Cellulose Nanofibril Hydrogel Tubes as Sacrificial Templates for

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Cellulose Nanofibril Hydrogel Tubes as Sacrificial Templates for Freestanding Tubular Cell Constructs Jose Guillermo Torres-Rendon, Marius Koepf, David Gehlen, Andreas Blaeser, Horst Fischer, Laura De Laporte, and Andreas Walther Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01593 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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Cellulose Nanofibril Hydrogel Tubes as Sacrificial Templates for Freestanding Tubular Cell Constructs Jose Guillermo Torres-Rendon, Marius Köpf†, David Gehlen, Andreas Blaeser†, Horst Fischer†, Laura De Laporte, Andreas Walther* DWI – Leibniz-Institute for Interactive Materials, Forckenbeckstr. 50, 52074 Aachen, Germany. † Dental Materials and Biomaterials Research, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany. KEY WORDS: additive manufacturing, nanocellulose, tissue engineering, biomaterials, enzymatic degradation.

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ABSTRACT The merging of defined nanoscale building blocks with advanced additive manufacturing techniques is of eminent importance for the preparation of multiscale and highly functional materials with de-novo designed architectures. Here, we demonstrate that hydrogels of cellulose nanofibrils (CNF) can be processed into complex shapes, and used as a sacrificial template to prepare freestanding cell constructs. We showcase our approach for the fabrication of hollow fibers using a controlled extrusion through a circular die into a coagulation bath. The dimensions of the hollow fibers are tunable, and the final tubes combine the nanofibrillar porosity of the CNF hydrogel with a sub-millimeter wall thickness and centimeter-scale length provided by the additive manufacturing technique. We demonstrate that covalent and supramolecular crosslinking of the CNFs can be used to tailor the mechanical properties of the hydrogel tubes within one order of magnitude and in an attractive range for the mechanosensation of cells. The resulting tubes are highly biocompatible and allow for the growth of mouse fibroblasts into confluent cell layers in their inner lumen. A detailed screening of several cellulases enables to degrade the scaffolding, temporary CNF hydrogel tube in a quick and highly cell-friendly way, and allows the isolation of coherent cell tubes. We foresee that the growing capabilities of hydrogel printing techniques in combination with the attractive features of CNFs – sustainable, globally abundant, biocompatible and enzymatically degradable – will allow to make plant-based biomaterials with hierarchical structures and on-demand degradation useful for instance to engineer complex tissue structures to replace animal models, and for implants.


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INTRODUCTION Biological tissues are slowly grown into hierarchical structures by precise orchestration of vastly different length scales, and incorporating periodic and aperiodic structures. While self-assembly and nanoscale building blocks can address rapid structuration on the nano- and mesoscale, the controlled structuring of large length scales up to the centimeter scale is predominantly performed by top down processing approaches. The bridging of length scales to reach materials structured from the nano- to the macroscale is of eminent technological importance for a range of fields, such as batteries, meta-materials, photonics, sensors, and moreover as 3D scaffolds for fundamental biological studies, tissue engineering and regenerative medicine.1-4 Advanced additive manufacturing techniques hold great promise to bridge structural length scales from nano-to-macro by merging top-down structuring processes with well-organized nanoscale building blocks and self-assembly. In particular for biomedical applications, it becomes of great importance to have a large flexibility in the processes to reach personalized medical products that can be prepared with great ease. Hydrogels are favorable candidates for additive manufacturing in the biomaterials world as their shear thinning behavior or rapid solidification in a coagulation bath can be used to prepare free-standing, highly biocompatible structures. However, it still remains a challenging task to develop simple and versatile pathways to design complex architectures for hydrogel-based biomaterials.5 If designed in a biocompatible manner, they can be loaded with cells prior to extrusion or cultured with cells after preparation. Additional complexity can be imparted into such materials by topographical or chemical patterning to bestow instructive cues to which cells respond,6-12 and by adapting the mechanical properties of the underlying building blocks to control cell behavior.13-17


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In this respect, it becomes obvious that the selection of hydrogels is crucial to be able to address challenges in biomaterials fabrication. Classical hydrogel building blocks used in additive manufacturing for tissue engineering are primarily based on either natural polymers (alginate, gelatin, elastin, fibrinogen, collagen, chitosan, hyaluronic acid and silk fibroin) or synthetic polymers (poly(ethylene glycol), polyacrylamide).18-23 However, such materials often offer only limited selection of mechanical properties, may be challenging to engineer towards cellular response or biodegradability, or may not contain topographical nano-to-mesoscale features. Therefore, hydrogels based on self-assembling/fibrillizing (bio)molecules or nanofibrils are attractive due to their ease of chemical modification, advanced and tunable mechanical properties, as well as the possible mesoscale alignment of colloidal scale nanofibrils to guide cell alignment in the future.24-26 Here, we will focus our attention on hydrogels formed by high aspect ratio cellulose nanofibrils (CNFs). These CNFs are typically isolated from plants, therefore, globally abundant, and a renewable feedstock, which is certain to be a mainstay of the forthcoming green materials revolution.27 This makes CNFs much more economic than for example, self-assembling peptides. Most importantly, the highly crystalline character of the nanofibrils is preserved during the extraction and yields nanofibrils based on one of the stiffest natural crystals (Ecellulose-I = 138 GPa).28 Hence, they complement classical polymer gels as they provide a stiff microenvironment for the mechanosensation of cells. These nanofibrils were previously used to prepare transparent nanopapers,29,

