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Bioprinting

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3D bioprinting of liver-mimetic construct with alginate/cellulosenanocrystal hybrid bioink

Yun Wua, Zhi Yuan (William) Linb, Andrew C. Wengera, Kam C. Tamc, Xiaowu (Shirley) Tanga,⁎

a Department of Chemistry & Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Ave West, Waterloo, Ontario, Canada N2L 3G1bOurotech, Inc., Waterloo, Ontario, Canadac Department of Chemical Engineering & Waterloo Institute for Nanotechnology, University of Waterloo, Canada N2L 3G1

A R T I C L E I N F O

Keywords:BioprintingAlginateCellulose nanocrystalsTissue engineeringLiver-mimetic structure

A B S T R A C T

3D bioprinting is a novel platform for engineering complex, three-dimensional (3D) tissues that mimic real ones.The development of hybrid bioinks is a viable strategy that integrates the desirable properties of the constituents.In this work, we present a hybrid bioink composed of alginate and cellulose nanocrystals (CNCs) and explore itssuitability for extrusion-based bioprinting. This bioink possesses excellent shear-thinning property, can be easilyextruded through the nozzle, and provides good initial shape fidelity. It has been demonstrated that the visc-osities during extrusion were at least two orders of magnitude lower than those at small shear rates, enabling thebioinks to be extruded through the nozzle (100 µm inner diameter) readily without clogging. This bioink wasthen used to print a liver-mimetic honeycomb 3D structure containing fibroblast and hepatoma cells. Thestructures were crosslinked with CaCl2 and incubated and cultured for 3 days. It was found that the bioprintingprocess resulted in minimal cell damage making the alginate/CNC hybrid bioink an attractive bioprinting ma-terial.

1. Introduction

3D printing, otherwise known as additive manufacturing, is a termused to describe the process of fabricating a three-dimensional (3D)object layer by layer [1,2]. 3D bioprinting is a subset of 3D printing,which dispenses bioinks to construct complex 3D tissue architecture[3]. It allows for recapitulations of cell-cell and cell-extracellular matrix(ECM) interactions that are absent in conventional two-dimensional(2D) in vitro cell culture but are crucial in modulating cellular behaviorfound under in vivo conditions [4]. The most commonly used bio-printing techniques are extrusion-based, particle fusion-based, light-induced, and inkjet-based bioprinting [1–3,5–9]. Extrusion-based bio-printing is one of the most popular techniques due to compatibility witha variety of bioink, ease of operation and relatively low cost [3,10].Among various bioinks, hydrogels represent a class of promising ma-terials because they provide a highly hydrated, biocompatible, 3D en-vironment [11–13].

Generally, a bioink should satisfy three requirements for extrusion-based bioprinting. First, the bioink should possess shear-thinningproperty, which ensures facile extrusion through a pressurized nozzleand good initial shape fidelity after extrusion [13–17]. Second, thebioink should have sufficient mechanical strength to support the

bioprinted structure and retain its shape [7]. Third, the bioink shouldprotect the encapsulated cells from mechanical stress during printing[18].

Alginate is a natural polymer that has been extensively studied as ahydrogel material for tissue engineering applications due to its favor-able properties, such as biocompatibility and easy processability[19–21]. Upon exposure to divalent cations such as Ca2+, alginateundergoes a rapid and robust gelation process where the polymerchains form an “egg-box structure” with the divalent ions [22,23].However, the rheological properties of pure alginate result in poorprintability and pattern fidelity, which greatly limit its use in 3D bio-printing [14,24].

Cellulose nanocrystals (CNCs) are rod-like or whisker-shaped na-noparticles extracted from the crystalline regions of cellulose fibers. Inrecent decades, its numerous favorable properties (e.g. renewability,low density, high mechanical strength, large and highly reactive sur-faces, and low cytotoxicity) have attracted a significant amount of re-search interests for a variety of applications, including tissue en-gineering [25–29]. CNC can be mixed into various polymer matrices toreinforce the mechanical strength and induce shear thinning behavior[28,30,31]. For example, Siqueira et al. developed a hybrid ink com-prising of CNC, 2-hydroxyethyl methacrylate (HEMA) monomer and

https://doi.org/10.1016/j.bprint.2017.12.001Received 25 August 2017; Received in revised form 7 November 2017; Accepted 4 December 2017

⁎ Corresponding author.E-mail address: tangxw@uwaterloo.ca (X.S. Tang).

