Printability of pulp derived crystal, fibril and blend nanocellulose-alginate bioinks for extrusion 3D bioprinting

Background: One of the main challenges for extrusion 3D bioprinting is the identification of non-synthetic bioinks with suitable rheological properties and biocompatibility. Our aim was to optimize and compare the printability of crystal, fibril and blend formulations of novel pulp derived nanocellulose bioinks and assess biocompatibility with human nasoseptal chondrocytes. Methods: The printability of crystalline, fibrillated and blend formulations of nanocellulose was determined by assessing resolution (grid-line assay), post-printing shape fidelity and rheology (elasticity, viscosity and shear thinning characteristics) and compared these to pure alginate bioinks. The optimized nanocellulose-alginate bioink was bioprinted with human nasoseptal chondrocytes to determine cytotoxicity, metabolic activity and bioprinted construct topography. Results: All nanocellulose-alginate bioink combinations demonstrated a high degree of shear thinning with reversible stress softening behavior which contributed to post-printing shape fidelity. The unique blend of crystal and fibril nanocellulose bioink exhibited nano- as well as micro-roughness for cellular survival and differentiation, as well as maintaining the most stable construct volume in culture. Human nasoseptal chondrocytes demonstrated high metabolic activity post printing and adopted a rounded chondrogenic phenotype after prolonged culture. Conclusions: This study highlights the favorable rheological, swelling and biocompatibility properties of nanocellulose-alginate bioinks for extrusion-based bioprinting.


Introduction
The ability to print biological 'inks' rather than traditional 3D printing of metals and plastic has resulted in the birth of the new bioprinting research field [1][2][3][4] which is gaining interest in engineering customized tissues for reconstructive surgery [5][6][7]. Developments in automative bioprinting technology, cell biology and material science has allowed production of an increasing range of 'printable' bioinks, consisting of cells and biocompatible materials, in an attempt to simultaneously replicate native tissue micro and macroarchitectures, overcoming the problems of repeatability and scalability of conventional tissue engineering strategies [6,8].
Of the three-main 3D bioprinting technologies: extrusion, inkjet and laser-assisted, extrusion is the most versatile, fast, scalable and cost-effective [9,10]. This technique relies on extruding bioinks with suitable mechanical properties (viscosity, elasticity, shear thinning) through a nozzle using either mechanical (piston or screw driven) or pneumatic forces. Suitable bioinks must also exhibit cellular viability, adhesion, proliferation and differentiation [8,11]. Although it is easier to tailor the biomechanical properties of synthetic bioinks such as polyacrylamides and polyethylene glycols to suit extrusion techniques, their biocompatibility and tissue regenerative potential are inferior to non-synthetic bioinks such as gelatin, agarose, alginate, hyaluronic acid and collagen, which mimic the natural extracellular matrix environment [12].
Contemporary research into 3D bioprinting of cartilage has identified several potential natural bioinks, including fibrin, alginate, gelatin, Matrigel, and nanocellulose [26,27], which have been used as scaffolds for facilitating cell homing from neighboring healthy tissue or deposition of extracellular matrix following the addition of cellular components such as chondrocytes or mesenchymal stem cells [28]. However, the application of the more commonly used bioinks can be a challenge due to suboptimal printability and pure alginate formulations, in particular, have been identified as providing poor post-printing shape fidelity even when viscosity is increased [8,29].
There have been recent reports in the literature concerning pulp biomass derived CNC and CNF [27,51,52] bioinks which rely on 'top-down' NC extraction using an expensive enzymatic treatment step and concentrated acids with poor chemical recovery, making clinical translation and 'up-scaling' problematic [53][54][55]. A lower cost, scalable solution has presented itself in the form of American Value-Added Pulping (AVAP ® ) technology, using a tunable pretreatment step without enzymes to produce CNF, CNC and a unique blend (NCB) formulation at low cost, high purity and with efficient, low cost chemical recovery [56]. Characterization of these formulations has revealed promising rheology for bioprinting and found that NCB exhibits porous fibrillar networks with interconnecting compact nanorods with favorable pore sizes for cellular ingress and maintenance [57]. This study optimizes pulp biomass derived CNF, CNC and NCB as bioinks for extrusion bioprinting and investigates their printability and biocompatibility using human nasoseptal chondrocytes.