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nanocomposites31 and fibers32,

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with outstanding mechanical and functional

properties.34 CNF hydrogels have shown to be highly biocompatible and support the growth of a range of cell types.35-38 In general, polysaccharide-based materials are interesting for biomaterials due to their low immunogenicity when implanted in vivo.39 We recently demonstrated how to make gyroidal scaffolds by a reverse micromolding technique using sacrificial synthetic resins, ACS Paragon Plus Environment

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and demonstrated their use for biomimetic bone tissue engineering using human mesenchymal stem cells.40 A structural analogue, bacterial cellulose (BC), has proven its utility in biomedicine41 and shown promising results for growth of artificial blood vessels/vascular grafts for bone42 and cartilage43 regeneration, but is very difficult to process as complex 3D shapes. Here we demonstrate a direct and facile approach using an additive manufacturing technique to prepare hollow tubes based on CNF hydrogels with flexible adjustment of the structural dimensions in a potentially continuous preparation (Scheme 1). We show that these tubes support the growth of fibroblast cells into continuous cylindrical cell layers in their inner lumen, and describe in detail how the CNF tubes can be sacrificed by non-toxic enzymatic degradation to furnish free-standing 3D cell constructs (tubes). We believe that the developed procedures will be applicable to flexibly generate other desirable biomaterials architectures with de-novo geometries, and that the enzymatic degradation may become a viable alternative to prepare free standing tissue cultures needed to replace animal models or for designed implantable tissue constructs.

Scheme 1. Schematic representation of the additive manufacturing of CNF tubes, their cultivation with cells in the inner lumen, and the release of cell tubes based on cell-friendly enzymatic degradation.


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EXPERIMENTAL SECTION Materials. Ethanol absolute (≥ 99.8 %), glutaraldehyde solution (GA) grade I, MTT stain (≥ 97.5 %), hydrochloric acid (HCl) standard solution 1 M, sodium phosphate dibasic (Na2HPO4, ≥ 99 %), citric acid (≥ 99.5 %), phosphate buffered solution (PBS), and calcium chloride anhydrous (CaCl2, ≥ 93 %) were purchased from Sigma Aldrich (Hamburg, Germany). All cellulases from trichoderma reesei, trichoderma species, trichoderma longibrachiatum, trichoderma viride and aspergillus niger (shown in Table 2) were also purchased from Sigma Aldrich (Hamburg, Germany). MTS-cell titer proliferation assay was purchased from Promega GmbH (Mannheim, Germany). Alexa Fluor 488 Phalloidin was purchased from New England Biolabs GmbH (Frankfurt, Germany). Hoechst 33342 and OCT compound were purchased from Thermo Fisher Scientific GmbH (Schwerte, Germany). Preparation of CNF suspensions. A 1.0 wt% suspension of CNF was prepared according to Isogai et al.27 by TEMPO-mediated oxidation under alkaline conditions (pH = 10.5) for 30 min of a softwood kraft pulp and subsequent homogenization at pH 8.5 with a pressure microfluidizer MRT model CR5, applying 2 passes at 1400 bar and 2 passes at 1000 bar. The resulting apparent viscosity degree of polymerization (DPv) is 600 and the content of carboxyl groups is 0.85 mmol/g. CNF tubes preparation. We used a custom built extruder comprised of a copper housing with fluid channel made of stainless steel and a changeable polyether ether ketone (PEEK) extrusion nozzle (Figure S2 and schematically represented in Figure 1a). The extruder is loaded with two disposable 5 mL syringes (Terumo Syringe 05SE1). A needle is fixed at the tip of the ethanol syringe (1 in Figure S2) through the central opening of the nozzle. This way, the needle is concentrically arranged with the steel channel, which is connected to the CNF containing syringe ACS Paragon Plus Environment

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on the other end (syringe 2 in Figure S2), enabling ethanol to be squeezed out into the core. Two independently controllable linear drives based on a stepping motor (ZweiPhasen Schrittmotor, Crouzet) and an M4 threaded rods were used to move the plungers. Before extrusion, the CNF hydrogel containing syringes were centrifuged at 2500 rpm for 15 min to remove entrapped air. CNF and ethanol were co-extruded into a pure ethanol bath to form the hollow hydrogel tubes. Two kinds of CNF tubes with different inner and outer diameters were extruded using nozzles with (i) 2 mm inner diameter with a G21 needle (0.8 mm outer diameter in the center of the nozzle) and (ii) 4 mm inner diameter (with an adapter of 2.4 mm diameter attached to a G21 needle). Cross sectional dimensions of the nozzles are shown in Figure 1b. The extrusion speed of the hydrogel tubes was ca. 0.5 cm/s. The extruded CNF tubes were deposited as a coil on the bottom of the ethanol bath. After extrusion into ethanol, the CNF tubes were dried at room conditions and then rehydrated when needed. GA crosslinking of CNF tubes. Rehydrated CNF tubes (1.5 cm length) were put in vials containing 20 mL of a 1 vol% GA aqueous solution and kept overnight at 4 °C. The next day, crosslinking was enforced by adding 1 M hydrochloric acid solution to reach pH = 3, and kept for another 24 h. Afterwards, all crosslinked samples (CNF-GA) were washed and kept in Milli-Q water. CaCl2 crosslinking of CNF tubes. Segments of 1.5 cm of CNF tubes were immersed in 30 mL of an aqueous CaCl2 solution (25 g/L) and kept for 48 h. Then, the crosslinked tubes (CNFCaCl2) were washed and kept in Milli-Q water. GA-CaCl2 crosslinking of CNF tubes. We combined the two crosslinking methods described above by first performing GA crosslinking, followed by CaCl2 complexation. CNF-GA samples were washed with Milli-Q water before continuing with the CaCl2 crosslinking. ACS Paragon Plus Environment

