sup\u003e) were yellow (which is typical for this ion complex) while the TOCNF gels cross-linked with Al\u003csup\u003e3+\u003c/sup\u003e (TOCNF-Al\u003csup\u003e3+\u003c/sup\u003e), Ca\u003csup\u003e2+\u003c/sup\u003e (TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e) and Mg\u003csup\u003e2+ \u003c/sup\u003e(TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e) remained colorless. The cross-linked gels were characterized with ATR-FTIR spectroscopy (Figure 1).\u003c/p\u003e\n\u003cp\u003eTable 1. IR Wavenumbers for CNF, TOCNF, and TOCNF-M\u003csup\u003en+\u003c/sup\u003e hydrogels.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"28%\"\u003e\n\u003cp\u003e\u003cstrong\u003eSamples\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"21%\"\u003e\n\u003cp\u003e\u003cstrong\u003eʋ\u003csub\u003eOH\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csub\u003e(H-bonded)\u003c/sub\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cbr /\u003e / cm\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15%\"\u003e\n\u003cp\u003e\u003cstrong\u003eʋ\u003csub\u003eC=O\u003c/sub\u003e\u003cbr /\u003e \u003cbr /\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e/ cm\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e\u003cstrong\u003eʋ\u003csub\u003eas, OCO\u003c/sub\u003e\u003cbr /\u003e \u003cbr /\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e/ cm\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e\u003cstrong\u003eʋ\u003csub\u003es, OCO\u003c/sub\u003e\u003cbr /\u003e \u003cbr /\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e/ cm\u003csup\u003e-1\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"28%\"\u003e\n\u003cp\u003eCNF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"21%\"\u003e\n\u003cp\u003e3335\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15%\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"28%\"\u003e\n\u003cp\u003eTOCNF\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"21%\"\u003e\n\u003cp\u003e3334\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15%\"\u003e\n\u003cp\u003e1725\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"28%\"\u003e\n\u003cp\u003eCNF-Fe\u003csup\u003e3+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"21%\"\u003e\n\u003cp\u003e3326\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15%\"\u003e\n\u003cp\u003e1725\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1601\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1412\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"28%\"\u003e\n\u003cp\u003eCNF-Al\u003csup\u003e3+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"21%\"\u003e\n\u003cp\u003e3330\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15%\"\u003e\n\u003cp\u003e1735\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1617\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1419\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"28%\"\u003e\n\u003cp\u003eCNF-Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"21%\"\u003e\n\u003cp\u003e3330\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15%\"\u003e\n\u003cp\u003e1744\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1590\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1418\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"28%\"\u003e\n\u003cp\u003eCNF-Mg\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"21%\"\u003e\n\u003cp\u003e3330\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"15%\"\u003e\n\u003cp\u003e1744\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1593\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e1418\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr /\u003e As shown in Figure 1, the spectrum of the initial and unmodified CNF exhibits the characteristic bands of the nanocellulose with bands localized at 3335\u0026nbsp;cm\u003csup\u003e‑1\u003c/sup\u003e (ʋ\u003csub\u003eOH\u003c/sub\u003e),\u0026nbsp; 2905\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e and 2860\u0026nbsp;cm\u003csup\u003e-1 \u003c/sup\u003e(ʋ\u003csub\u003eC-H\u003c/sub\u003e), 1637\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e (\u0026delta;\u003csub\u003eOH\u003c/sub\u003e), 1429\u0026nbsp;cm\u003csup\u003e-1 \u003c/sup\u003e(\u0026delta;\u003csub\u003eCH2\u003c/sub\u003e), 1369\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e (\u0026delta;\u003csub\u003eC‑H\u003c/sub\u003e) and 1335\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e (\u0026delta;\u003csub\u003eO-H\u003c/sub\u003e). In addition to those characteristic bands, the TOCNF spectrum shows a strong additional absorption band localized at 1725\u0026nbsp;cm\u003csup\u003e‑1\u003c/sup\u003e which is attributed to the vibration of the carbonyl bond (ʋ\u003csub\u003eC=O\u003c/sub\u003e) in the carboxylic group. The presence of this new band confirms the successful chemical conversion of CNF into TOCNF. After cross-linking of the TOCNF with a metal cation, new bands appear in the region 1650\u0026ndash;1400\u0026nbsp;cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eVibration assignments for the most relevant bands are listed in Table 1. With or without cross-linking, the broad bands localized between 3297-3335 cm\u003csup\u003e-1\u003c/sup\u003e (ʋOH stretching vibrations) remain unchanged.