Extrusion 3D bioprinting set up
A custom-built extrusion bioprinter with a variable speed control syringe driver mounted on a motordriven XY-Z system featuring custom RepRap firmware was used to print 3D structures from NC/ alginate hydrogel bioinks. Bioink was loaded into 5 ml syringes (Sterile BD Plastipak slip tip) and extruded through 610 μm precision nozzles (Adhesive Dispensing Ltd). Slic3r (open source software) allowed adjustment of print settings to suit the individual bioinks; including print-head movement speed, extrusion speed, layer height, infill pattern and density. The 3D model was sliced and exported as G-code to command the bioprinter. Initial calibration of the printer and software resulted in a minimum prefabrication 'single layer' of 1.7 mm to allow for continuous, uniform printing. Sterile printing was achieved in a class II laminar flow hood following cleaning of all external bioprinter components using 70% ethanol and UV treatment for 60 min.
CNC, NCB, and CNF were centrifuged at 1500G for 5 min to remove any residual bleaching chemicals and lignin fragments and steam sterilized in an autoclave (100 kPa, 121°C, for 30 min). Alginic acid sodium salt (medium viscosity from brown algae, Sigma-Aldrich, Poole, UK) was UV sterilized for one hour prior to being dissolved in sterile culture medium to make up 0.625%, 1.25%, 2.5%, 5%, 7.5% and 10% alginate concentrations (w/v). One part alginate was combined with four parts nanocellulose (to create CNC-AG, NCB-AG and CNF-AG bioinks) as described by Markstedt et al [27] to allow crosslinking with calcium chloride (CaCl 2 ) following bioprinting. Alginate solutions from 0.625% to 10% were qualitatively assessed against four criteria set to define optimal bioink preparations; ease of dissolution, ease of flow at room temperature, ease of mixing with NC, and structural retention when printed with NC (table 1).

Crosslinking optimization
Different concentrations (0.1, 0.5, and 1 M) of aerosolized CaCl 2 solutions (anhydrous CaCl 2 powder Sigma-Aldrich, Poole, UK, dissolved in distilled water) were tested for crosslinking time (from print completion to cessation of structural changes on application of an indentation force). All solutions were sterilized and filtered by a 0.22 μm cell filter (Merck Millipore, Watford, UK) and stored in an incubator at 37°C before use. Cylindrical constructs measuring 27 mm in diameter and 7 single layers (11.9 mm) in height were printed using alginate concentrations of 2.5% and 5% mixed with nanocellulose in 1:4 ratio respectively as described above. Crosslinking was performed by aerosolized CaCl 2 between each layer.

Transmission electron microscopy (TEM)
Each sample (2 mg) was dispersed in 5 ml of deionized water and sonicated for 30 min. After sonication 50 μl of the sample was immediately taken and further dispersed into 1 ml of deionized water to prevent coalescence. This solution (10 μl) was added to 300 mesh copper grids coated with lacey carbon film. The grid was allowed to air dry prior to staining with a 1.5% uranyl acetate solution. For staining, a drop of the uranyl acetate solution was placed on a parafilm strip and the grid inverted onto the droplet for a few seconds. The samples were then allowed to air dry. Analysis was performed on a Jeol 2100 JEM operating at 200 KV.
2.3. Printability testing 2.3.1. Resolution Print resolution using the three different formulations of NC (CNC-AG, CNF-AG and NCB-AG) was tested by printing 40×40 mm square grids a single layer tall (1.7 mm) with 27% rectilinear infill and crosslinked with 0.5 M CaCl 2 (figure 2), adapted from Markstedt et al [58]. Grid line thickness was measured at the halfway points of the infill diagonal lines using a digital caliper ( figure 2(B)). Shape fidelity of bioprinted and crosslinked structures using the three formulations of NC (CNC-AG, CNF-AG and NCB-AG) was assessed by bioprinting 27 mm diameter cylinders with 7-layer height (11.9 mm) and measuring diameter and height immediate post-printing, at 24 and 72 h following immersion in culture media at 37°C.