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Fourier Transform Infrared Spectroscopy (FTIR). All FTIR spectra were obtained using a spectrometer Thermo Nicolet Nexus 470 equipped with a smart split ATR single reflection Si crystal using a frequency range from 1800 cm-1 to 1500 cm−1 and a resolution of 4 cm−1. Field-Emission Scanning Electron Microscopy (SEM). SEM was performed using a Hitachi S4800. All samples were sputter-coated with a thin layer of Au/Pd. Supercritical drying to prepare the SEM samples was performed for selected samples using an E3000 Critical Point Dryer (Quorum Technologies) and ethanol as initial solvent. In the case of the CNF tubes containing cells, the cells were previously fixed using a 4 wt% paraformaldehyde solution in PBS for 15 min. Mechanical tests in wet state. Tensile tests were performed on a DEBEN minitester with a 20 N load cell at a strain rate of 1.5 mm/min and a gauge length of 1 cm. All samples were previously submerged in Milli-Q water for at least 2 h. At least 4 samples were used to build the average. The calculated elastic moduli are the tangent moduli from the elastic region starting from zero to the first yield point of the curves. Screening of enzymatic degradation on non-crosslinked (untreated) CNF films and crosslinked CNF-GA films (5 cellulases). Pieces of hydrated, non-crosslinked and CNF-GAcrosslinked films, prepared by film casting (ca. 1.5 cm x 1.5 cm, 20 µm thickness), were placed in vials containing 10 mL of enzyme solutions at different concentrations (5, 1, 0.5 g/L) in a Na2HPO4-citric acid buffer adjusted to pH 5.5. Degradation was carried out by incubation at 37 °C and monitored by macroscopic observation every 24 h. Table 2 shows the cellulases used in this work.


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Enzymatic degradation test on CNF-GA-CaCl2 films in cell media (cells not present). CNFGA-CaCl2 crosslinked CNF films with dimensions of ca. 0.5 cm x 0.5 cm (20 µm thickness) were subjected to enzymatic hydrolysis using only cellulase 1 (from Trichoderma reesei) in a 96-well TCPS plate at several concentrations (0-45 g/L) in RPMI cell media (pH 7.4) (n=3). All enzyme solutions were sterile filtered using a syringe filter with a pore size of 0.45 µm. The degradation study was performed in an incubator set 37 °C and 5% CO2. Degradation was followed up by microscopic observation every 24 h. Enzymatic degradation test on CNF-GA-CaCl2 films in cell media (cells present). CNF-GLCaCl2 crosslinked CNF films of approximately 0.5 cm x 0.5 cm (20 µm thickness) were first sterilized by immersing the films in 70 vol% ethanol, and washing them twice with 1X PBS. Before adding the enzyme (cellulase 1), L929 mouse fibroblasts were seeded on the surface of the films (16,000 cells per well) and cultured in a 12 well plate in an incubator (37 °C, 5 % CO2) for 5 days until confluency was reached. The degradation tests were carried out in a 12-well TCPS plate containing RPMI cell media (2 mL, pH 7.4) inside of an incubator at 37 °C and 5 % CO2. The enzyme concentrations were 23, 11 and 0 g/L (two films per concentration) and all enzyme solution were sterile filtered prior addition to the films. Degradation was followed by microscopic observation every 24 h. Cell cytotoxicity tests. In a 96-well TCPS plate, we seeded 10,000 cells per well and then added sterile filtered enzyme solutions with concentrations ranging from 45 to 0.02 g/L. No enzyme was added as reference. We performed a MTS-cell assay to measure cell proliferation after 24 hours. A Zeiss Axio Observer Z1 microscope equipped with an AxioCam MRm camera was used to observe the cells before adding the enzyme solution and after 24 hours (n=2).