\u003c/p\u003e\n\u003cp\u003eAs mentioned earlier, the TOCNF spectrum exhibits a strong additional absorption band localized at 1725\u0026nbsp;cm\u003csup\u003e‑1\u003c/sup\u003e (ʋ\u003csub\u003eC=O\u003c/sub\u003e). A shift of this carbonyl band is observed after metal ion cross-linking of the TOCNF. The bands for these vibrations in TOCNF-M\u003csup\u003en+\u003c/sup\u003e spectra are attributed to un-complexed carboxylate groups that still exist in the carboxylic acid form. Thus, the divalent cations Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e incorporated better with deprotonated TOCNF than the trivalent cations Fe\u003csup\u003e3+\u003c/sup\u003e and Al\u003csup\u003e3+\u003c/sup\u003e, due to the relatively stronger ʋ\u003csub\u003es, OCO\u003c/sub\u003e stretching vibration of TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e and TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e, whereas the hydrogels with trivalent cations Fe\u003csup\u003e3+\u003c/sup\u003e and Al\u003csup\u003e3+\u003c/sup\u003e had less incorporation and more un-complexed C=O groups that exist as carboxylic acid form.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn the TOCNF-M\u003csup\u003en+ \u003c/sup\u003espectra, the symmetric and asymmetric bands (ʋ\u003csub\u003eas/s, OCO\u003c/sub\u003e) are also shifted towards lower wavenumbers, which is due to the formation of ionic bonds between the cations and the carboxylate groups of the surface-modified cellulose.\u003csup\u003e25\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eVarious cations and various M\u003csup\u003e2+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratios were investigated (Table 2). The gelation process was faster with the addition of trivalent cations (Fe\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e) than with divalent cations (Ca\u003csup\u003e2+\u003c/sup\u003e, Mg\u003csup\u003e2+\u003c/sup\u003e). Lower yields were observed when divalent ions were used for the cross-linking. The amounts of TOCNF-Fe\u003csup\u003e3+ \u003c/sup\u003eand TOCNF-Al\u003csup\u003e3+\u003c/sup\u003e gels were similar and about twice as high as the yields of TOCNF-Ca\u003csup\u003e2+ \u003c/sup\u003eand TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHydrogels synthesized with a M\u003csup\u003en+\u003c/sup\u003e:TOCNF ratio of 1:1 and without water dilution did not exhibit any fluidity and were mechanically too robust and rigid to pass through the 3D printer nozzle and could therefore not be 3D printed. The same was observed for M\u003csup\u003en+\u003c/sup\u003e:TOCNF gels with a M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratio of 1:1:1, regardless of the valency of the cation.\u003c/p\u003e\n\u003cp\u003eTo obtain 3D printable gels, the hydrogels were swollen through the addition of water. The addition of water during gel preparation (M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratios ranging from 1:1:10 to 1:1:25) influenced the rheological behavior of the gels and the rigidity decreased in the order TOCNF-Fe\u003csup\u003e3+ \u003c/sup\u003e\u0026gt; TOCNF-Al\u003csup\u003e3+\u003c/sup\u003e\u0026gt; TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e \u0026gt; TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e (Figure 4). The synthesized hydrogels obtained with trivalent cations were unprintable regardless of the M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratio, probably due to the high gel density that does not meet the specific rheological requirements (for example shear thinning) and therefore could not be pneumatically extruded since they blocked the 3D printer nozzle.\u003c/p\u003e\n\u003cp\u003eOn the contrary, hydrogels cross-linked with divalent cations could be 3D printed, however, the printed objects became inhomogeneous (heavy structural defects) at M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratios ranging from 1:1:10 - 1:1:20. When the ratio was 1:1:25, the gels of TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e offer the best printing performance while the TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogel was not firm enough for shape retention when printed in the form of a cube. When the ratio was kept at 1.5:1:25, the TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e-gel was inhomogeneously printed and the TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e-gel was still too fluid, although this would be the perfect ratio according to the Derjaguin\u0026ndash;Landau\u0026ndash;Verwey\u0026ndash;Overbeek (DLVO\u003ca href=\"#_ftn1\" name=\"_ftnref1\"\u003e\u003csup\u003e\u003csup\u003e[1]\u003c/sup\u003e\u003c/sup\u003e\u003c/a\u003e) theory, based on calculations from Fukuzumi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e in a study on the dispersion stability and aggregation behavior of TEMPO-oxidized cellulose nanofibrils in water as a function of salt addition.