Shape fidelity
Complex structure testing utilized 3D geometrical shapes and anatomical STL models of auricular    Cylindrical shapes were created using Microsoft 3D Builder software (Microsoft Corporation, New Mexico, USA) and exported in STL file format for 3D bioprinting. The volume was calculated by measuring the diameter (2r) and height (h) of the 3D printed cylindrical constructs using V=πr2h to assess post printing shape fidelity after 24 and 48 h under culture conditions.

Rheology
The rheological properties of bioink mixtures (alginate 1.25%/2.5%/5% and NC mixtures with alginate 1.25%, 2.5% and 5% to create CNC-AG, NCB-AG and CNF-AG bioinks (with overall alginate concentrations of 0.25%, 0.5% and 1% (w/w) respectively) were measured using an AR-G2 (TA instruments, UK) Controlled Stress Rheometer fitted with a 40 mm diameter parallel plate geometry. Solutions were mixed on a rolling rocker for ∼15 min before sample loading. Prior to each experiment, the zero gap was set on the rheometers and the geometry was calibrated using rotational mapping. Approximately 0.65 ml of sample was carefully loaded using a spatula onto the center of the lower plate of the rheometer and the upper plate was gradually lowered onto the sample until the gap was totally filled (gaps ranged from 350 to 500 μm). Any excess sample around the edge of the geometry was trimmed using a spatula. The normal force measured at the lower plate was set at a maximum of 0.1 N to ensure that any mechanical damage to the sample during gap setting procedure was minimized. The rheometers lower plate was controlled at a temperature of 22°C and a low viscosity silicon oil (Fisher Scientific: Brookfield, Viscosity standard 0.920 specific gravity silicone oil 5 mPa s-Ref: 10543108) was used to surround the outer edges of the sample, in order to reduce sample evaporation. Following a sample equilibration period of 2 min, a frequency sweep (0.1-10 Hz) was performed at a constant stress of 0.5 Pa. Each measurement was within the linear viscoelastic range of the sample as confirmed by stress sweeps (data not shown). The values of storage modulus (G′) and loss modulus (G″) were recorded over the entire frequency range employed. Following the completion of the frequency sweep, the sample was allowed to equilibrate for a period of 10 s before a shear flow ramp was carried out over logarithmically increasing shear rates from 0.1 to 100 s −1 for a period of 2 min. Both frequency sweep and shear flow ramp experiments were repeated at least three times for each sample. Cartilage tissue was minced into 1 mm 3 pieces and digested by 0.4% pronase (Roche, West Sussex, UK) at in culture media for 1 h at 37°C (5% CO 2 ) with gentle agitation, followed by digestion with 0.2% collagenase type I for 18 h [59]. The solution was filtered through a 40 μm cell strainer (VWR, Leicestershire, UK) and then centrifuged at 500G for 5 min to replace the enzyme mixture with culture media. Cells were grown in 5% CO 2 at 37°C and culture medium was changed every 2-3 d. Culture medium used consisted of Dulbecco's Modified Eagle Medium without glucose (Sigma-Aldrich, Poole, UK) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Poole, UK), 100 μg ml −1 penicillin and 100 U ml −1 streptomycin (Sigma-Aldrich, Poole, UK), 1 mM glucose (Sigma-Aldrich, Poole, UK), and 0.1% non-essential amino acids (Thermo Fisher Scientific, Paisley, UK). Cells were passaged using 0.05% trypsin-EDTA (Thermo Fisher Scientific, Paisley, UK) when they reached 80%-90% confluency. The number of total and viable cells was estimated by 0.4% trypan blue staining using the Invitrogen™ Countess™ II automated cell counter (Thermo Fisher Scientific, Paisley, UK).
Passages 5-7 were chosen for subsequent experiments because it yielded the adequate number of chondrocytes for extrusion 3D bioprinting using 2×10 6 cells ml −1 of bioink [60]. Immediately prior to bioprinting of cell-laden bioinks, cell suspensions were drawn up into a syringe and gently mixed with a syringe of NC/Alginate bioink using a two-way tap under sterile conditions for one minute to ensure uniform distribution. Immediately post printing the cellbioink constructs were washed in culture medium to remove the excess CaCl 2 before culturing in the incubator.