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Cell seeding on CNF-GA-CaCl2 tubes. First, 5 mm of ethanol-sterilized segments of CNF-GLCaCl2 tubes were placed in 500 µL Eppendorf tubes (1 tube per tube). Then 10 µL of cell solution in media (L929 mouse fibroblasts) were added having different amounts of cells, from 10,000 to 500,000 cells per CNF tube. Afterwards, they were put into 50 mL centrifuge tubes and placed on a roller mixer for 1 h (12 rounds per minute). Finally, we transferred the tubes to a 12well TCPS plate, with 3 mL of cell media and incubated them for different time periods. At the third and seventh day after cell seeding, cell cultured CNF-GA-CaCl2 tubes were stained using a MTT stain and visualized using optical microscopy (Zeiss Axio Observer Z1 with a AxioCam MRm camera). Nucleus and cytoskeleton fluorescent staining of cross sections of cell cultured CNF tubes and cell layers. Prior to staining, cell cultured CNF-GA-CaCl2 tubes and cell layers were fixed with 4 wt% paraformaldehyde for 15 min, embedded in the OCT compound, and slowly frozen with liquid nitrogen. Cross sectional slices (thickness of 6 µm) were obtained using a cryostat microtome (CM-2850). The sections were blocked with 2 % bovine serum albumin for 30 min at room temperature and permeated with 0.1 % Triton X-100 in PBS buffer for 5 min. The cell nuclei and the tubes were stained using Hoechst 33342 stain (2.5 µg/µL) for 5 min. The actin filaments of the cells were stained with Alexa Fluor 488 Phalloidin (0.5 µg/µL) for 30 min. The samples were washed several times with PBS buffer between the staining procedures and finally stored at 4 °C. Fluorescence images were taken using a camera AxioCam MRm attached to a Zeiss Axio Observer Z1.


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RESULTS AND DISCUSSION The cellulose nanofibrils (CNFs) were prepared by TEMPO/NaBr/NaOCl oxidation of wood pulp and subsequent disintegration into nanofibrils with average lengths of up to 3 µm at diameters around 2 nm (atomic force microscopy in Figure S1). The TEMPO oxidation oxidizes parts of the C6 hydroxyl groups at the surface of the CNFs to carboxyl groups to impart electrostatic stabilization of the nanofibrils in water, while it leaves the crystalline interior majorly intact.29, 40, 44

Such CNFs form free-standing hydrogels above concentrations of ca. 0.4 wt.-%, which are

shear thinning and can be readily extruded through a syringe. The preparation of the sacrificial CNF tubes starts with the controlled co-extrusion of an inner ethanol stream with a sheath of CNF hydrogel (1 wt.-%) into a coagulation bath of ethanol using a custom-built bioextruder with a concentric die (Figure 1a, d; Figure S2). This yields CNF tubes with different inner (0.8 or 2.4 mm) and outer (2 or 3.8 mm) diameters. The outer diameter of the larger tubes decreases by ca. 10 % after normal drying and rehydration, which is due to the formation of strong interfibrillar hydrogen bonds, and potentially some hemiacetal linkages between hydroxyl groups of the CNF and some incompletely oxidized aldehyde surface functions originating from the original TEMPO oxidation of the pulp in the dehydrated state.45,

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amount of aldehyde groups can be estimated to be around 0.05-0.1 mmol/g based on earlier work on TEMPO oxidation of similar pulp under the same conditions.44 These new bonds act against the recovery of the original dimensions during re-hydration, and provide a simple way to generate a mechanically more robust structure compared to the original CNF hydrogel. The inner diameters remain unchanged, and hence we find an overall very good confidence of the additive manufacturing step, enabling to overlay a sub-millimeter wall thickness and centimeter-scale length with the nanoscale network porosity of the CNFs. For ease of handling of the objects, and


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first proof of principle, we performed all following experiments with tubes of the larger diameter (Figure 1b). Scanning electron microscopy (SEM) images show the almost perfect circumference of supercritically dried CNF tubes with a nanofibrillar structure in the walls at higher magnification (Figure 1e,f and dried sample in d). The average pore radius of the nanofibrillar gel can be estimated to be in a range of 20 nm for 1 wt.-% CNF hydrogel using a theoretical model for infinitely long fibers.40, 47 Such a porosity is sufficient for nutrient and protein diffusion, and potentially allows intercellular communication through the walls in future biomaterials.

Figure 1. Preparation and structural characterization of the CNF hydrogel tubes. (a,b) Schematic representation and nozzle dimension of the bioextruder. (c) Photographs of the extruded hydrogel tubes. The inset shows a long segment. (d) Photograph and (e,f) SEM micrographs of the surface and crosssection of a supercritically dried CNF tube. The small dimensions of the sample in (f) are due to shrinkage during supercritical drying.