\u003c/p\u003e\n\u003cp\u003eThe initial TOCNF suspension is opaque, nearly transparent and very fluidic. The direct 3D printing of the initial TOCNF suspension in a bath what contains the metal cation solution for post-printing cross-linking was investigated, but was unfortunately not successful.\u003c/p\u003e\n\u003cp\u003eCentrifugation with higher rotation speed (Table 3) had no significant impact on the performance of TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e (solid content remained unchanged: 1.39\u0026nbsp;wt%) but affected the TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogel with an increase of the solid content to 1.39 wt%. With this centrifugation step, TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogels could be 3D printed as a cube with a good shape fidelity. After the freeze-drying process, the 3D printed TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogels maintained good structural integrity (Figure 2).\u003c/p\u003e\n\u003cp\u003eThe CNF, TOCNF, and the 3D printed and freeze-dried TOCNF-M\u003csup\u003e2+\u003c/sup\u003e samples were also analyzed by Scanning Electron Microscopy (SEM) and the SEM images are shown in Figure 3.\u003c/p\u003e\n\u003cp\u003eThe SEM images of CNF and TOCNF in Figure 3 show an agglomerated network of isolated fibrils. The images of the 3D printed and freeze-dried cubes of the cross-linked samples TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e and TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e show a highly porous structure with dense pore walls made from the cross-linked TOCNF. Cross-linking in combination with freeze-drying leads to a very high degree of interfibril interaction and the formation of dense sheets as observed in the TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e sample (Figure 3, middle right).\u003c/p\u003e\n\u003cp\u003eTable 2. Solid contents of 3D printed TOCNF-Ca\u003csup\u003e2+ \u003c/sup\u003eand TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogels at various ratios of M\u003csup\u003e2+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e\u003cstrong\u003eM\u003csup\u003e2+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e\u003cstrong\u003eTOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ewt%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e\u003cstrong\u003eTOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ewt%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e1:1:1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.17\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e1:1:10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e0.79\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e1:1:20\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e0.52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e0.95\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e1:1:25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e0.92\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e1.5:1:25\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.19\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr /\u003e Table 3. Solid contents of 3D printed TOCNF-Ca\u003csup\u003e2+ \u003c/sup\u003eand TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogels at the ratio of 1:1:25 with various centrifugation intensities.\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e\u003cstrong\u003eCentrifugation Intensities\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33%\"\u003e\n\u003cp\u003e\u003cstrong\u003eTOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ewt%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e\u003cstrong\u003eTOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ewt%\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e1\u0026times;G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33%\"\u003e\n\u003cp\u003e1.39\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e0.92\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e2\u0026times;G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33%\"\u003e\n\u003cp\u003e1.40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.33\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"34%\"\u003e\n\u003cp\u003e3\u0026times;G\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"33%\"\u003e\n\u003cp\u003e\u0026ndash;\u0026ndash;\u0026ndash;\u003ca href=\"#_ftn2\" name=\"_ftnref2\"\u003e\u003csup\u003e\u003csup\u003e[2]\u003c/sup\u003e\u003c/sup\u003e\u003c/a\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"32%\"\u003e\n\u003cp\u003e1.39\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr /\u003e Table 2 and Table 3 list the solid contents of the 3D printed TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e and TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogels after freeze-drying. The solid content of pristine TOCNF (2.64\u0026nbsp;wt%) decreases after the cross-linking process (TOCNF-M\u003csup\u003e2+\u003c/sup\u003e) probably due to the insufficient interaction between the metal cations and deprotonated TOCNF dispersions. Higher water contents within the TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e (ratio 1:1:25) were decreased by more intensive centrifugation (Table 3), thus leading to a higher solid content and a better 3D printing performance of the resulting TOCNF-Mg\u003csup\u003e2+ \u003c/sup\u003ehydrogels.