Biocompatibility
A LIVE/DEAD ® Cell Viability Kit (Thermo Fisher Scientific, Paisley, UK) was used for assessment of cell viability according to the manufacturer's instructions at 24 and 48 h after bioprinting. The samples were stained with 2 μM calcein AM (green) and 1 μM ethidium homodimer-1 (red). Labeling was examined using confocal microscopy (Zeiss LSM 710 inverted confocal microscope), where green labeled cells represented live cells and red labeling indicated dead cells. Images were taken from at least six different areas of three bioprinted constructs for each condition, the number of live and dead cells were counted using NIH ImageJ software, and cell viability was then expressed as the percentage of the number of live cells to total cells.
Cell metabolic activity was quantified using 10% (v/v) AlamarBlue ® cell viability reagent (Thermo Fisher Scientific, Paisley, UK) at 4, 12 and 24 h after bioprinting. The fluorescence was measured (at 530-560 nm excitation wavelength and 590 nm emission wavelength) with plate reader (POLARstar Omega spectrophotometer, BMG LABTECH, Ortenberg, Germany). Matched concentration of cells in media were used as controls. The cross reactivity of Alamar Blue with nanocellulose in the medium was also tested. The OD values were normalized against that at t=0. The cell viability was determined by plotting fluorescence emission intensity versus cell concentration.

Cytotoxicity
Cytotoxity of NCB bioink was assessed using lactose dehydrogenase (LDH) cytotoxicity assay. The plate containing cells with NCB-AG (n=6) as well as cells only (n=6), media only (n=3) and NC controls (n=3) were incubated at 37°C, 5% CO 2 overnight. Then 50 μl of each sample medium was transferred to a 96-well plate and mixed with 50 μl Reaction Mixture for 30 min at room temperature, protected from light, at which point Stop Solution was added. The absorbance measurements (at 490 and 680 nm) was used to determine LDH activity.

Scanning electron microscopy (SEM)
Bioprinted and crosslinked cellular constructs grown for 3 weeks in culture were washed three times with 50 mM Sodium Cacodylate-HCl Buffer solution (pH 7.2-7.4, SPI Supplies) at 10-20 min intervals to remove excess salt. The sample were fixed overnight in 2% Glutaraldehyde (Sigma Aldrich, UK) and  dehydrated with a series of graded concentrations (30%-100%) of ethanol. The dehydrated sample was then rinsed with 50% hexamethyldisilazane solution (HMDS) in 100% ethanol for 10 min in a fume hood and then three times in 100% HMDS and left overnight to dry. The sample was coated with a thin layer of gold (∼15 nm) using sputter coating and was imaged using SEM (Hitachi 4800s).   . Rheological properties of nanocellulose-alginate bioinks at 1 Hz. G′, storage moduli (dark gray); G″, loss moduli (light gray) for blend (NCB), crystal (CNC) and fibril (CNF) nanocellulose bioinks combined with 1.25%, 2.5% and 5% wt alginate. Data is expressed as the mean±SD (N=3 for all groups). Statistical differences calculated by one-way ANOVA with Tukey's multiple comparison post-hoc test. * P<0.05.

Compression testing
Mechanical tests were performed on bioprinted NCB-AG cylinder discs (5 mm diameter and 4 mm height), crosslinked with 0.5 M of CaCl 2 for 5 min and incubated in phosphate buffered saline (Thermo Fisher Scientific, Paisley, UK) at room temperature for 1 h to reach swelling equilibrium, using the BOSE ElectroForce ® 3200 mechanical loading machine (Bose Corporation, ElectroForce ® Systems Group, Minnesota, USA). The compressive strength of the samples was tested at 1 Hz. The compression (mm) and load (N) were recorded and Young's modulus was determined as the slope of the linear region of the stress/ strain curve:

Statistical analysis
Quantitative results are expressed as mean±standard deviation (SD), and the statistical analyses were performed using the GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Statistical analyses for single comparisons were performed using the Mann-Whitney U test for non-Gaussian distributions and the t test for Gaussian distribution. Multiple comparisons were performed by one-way ANOVA, followed by a Tukey's post hoc test. Number of biological replicates are indicated in each figure legend. A value of p<0.05 was considered statistically significant.