Aside providing biochemical cues through appropriate surface modifications, the mechanical properties are decisive in instructing cellular response in biomaterials.13-15 To this end, CNFs are interesting because they are based on one of the stiffest natural materials. The bulk mechanical properties of the hollow CNF hydrogel tubes can be manipulated in a facile way using different types of crosslinking procedures. It is important to point out that all samples are dried after ACS Paragon Plus Environment

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extrusion into ethanol and then re-hydrated. This drying step is important to augment the mechanical properties of the extruded tubes as pointed out above. In comparison, tubes that were never dried can hardly be tested in tension as their physical integrity is too low. The red curve in Figure 2 displays the mechanical properties of untreated (no additional crosslinking), rehydrated CNF tubes. They exhibit a stiffness of 55 kPa, and a tensile strength of ca. 5 kPa at a total elongation of 14 %. To further augment the mechanical properties, we investigated both covalent and supramolecular crosslinking approaches. Mechanical parameter for all samples are listed in Table 1. Toward covalent crosslinking, we used glutaraldehyde (GA), which can link hydroxyl groups present on the surface of the CNF by forming acetal bridges under acidic conditions.48 Figure S3 shows the FTIR spectra with a decrease of hydroxyl groups in CNF-GA confirming GA crosslinking. After covalent crosslinking, there is a slight contraction of 5% in the outer diameter of the samples, indicating lower susceptibility to swelling by incorporation of hydrophobic GA and blocking of some hydroxyl groups, thereby reducing the plasticizing effect of water.29 The GA crosslinking (CNF-GA tubes) increases all mechanical parameters, leading to a doubling of the stiffness and substantial improvements in tensile strength and elongation at break (blue curve in Figure 2). Table 1. Mechanical properties of CNF tubes Young’s modulus, Tensile strength, Sample E (kPa) σUTS (kPa) Untreated (CNF) 55 ± 4 5.3 ± 0.5 CNF-GA 120 ± 26 16.1 ± 3 CNF-CaCl2 504 ± 38 54 ± 5 CNF-GA-CaCl2 223 ± 3 36.5 ± 7

Strain at failure εmax (%) 14 ± 1 24 ± 4 33 ± 6 30 ± 5

In the case of supramolecular crosslinking, we employed a treatment with CaCl2, in which the Ca2+ ions serve as strong, divalent ionic crosslinkers between the carboxylates present on the


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surface of the CNFs, a process similar to the gelation of alginate-based materials.49, 50 In contrast to covalent bonds, such supramolecular bonds can in principal be dynamic, and allow for combinations of high stiffness and toughness by the formation of reversible, sacrificial bonds.51-53 This Ca2+ induced crosslinking can be done in addition to the GA crosslinking, or separately, leading to samples termed CNF-GA-CaCl2 and CNF-CaCl2, respectively. Corresponding FTIR spectra of the non-crosslinked (CNF) and CNF-CaCl2 samples in the region of 1450 to 1750 cm-1 (Figure S3c) show that the characteristic C=O vibration band related to carboxyl groups (salt type) is located at nearly the same position at ~1606 cm-1. This result is consistent with a recent study concerning ionically crosslinked CNF films using metal ions.54 However, the profound change in mechanical properties confirms the successful crosslinking.

Figure 2. Tensile mechanical properties of crosslinked and non-crosslinked hydrogel tubes.

In both cases, an increase in mechanical properties with higher stiffness, strength and toughness (elongation at break) can be observed. The enhanced toughness can be attributed to re-zipping of such sacrificial ionic crosslinks under dynamic hydrated conditions.52,


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exclusively treated with CaCl2 display the best mechanical properties with substantial improvements in the mechanical characteristics (E = 504 kPa, σUTS = 54 kPa, εmax = 33 %; CNFCaCl2; black curve in Figure 2). To explain their better mechanical behavior, we hypothesize that the treatment with HCl during the GA crosslinking leads to some unwanted flocculation and mesoscale aggregation of electrostatically stabilized CNFs inside the hydrogel walls. From earlier studies, it is known that such agglomeration and improper colloid stabilization at low pH have a negative effect on the mechanical properties of analogous TEMPO-oxidized CNF nanopapers, leading to substantially lower stiffness therein.29 A second explanation for the lower mechanical properties in CNF-GA-CaCl2 may be the lower efficiency of Ca2+ crosslinking in presence of GA. The multiple yield points of the stress-strain curves (Figure 2) are caused by relaxation of different structural entities (e.g. fibrillar entanglements) as described by us previously.29 In summary, the appropriate crosslinking of the pristine CNF tubes leads to an increase of stiffness, strength, and toughness up to one order of magnitude. Consequently, the mechanical properties can be tuned in an attractive range where mechanosensation of cells occurs.13-15 Compared to tubes based on bacterial cellulose (BC), our stiffest materials (CNF-CaCl2 samples) are at the lower range of reported values (E = 60-8,250 kPa,56-61 σUTS= 64-5,000 kPa56-58, 61-64). While the mechanical properties of BC-based structures have a large variability due to different culturing conditions60-62, the higher degree of crystallinity in BC (80-90 %)65 is one of the primary factors allowing to reach higher mechanical properties. However, the growth of such BC-based tubes requires specific reactor design and is a lengthy and tedious process, while the tubes described here can be rapidly fabricated with more ease, and potentially even in a continuous manner.