\u003c/p\u003e\n\u003cp\u003e\u0026shy;\u003c/p\u003e\n\u003cp\u003eViscoelastic properties of the hydrogels, storage modulus (G') and loss modulus (G''), are shown in Figure 4. Moduli were measured as a function of a dynamic frequency sweep between 0.1 and 100\u0026nbsp;rad/s. The G' values of the hydrogels are consistently larger than the G'' values in the entire angular frequency range. Moreover, both G' and G'' values show similar small variations with frequency in the defined range, which indicate a stable gel state of TOCNF-M\u003csup\u003en+\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe dynamic moduli of TOCNF-Fe\u003csup\u003e3+\u003c/sup\u003e and TOCNF-Al\u003csup\u003e3+\u003c/sup\u003e are clearly higher than for TOCNF-Ca\u003csup\u003e2+ \u003c/sup\u003eand TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e, and they present a significant declining trend after dilution with water during the gelation process. The highest storage modulus of the hydrogels with the ratio of M\u003csup\u003en+\u003c/sup\u003e:TOCNF at 1:1 (up to Gʹ = 40\u0026nbsp;kPa for Fe\u003csup\u003e3+\u003c/sup\u003e:TOCNF, 1:1) demonstrated the high rigidity and unprintability of those gels. Interestingly, increasing the proportion of metal cations to a M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratio of 1.5:1:25 decreased the dynamic modulus, if compared with the ratio of 1:1:25, at which TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e had the best 3D printing performance. It is possibly due to more substantial intra-fibril interactions rather than an inter-fibril cross-linking, at higher amounts of metal cations and wider dispersed TOCNFs.\u003csup\u003e27\u003c/sup\u003e Additionally, the rheological measurements of hydrogels with M\u003csup\u003en+\u003c/sup\u003e:TOCNF ratios of 1:1 and the TOCNF-Al\u003csup\u003e3+\u003c/sup\u003e hydrogel (M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratio 1:1:25) present some deviations, and the hydrogel performance in the viscoelastic area under the strain sweep at a frequency of 6.28\u0026nbsp;rad/s should be further ensured.\u003c/p\u003e\n\u003cp\u003eThe G' value of TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e, at an M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratio of 1:1:25, is one order of magnitude higher than G\u0026rsquo; for the original deprotonated TOCNF dispersion, indicating a better elasticity of the hydrogels due to the incorporation of the metal cations (cross-linking). The impact of centrifuging intensity on hydrogels was further studied (Figure 5). An increase of the centrifugation force increases the viscoelasticity of the hydrogels to a certain extent. The centrifugation effect on the TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e hydrogel (0.92 to 1.39\u0026nbsp;% solid content) is more significant than on the TOCNF-Ca\u003csup\u003e2+ \u003c/sup\u003ehydrogel (solid content remains constant).\u003c/p\u003e\n\u003cp\u003eThe viscoelastic properties of the TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e after intensive centrifuging, were in the similar range as of the optimal TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e and also had a comparable good 3D printing performance as of the TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003ca href=\"#_ftnref1\" name=\"_ftn1\"\u003e[1]\u003c/a\u003e This theory explains that chemical factors, such as pH and electrolyte concentration, can reduce the thickness of the electrical bilayers of colloids and cause an aggregation of colloids through Brownian motion.\u003c/p\u003e\n\u003cp\u003e\u003ca href=\"#_ftnref2\" name=\"_ftn2\"\u003e[2]\u003c/a\u003e Not measured, because no need for 3\u0026times;G at TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion And Outlook","content":"\u003cp\u003eIn this study, CNF-based hydrogel inks for 3D printing were prepared from TEMPO-oxidized CNF (TOCNF) with a solid content of 2.64\u0026nbsp;wt% and a carboxylate content 1.94\u0026nbsp;mmol/g.\u0026nbsp; Divalent and trivalent metal cations were introduced to cross-link the deprotonated TOCNFs to form the corresponding hydrogels. The chemical functional groups of the original CNF suspension, the TOCNFs and TOCNF-M\u003csup\u003en+ \u003c/sup\u003ehydrogels were analyzed with FT-IR, which demonstrated a better interaction between carboxylate anions and the divalent cations Ca\u003csup\u003e2+\u003c/sup\u003eand Mg\u003csup\u003e2+\u003c/sup\u003e than with the trivalent cations Fe\u003csup\u003e3+\u003c/sup\u003e and Al\u003csup\u003e3+\u003c/sup\u003e. The storage modulus (G') and loss modulus (G'') of hydrogels incorporating with trivalent cations Fe\u003csup\u003e3+\u003c/sup\u003e and Al\u003csup\u003e3+\u003c/sup\u003e were significantly higher than thoughts with divalent cations Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e. Hydrogel 3D printing performance was evaluated and showed that gel cross-linked with the