Results
3.1. Nanocellulose bioink formulation and crosslinking optimization 0.625% alginate solution exhibited superior properties with regards to dissolution and mixing but quickly collapsed at room temperature post printing. 7.5% and 10% alginate solutions were difficult to dissolve and too viscous to mix evenly with NC. Concentrations 1.25%, 2.5% and 5% were found to have promising viscosities and mixing potential and went on to rheological testing. Optimum alginate concentration 2.5% was chosen for further experiments due its ability to balance ease of dissolution and mixing with uncrosslinked structure stability post printing (table 1). Crosslinking optimization is summarized in table 2. 1 M CaCl 2 was crosslinked in the shortest time (immediately, compared to 2 min for 0.5 M and 4 min for 0.1 M), with final constructs able to withstand larger mechanical force compared to 0.5 and 0.1 M. However, it was found that the immediate crosslinking time of 1 M CaCl 2 resulted in crosslinking of the filament during deposition, causing dragging and ultimately spoiling the final printed structure. Figure 2(A) illustrates crosslinked lattice structures using 0.5 M CaCl 2 withstanding agitation using a spatula.
All three formulations displayed varying degrees of nanofiber entanglements and extensive porous networks as seen on TEM prior to crosslinking due to agglomeration (figures 4(A), (C), (E)) as reported in other studies [61]. NCB-AG shows fibrillar entanglements of NC nanofibrils interspersed with CNCs ( figure 4(E)). On crosslinking with CaCl 2 the alginate pulls the nanofibrils and crystals together to reduce voids within the material and produce a firm structure that can be manipulated and withstand cell culture conditions (figures 4(B), (D), (F)).

3D bioprinting of macrostructures using nanocellulose bioinks
NCB-AG had the highest resolution demonstrated by the median filament thickness of 1.01 mm compared to 1.07 mm and 1.04 mm for CNC-AG and CNF-AG respectively ( figure 2(D)), however, this was not found to be statistically significant (p=0.42, one-way ANOVA). Crosslinked CNC-AG and NCB-AG cylindrical constructs swelled, especially at low crosslinker concentrations, over 24 h under culture conditions (in media at 37°C), but these changes were not statistically significant (figures 5 (A) and (B)). After 72 h there was a significant increase in volume for CNC-AG (697.6 mm 3 , p-value 0.003) and NCB-AG (523.9 mm 3 , p-value 0.021) constructs crosslinked with 0.1 M versus 0.5 M CalCl 2 concentrations whereas constructs printed using CNF-AG decreased in volume, with 0.5 M crosslinked constructs having a greater reduction  Since NCB-AG crosslinked with 0.5 M CaCl2 demonstrated the most stable construct volume under culture conditions this was the formulation chosen for complex shape testing, including hollow cylinders and anatomical auricular cartilage (figure 6) and biocompatibility experiments.