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Compared to other biobased hydrogel tubular constructs, the CNF-CaCl2 tubes exhibit a higher Young’s modulus than fibrin (0.7-289 kPa),66, 67 gelatin (4.4 kPa),66 hyaluronic acid (58.5 kPa)66 or collagen (140-200 kPa)68, 69 ones, but a lower modulus than alginate tubular constructs (60010,800 kPa),66 which also crosslink supramolecularly, yet in a much denser fashion. Note that the large variations within those material classes are due to different concentrations, preparation methods or crosslinking conditions. More interestingly, the range of Young’s moduli covered by our differently modified CNF tubes are comparable to those of some native tubular tissues, such as the porcine carotid arteries (200 kPa),70 rat abdominal aorta (170 kPa)71 and spinal cord (depending on the species, type of mechanical characterization and time after death: 250-600 kPa).72-76 Hence, overall, the elastic properties achieved so far are in range with other biomaterials and relevant, selected native tissues. We foresee further improvements by increasing the concentration of CNFs, blending them with appropriate polymers, reinforcing the hydrogels using inorganic nanoparticles (e.g. hydroxyapatite) and by aligning the nanofibrils during shear extrusion.32, 77 Next, we will demonstrate the use of the hollow fibers as sacrificial biomaterial scaffolds to create freestanding cell constructs with de-novo designed geometries. We and others recently reported excellent biocompatibility of native and TEMPO-oxidized CNFs.38,

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instance, simple films made from TEMPO-CNFs, bearing carboxylate groups at their surface (as used herein), exhibit excellent adhesion properties for a wide variety of cells, e.g. human mesenchymal stem cells and mouse fibroblasts, and lead to similar proliferation rates compared to classical tissue culture polystyrene (TCPS).40 Here, we focus on using TEMPO-CNF constructs as geometrically defined, sacrificial template to enable the isolation of free-standing, confluent cell structures via a mild enzymatic dissolution of the CNFs, which opens the possibility to fabricate 3D tissues, such as vascular grafts, in a new controlled manner. ACS Paragon Plus Environment

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To determine cell-friendly conditions for enzymatic degradation of CNF materials, we investigated five different, commercially available cellulases (or mixtures as detailed below; Table 2). Experimentally, we exposed CNF model films to different cellulases at 37 °C and pH 5.5, which is near the optimum pH for cellulases78, yet at the lower end of what many cell lines can potentially tolerate. For ease of operation, we performed the experiments at different mass concentrations rather than normalized to the catalytic unit. Table 2. Commercial cellulases used in this work Label Source 1 Trichoderma reeseib 2 Trichoderma speciesc 3 Trichoderma longibrachiatum 4 Trichoderma viride 5 Aspergillus niger a

Activity (units/mg)a ~ 1 unit/mg ~ 5 units/mg ~ 1 unit/mg 0.3~1 units/mg ~ 0.3 units/mg

Activity of the cellulases to liberate 1 µmol of glucose at pH 5 and 37 °C in 2 hours of incubation (product specification). Lyophilized version of Celluclast® 1.5 L. c not further specified.

b

Once exposed to the cellulases, both non-crosslinked (untreated) and crosslinked samples exhibit a similar physical degradation behavior. At early stages of degradation, CNF films start losing rigidity and degradation continues with a loss of physical integrity. The films break into pieces, and depending on the type of cellulase, the degradation continues until no more remaining pieces can be identified. The degradation rate and the total extent of degradation depend on the type of cellulase and its concentration. Figure S4 summarizes the observed degradation at three cellulase concentrations. In the case of non-crosslinked films, cellulase 1 (from Trichoderma reseei, ~1 unit/mg) and cellulase 2 (from Trichoderma species, ~5 units/mg) are able to completely degrade the films. They require one to eleven days depending on the type of cellulase and its concentration. Samples treated with cellulase 3 (from Trichoderma longibrachiatum, ~ 1 unit/mg) and cellulase 4 (from Trichoderma viride, 0.3~1 units/mg) exhibit degradability during the first 7


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days, however, full degradation is not observed, even after 15 days. Cellulase 5 (from Aspergillus niger, ~ 0.3 units/mg) does not visibly affect the samples under the tested conditions. Based on the mentioned observations, we also applied cellulase 1 and 2 to crosslinked CNF-GACaCl2 films to demonstrate that cellulases can degrade ionically and covalently linked CNF samples. We find an overall comparably fast degradation, thus confirming good flexibility in terms of applying chemical and supramolecular modifications to the CNF materials. Figure 3d,e shows the complete degradation of a CNF-GA-CaCl2 film after 24 h using cellulase 1. Overall, cellulase 1 (cellulase from Trichoderma reesei, also known as Celluclast® 1.5 L) leads to the most rapid degradation, despite the presence of COOH surface groups, as well as ionic and covalent crosslinking points. This cellulase mixture preparation is specifically formulated to break down lignocellulosic materials.79,

80

It predominantly contains endo- and exoglucanases, which act

synergistically during the degradation of native celluloses.81-83 Endoglucanases create free ends by attacking random parts of cellulose chains (mostly in the amorphous parts), allowing the exoglucanases to act in those free ends, liberating cellobiose and oligosaccharides units.84 Our observations are in line with the degradation of native BC sheets without surface carboxylates,85 and we refer to the successful recovery of stem cells from hydrogel cultures of native, nonmodified CNFs, where however the type of cellulase was not specified.37 Based on these results, we continued the enzymatic degradation studies using only the cellulase 1.