divalent cations Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e had good printability and that the TOCNF-Ca\u003csup\u003e2+\u003c/sup\u003e prepared with an M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO ratio of 1:1:25 under 1\u0026times;G centrifugation was the best. This gel had a solid content of 1.39\u0026nbsp;wt% and a storage modulus of Gʹ = 2\u0026nbsp;kPa. A comparable performance was achieved with TOCNF-Mg\u003csup\u003e2+\u003c/sup\u003e at the same ratio by 2\u0026times;G centrifugation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB. Saake and B. Mietner thank the Fachagentur Nachwachsende Rohstoffe e.V. for financial support (FNR Project number BMEL-22004518). J. Navarro thank the Fachagentur Nachwachsende Rohstoffe e.V. for financial support (FNR Project number BMEL-2200HV024X).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.N. and J.B.M. designed the experiments, X.J. performed the experiments, all authors contributed to the synthesis of results and the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThomas, B. \u003cem\u003eet al.\u003c/em\u003e Nanocellulose, a Versatile Green Platform: From Biosources to Materials and Their Applications. \u003cem\u003eChem. 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Relationship between Length and Degree of Polymerization of TEMPO- Oxidized Cellulose Nanofibrils ; Supporting Information. \u003cem\u003eBiomacromolecules\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1\u0026ndash;3 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cellulose nanofibrils, TEMPO mediated oxidation, Metal-ion crosslinking, Hydrogel, 3D printing, Nanocellulose ","lastPublishedDoi":"10.21203/rs.3.rs-122100/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-122100/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this work, we present an approach to cross-link cellulose nanofibrils (CNFs) with various metallic cations (Fe\u003csup\u003e3+\u003c/sup\u003e, Al\u003csup\u003e3+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, and Mg\u003csup\u003e2+\u003c/sup\u003e) to produce inks suitable for three-dimensional (3D) printing application. The printability of each hydrogel ink was evaluated, and several parameters such as the optimal ratio of M\u003csup\u003en+\u003c/sup\u003e:TOCNF:H\u003csub\u003e2\u003c/sub\u003eO were discussed. CNF suspensions were produced by mechanical disintegration of cellulose pulp with a microfluidizer and then oxidized with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). Finally, metal cations were introduced to the deprotonated TEMPO-oxidized CNF (TOCNF) suspension to cross-link the nanofibrils and form the corresponding hydrogels. The performances of each gel-ink were evaluated by rheological measurements and 3D printing. Only the gels incorporated with divalent cations Ca\u003csup\u003e2+\u003c/sup\u003e and Mg\u003csup\u003e2+\u003c/sup\u003e were suitable for 3D printing. The 3D printed structures were freeze-dried and characterized with Fourier transform infrared spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM). The better interaction of the TOCNFs with the divalent metallic cations in terms of printability, the viscoelastic properties of the inks, and the variation trends owing to various metal cations and ratios are discussed.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"3D Printing of a Bio-Based Ink Made of Cross-Linked Cellulose Nanofibrils With Various Metal Cations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-09 17:01:07","doi":"10.21203/rs.3.rs-122100/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-02-02T04:24:36+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"80097321-e20f-48f9-8825-e5d69e305ef4","date":"2021-01-09T10:24:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-01-08T09:27:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11aca0bf-907f-40a0-b121-14b88aaf0bf4","date":"2020-12-28T08:17:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-12-07T12:21:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-12-07T12:18:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-12-07T12:16:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-12-07T10:36:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2020-12-04T19:58:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d5007fb5-d594-4624-9b96-e828ee0a74fa","owner":[],"postedDate":"December 9th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1396044,"name":"Biomaterials"},{"id":1396045,"name":"Materials Chemistry"}],"tags":[],"updatedAt":"2021-08-18T19:18:54+00:00","versionOfRecord":{"articleIdentity":"rs-122100","link":"https://doi.org/10.1038/s41598-021-85865-4","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2021-03-19 19:04:08","publishedOnDateReadable":"March 19th, 2021"},"versionCreatedAt":"2020-12-09 17:01:07","video":"","vorDoi":"10.1038/s41598-021-85865-4","vorDoiUrl":"https://doi.org/10.1038/s41598-021-85865-4","workflowStages":[]},"version":"v1","identity":"rs-122100","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-122100","identity":"rs-122100","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}