Rheological properties of pulp-derived nanocellulose enable bioprinting
Pure alginate bioinks showed increasing G′ and G″ with increasing frequency (figure 7). The mean loss moduli (G″) were consistently higher than mean storage moduli (G′) for all concentrations (1.25%, 2.5%, 5% and 7%) demonstrating a dominance of viscous over elastic properties in pure alginate bioinks ( figure 7). Shear rate ramps were performed to investigate the flow properties of bioinks by measuring the viscosity as a function of increasing shear rate. This revealed limited shear thinning behavior at 5% and 7.5% and near Newtonian behavior at 1.25% and 2.5% concentrations of pure alginate bioinks over the entire shear rates studied ( figure 8).
However, unlike alginate bioinks, the mean G′ for all NC bioinks is greater than the G″ over all frequencies studied indicating a dominance of elasticity and demonstrating strong interconnecting networks between nanostructures (figure 9). Shear rate ramps were performed to investigate the flow properties of CNF-AG, CNC-AG and NCB-AG bioinks by measuring the viscosity as a function of increasing shear rate ( figure 10(A)). All nanocellulose containing bioinks exhibited a higher degree of non-Newtonian, shear thinning (pseudoelastic behavior) compared to pure alginate bioinks. CNF-AG and NCB-AG formulations demonstrated greater and statistically different viscosities compared to all CNC formulations. This difference is best seen at shear rate=0.12 s −1 but is still present at higher shear rates ( figure 10(B)). This implies that there are stronger particle-particle interactions or increased entanglements between nanoand microstructures within the NCB-AG and CNF-AG compared to CNC-AG bioinks, providing better shape fidelity post printing [27].
Constructs bioprinted using NCB-AG and crosslinked with 0.5 M CaCl 2 demonstrated the greatest volume stability and favorable cell viability and was therefore used for further biocompatibility experiments. Cell viability in NCB-AG (90.1% ±13.8%) after 24 h in culture was comparable to cells on tissue culture plastic alone (85.3%±3.4%). However, by 48 h cell viability was significantly higher for cells cultured in NCB-AG (97.3%±4.5%) compared to cells on cultured on plastic alone (94.0%±6.4%, P=0.0326) ( figure 12).
Median LDH activity, an indicator of cytotoxicity, was not found to be significantly different between cells cultured on plastic versus bioprinted and cultured 3D in NCB-AG (0.05 versus 0.049 respectively, P=0.1138) (figure 13). Human nasoseptal chondrocytes had significantly increased metabolic activity between 4 and 12 h in culture at all cell densities and conditions tested (P<0.0001) but not between 12 and 24 h ( figure 14). Chondrocytes demonstrated significantly increased