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Figure 3. Cytotoxicity of cellulase 1 and enzymatic degradation. (a) MTS proliferation assay for L929 mouse fibroblasts in the presence of different cellulase 1 concentrations. (b,c) Brightfield micrographs of mouse fibroblasts in a 45 g/L cellulase 1 solution at (b) 30 min and (e) 24 h after seeding 10,000 cells onto TCPS. (d,e) Photographs of a CNF-GA-CaCl2 film immersed in a 5 g/L enzyme solution (pH 5.5) at (d) 5 min and (e) 24 h. (f-i) Photographs of a CNF-GA-CaCl2 film inside of a 23 g/L enzyme solution (culture media pH 7.4, 16,000 cells seeded) at (f) 30 min, (g) 24 h, (h-i) 48 h. The cells were cultured for five days prior addition of the enzyme. The inset in (i) shows the remains of the MTT-stained folded cell layer, scale bar is 3 mm.

Next, we assess the cytotoxicity of cellulase 1 by measuring the viability and proliferation of L929 mouse fibroblasts cultivated on TCPS at different cellulase 1 concentrations using an MTS assay. Figure 3a monitors the amount of viable cells by absorption spectroscopy of the liberated dye at 490 nm. The near constant absorption as a function of enzyme concentration confirms that the cell proliferation and viability are not crucially affected by the presence of cellulase 1. No significant changes and adverse effects are observed from the positive control up to very high concentrations of 23 g/L. A morphological observation of the cells in Figure 3b,c also indicates that the cells are unaffected, and well spread on the TCPS plate after 24 h of cultivation, even when using the highest enzyme concentration (45 g/L).


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A surprising phenomenon was observed when merging the cell culturing conditions with enzymatic degradation. To this end, we exposed CNF-GA-CaCl2 films and films cultured with L929 mouse fibroblast cells on top (both in RPMI media at pH 7.4 and at 37 °C) to cellulase 1. We use CNF-GA-CaCl2 samples for the cell culture experiments to show that both GA and CaCl2 crosslinking procedures are not cytotoxic and fully compatible with the overall strategy. Interestingly, complete disintegration of the CNF-GA-CaCl2 films occurs only in the presence of cells. Pure films without cells do not reach complete disintegration in the presence of culture media, even at the highest enzyme concentration of 45 g/L and after 11 days of exposure. This reduced degradation behavior can most likely be explained by the pH of the culture media (pH 7.4), which is slightly outside the optimum pH of the cellulase (pH 5.5).86 However, when cells are present on the film, the media becomes slightly more acidic. For instance, solutions with fibroblast cultured for 5 days and subsequently treated with enzymes for 7 days display a pH value of 6. This acidification is due to secretion of acidic proteins.87 As the cells are in contact with the CNF surface, the local pH near the films may be even lower than the overall media. In this case, full degradation of a 20 µm thick CNF sheet occurs within 48 h at an enzyme concentration of 23 g/L (Figure 3f-i and observations in Figure S4). Lower concentrations lead to longer degradation times, which demonstrates that the degradation rate can be adjusted in a desirable window. The unexpectedly occurring differences confirm that the cells themselves may play an important role for the efficacy of cellulase 1 by lowering the pH of the media. The combined cell culturing and CNF degradation studies enable a first mechanistic understanding of the sacrificial nature of the substrate. Confluent cell layers formed on top of the CNF films detach at early stages of the degradation (~ 6 h), and the resulting 2D cell layer folds into an arbitrary object (Figure 3g-i). Obviously, the cells have a higher affinity to each other than to the media, and some mechanical stress due to cell spreading on the surface is released ACS Paragon Plus Environment

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leading to the observed contraction. The release of the confluent cell layer confirms that the enzymes are able to quickly erode CNF directly at the cell/CNF film interface via surface degradation. This cell detachment from the surface using readily available and cheap enzymes is conceptually different and much milder compared to classical trypsination, which cleaves off the integrin anchors and destroys cell-cell interaction. Furthermore, it also offers an alternative pathway towards the detachment of confluent cell sheets compared to thermo-responsive Poly(Nisopropyl acrylamide) surfaces, for which a temperature decrease from 37 °C to room temperature is used to release intact cell sheets from a surface.88-90 More importantly, however, the sacrificial nature of the CNF, meaning its complete dissolution, also allows much more complex geometries, such as bicontinuous cell structures, to be generated, which is not possible for the aforementioned methods. To generate tubular cell constructs based on the printed tubes, we first developed a procedure to grow a confluent and homogeneously distributed cell layer of L929 mouse fibroblasts in the inner lumen of the CNF-GA-CaCl2 tubes. To examine the necessary amount of cells needed to form a confluent and homogeneous cell layer, we seeded different amounts of cells (10,000 to 500,000 cells) into tubular segments (5 mm length) of CNF-GA-CaCl2 samples. Initial cell seeding of 150,000 to 250,000 cells and subsequent cultivation between 3 to 14 days leads to confluent and homogeneous cell layers (Figure 4a-c). Lower numbers of added cells do not produce a homogeneous layer in such an appropriate time frame (data not shown), while higher amounts of cells result in inhomogeneous distributions (e.g. 500,000 cells, Figure S5a-c). SEM micrographs of cultured CNF-GA-CaCl2 tubes (Figure 4d-f) confirm the formation of a homogeneous cell layer with a thickness of ca. 9 µm after 9 days at an initial seeding of 150,000 cells. We believe this developed approach can easily be extended to other types of cells.


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To sacrifice the CNF template structure, we used the enzymatic degradation conditions developed above and exposed the tubes containing a confluent fibroblast layer in the lumen after extended cultivation to 45 g/L of cellulase 1. Figure 4g, h depicts fluorescence microscopy images of cross sections of CNF-GA-CaCl2 tubes (microtomed into 6 µm thin slices after embedding into OCT (optimal cutting temperature) resin), before and after degradation. Before degradation of the CNFs, a thick wall (stained in blue) of CNF can be observed that carries a dense cell layer at the inner lumen (left). Note that some cells also attached to the outside of the tube. The cytoskeleton is stained in green using a phalloidin stain, while the cell nuclei are stained in blue using a Hoechst stain. Interestingly, the Hoechst stain also has a certain affinity to the CNF due to its cationic aromatic structure, and also enables a distinct visualization of the walls. After degradation, the blue CNF wall completely disappears and only the connected cell layer remains in a soft tubular shape (Figure 4h). The microtomed cross section are deformed because the tubes do not have enough bending stiffness to maintain a perfect circular shape during embedding in the OCT resin and subsequent microtoming. Note that contraction of the confluent cell tubes is less pronounced when the fibroblast layers are allowed to grow into confluent layers over extended periods of time.


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Figure 4. Freestanding tubular cell constructs by sacrificing the printed, temporary CNF support shown for CNF-GA-CaCl2 tubes cultured with L929 mouse fibroblasts (150,000 cells seeded). (a-c) Bright-field micrographs of CNF-GA-CaCl2 samples cut in half and MTT-stained after 7 days of cell seeding. Scale bar in the inset of (c) is 100 µm. (d-f) SEM images showing a (d-e) confluent cell layer and (f) morphology of cultured fibroblasts inside a CNF-GA-CaCl2 tube (7 days after cell seeding). (g, h) Fluorescence microscopy of microtomed cross sections of a cell cultured CNF-GA-CaCl2 tube (g) before and (h) after enzymatic degradation of CNF. Fluorescent staining was performed 9 days after cell seeding in cross sectional slices (6 µm thickness). Alexa Fluor 488 Phalloidin was used to stain the actin filaments and Hoechst 33342 to stain the cell nuclei. The CNF tube is also stained by Hoechst. To our knowledge, this is the first report that uses a printed CNF scaffold in combination with enzymatic degradation to detach confluent cell layers and create macroscale 3D cell constructs. Overall, these results demonstrate that even complex shapes of confluent cells layers can be templated, and we believe that cell constructs containing multiple cell layers with higher mechanical rigidity can be further created by mediating the interplay between the cell culture ACS Paragon Plus Environment

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time and the degradation rate of the CNF substrates allowing for cell migration and proliferation inside the slowly eroding CNF material.

CONCLUSIONS We established a generic and versatile principle to prepare 3D cell constructs with de-novo designed geometries by combining additive manufacturing of CNF hydrogels, subsequent cell culturing, and mild enzymatic degradation of the sacrificial CNF templates. We showcased the approach for the formation of fibroblast tubes based on CNF tubes. The Young’s moduli of the CNF tubes can be tuned within an attractive range for biomaterials, and the mechanical robustness exceeds most other classical, natural biopolymers such as gelatin, hyaluronic acid, fibrin, and collagen. Furthermore, our in-depth enzymatic degradation screening demonstrates the importance of selecting appropriate degradation conditions, for which the presence of cells is an influential component in cellulase-mediated CNF degradation. Optimized conditions enable rapid and highly cytocompatible degradation of the CNF materials. While here, we focused on rapid degradation, we foresee that adjusted degradation times may be beneficial for in-growth of cells into the CNF template to reach multilayered cell structures. Additionally, the increasing capabilities of additive manufacturing techniques provide a suitable framework to approach more complex hydrogel geometries, and the cultivation of different cell types on different surfaces or compartments will allow advancing toward hierarchical and communicating cell structures in the future. Overall, the easy, global, and cheap availability of CNFs, together with their biocompatibility, ease of chemical functionalization and versatile structuring via additive manufacturing provides a new, attractive, and versatile toolbox for preparing complex 3D tissue structures.


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ASSOCIATED CONTENT AFM micrograph showing the CNFs (Figure S1), a scheme showing the components of the bioextruder (Figure S2), FTIR data (Figure S3), degradation observations (Figure S4) and multiscale characterization of cell cultured CNF-GA-CaCl2 tubes (500,000 cells seeded and negative control, Figure S5) are supplied as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS We thank R. M. Ortíz de la Morena for early extrusion experiments, B. Wang for AFM measurements and S. Rütten for supercritical drying. We acknowledge the BMBF for funding the AQUAMAT research group. This work was performed in part at the Center for Chemical Polymer Technology CPT, which is supported by the EU and the federal state of North RhineWestphalia (grant no. EFRE 30 00 883 02). A.W. gratefully acknowledges continuous support from Martin Möller.

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