Discussion
Nanocellulose is rapidly gaining interest for biomedical applications due to its unique physical, chemical and biological properties [50]. Results demonstrate that our optimized crystal, fibril and blend nanocellulose-alginate bioink formulations have comparable resolution, suitable rheology and biocompatibility with human nasoseptal chondrocytes to enable utility for cartilage bioprinting using extrusion-based techniques. The unique blend nanocellulose-alginate bioink (2.5% alginate, crosslinked with 0.5 M CaCl 2 ) offers the advantages of exhibiting nano-as well as microroughness for cellular functionality, as well as maintaining the most stable construct volume, to more reliably maintain the shape of patient specific cartilage constructs under culture conditions in the future. It was therefore chosen as the optimum bioink for our metabolic and topographical studies.
The few studies investigating bioprinting with pulp based nanocellulose have assessed crystal or fibril formulations individually [27,51,62] with none directly comparing CNC, NCB and CNF. Our previous work has demonstrated that AVAP technology produces nanocellulose with promising pore and fiber networks for tissue engineering applications [57]. TEM and SEM data in this study shows that even with addition of alginate all three NC formulations continue to be highly porous both in the nano and micro range, unlike pure alginate bioinks which are typically only nanoporous (approx. 5 nm) [63]. NCB hydrogels exhibited a combination of fibrillar networks and interconnecting compact nanorods and have been shown to have a broader variation in pore sizes (55 nm to over 12 μm, mean 934 nm) compared to those from CNC alone, which would allow chondrocyte migration (approx. 10 μm diameter) and provide a tunable nanotopography for a greater variety of bioactive signals [57].
The addition of alginate to allow crosslinking capability did not adversely affect the elasticity or shear thinning behavior of nanocellulose bioinks, which depends on the dry content of the ink rather than the ratio between the two components [27]. The storage modulus was greater than the loss modulus for all combinations of nanocellulose-alginate bioinks, over all frequencies studied, and therefore dominance of elasticity similar to pure nanocellulose materials [57]. This was in contrast to pure alginate bioinks, which are predominantly viscous. The large aspect ratio and ability to form interconnected network structures through hydrogen bonding makes nanocellulose both stiff due to the ordered (crystalline) regions and flexible due to the disordered (amorphous) regions of the nanoparticle [50]. These nanocellulose network structures can disentangle and align parallel to the direction of flow when placed in suspension which also accounts for the high degree of shear thinning exhibited in our study and has also been confirmed for other types of nanocellulose formulations in the literature [27,62,64]. Shear thinning enables bioprinting through fine deposition nozzles at low shear rates with reduced mechanical forces exerted on cells [27]. This is in contrast to the more commonly used pure alginate bioinks, which exhibit only limited shear thinning even at high shear rates requiring higher forces (shear stress) for extrusion which can impact post-printing cell viability [65][66][67]. Reversible stress softening behavior of nanocellulose means that post printing the storage modulus (G′) is recovered under static conditions and is higher than that for pure alginate bioinks (in the 1.25%-7.5% range) contributing to shape fidelity and preventing collapse of complex 3D bioprinted constructs [47]. The rigidity of NCB was significantly higher than for CNC but similar to CNF indicating that the double network hydrogel structure previously reported for NCB is not disrupted by the addition of alginate [57].
Nanocellulose is broadly considered to be biocompatible using in vivo animal studies of BNC demonstrating no foreign body reaction or inflammation [68,69]. Although it is well known that cellulose is not readily degraded by the human body due to the lack cellulolytic enzymes, non-enzymatic, spontaneous hydrolysis of cellulose chains have been suggested to account for slow breakdown which can be enhanced through oxidation [34,70]. Even if not fully broken down, cellulose is biologically inert and theoretically should not interfere with homeostatic processes such as matrix remodeling [71]. Most reports on the nanotoxicology of nanocellulose use BNC and show no evidence of damage at the genetic, cellular and in vivo animal level [72] but few studies use human cells and none directly compare pulp based crystal, fibril and blend formulations [50]. Our study shows that pulp based nanocellulose is non-cytotoxic to nanoseptal chondrocytes with no significant differences in cell viability post-printing with CNC, NCB and CNF. NCB not only significantly increased the proportion of viable nasoseptal chondrocytes in culture after two days, it also encouraged them to be more metabolically active after the first four hours post printing, whilst maintaining a rounded chondrogenic phenotype after three weeks in culture, suggesting a suitable 3D environment, nanotopography and porosity for cartilage matrix formation. These findings are consistent with previous studies indicating that maintenance of a round cell morphology is a prerequisite for overt chondrogenic differentiation [73]. The nanocellular fibrillary structures can mimic the properties of extracellular matrix components, which are also nanofibrillar networks composed of glycosaminoglycans and fibrous proteins, such as collagen, elastin, laminin and fibronectin, thereby encouraging cells to differentiate [74,75]. The 3D bioprinted constructs themselves are immature and do not have the mechanical properties (Young's modulus 52.6 kPa) of native nasoseptal cartilage (in the region of 2-4 MPa [76]) and would require maturation via cartilage matrix secretion in a bioreactor before implantation to determine utility as cartilage tissue substitutes.
The AVAP process does not introduce posthydrolysis modifications to the nanocellulose preparations which provides exiting potential for functionalization [56,57]. The three available hydroxyl groups and negative surface charge allow the possibility of protein immobilization based on chemical conjugation and electrostatic adsorption respectively, in order to enhance cell attachment, migration, proliferation and differentiation [77].

Conclusion
3D bioprinting research has been gaining traction to transition from theory into practice and to ultimately allow biofabrication of customized and anatomically accurate replacement tissue using the patient's own cells for surgical reconstruction. Pulp derived nanocellulose was found to be extremely shear thinning with reversible stress softening behavior, thereby contributing to post-printing shape fidelity. The unique blend of crystal and fibril nanocellulose bioink exhibited nano-as well as micro-roughness for cellular survival and differentiation, as well as maintaining the most stable construct volume in culture. One of the major challenges in bioprinting any tissue type are bioinks that are printable, capable of maintaining complex macrostructures structures and able to provide an environment in which cells can thrive and differentiate, making pulp based nanocellulose a promising addition to the bioink research field.
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