Bioprinting 9 (2018) 1–6

Available online 14 December 20172405-8866/ © 2017 Published by Elsevier B.V.

T

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polyether urethane acrylate (PUA). They found that the CNC particlesalign along the printing direction by shear flow, and the CNC inksdisplay shear-thinning behavior [32]. Hence, CNC is a suitable additivefor enhancing the shear-thinning property of bioink.

In this work, we prepared a hybrid bioink by blending alginate andCNC, which combines the desirable properties and overcomes the in-herent disadvantages of the individual components. Rheological prop-erties such as shear-thinning, extrudability, and shape fidelity wereinvestigated. An optimized bioink formulation was then selected toprint a liver-mimetic 3D honeycomb structure, which was cultured withfibroblasts and hepatoma cells. It has been demonstrated that viabilityof cells encapsulated in this bioink is unaffected during printing.

2. Materials and methods

2.1. Materials

Pharmaceutical grade sodium alginate (PROTANAL LF 10/60 FT)containing 60–70% G residues was acquired from FMC (Philadelphia,PA). CNC hydrolyzed from wood was supplied by Celluforce Inc.Phosphate-buffered saline was purchased from EMD Chemicals(Darmstadt, Germany). Calcium chloride was purchased from Sigma-Aldrich (St. Louis, Missouri). NIH/3T3 Fibroblast and human hepatomacells, Eagle's Minimum Essential Medium (EMEM) and penicillin/streptomycin were purchased from ATCC (Manassas, VA). Dulbecco'sModified Eagle's Medium (DMEM) and trypsin/EDTA solution wereobtained from Lonza (Walkersville, MD). Fetal bovine serum (FBS) andtrypan blue stain (0.4%) were provided by Gibco (Carlsbad, CA). LIVE/DEAD viability/cytotoxicity kit was purchased from Life Technologies(Carlsbad, CA).

2.2. Bioink preparation

Dry CNC powder was dispersed in Milli-Q water to prepare 2%, 4%,6%, and 8% (w/v) CNC solutions. Similarly, dry alginate powder wasweighed and dissolved in Milli-Q water to prepare 4% (w/v) and 6%(w/v) alginate solutions. To prepare the alginate/CNC hybrid pre-gelsolutions, equal volumes of CNC and alginate solutions were mixed.Seven formulations of bioinks with different ratios of alginate and CNCas listed in Table 1 were prepared. The pre-gel solution was sterilized byautoclaving at 121 °C for 20 min for cell culture.

2.3. Rheological properties of the hybrid bioinks

The rheological properties of the inks were analyzed using a Bohlin-CS Rheometer with a cup and bob geometry (C25, bob diameter of25 mm and gap width of 150 µm). All measurements were conducted atroom temperature.

2.3.1. Determination of shear-thinningUsing a constant rate testing mode, the steady-state shear viscosities

of pure alginate (20/00, 40/00, and 60/00), and hybrid bioinks (20/10,20/20, 20/30, and 20/40) were conducted over a range of shear rate of0.1–500 s−1.

2.3.2. Determination of shear ratePower-law index (n) was calculated by curve-fitting the viscosity vs.

shear rate curves using the power-law model:

= −η Kγ ̇n 1 (1)

where K is the consistency, η is the viscosity, and γ ̇ is the shear rate. Theshear rate at the nozzle wall can be obtained from the equation:

= ⎛⎝

+ ⎞⎠

γ Qπr n

̇ 4 34

14w 3 (2)

where Q is the volumetric flow rate (16 µL/s) and r is the nozzle radius(50 µm).

2.3.3. Determinations of elastic and viscous moduliA strain sweep from 0.05% to 10% was performed at a frequency of

1 Hz to determine the linear viscoelastic region (LVR). Subsequently, anoptimized LVR strain of 0.1% was chosen for the oscillation frequencymeasurements performed from 0.1 to 10 Hz. The frequency sweepswere then used to define the elastic modulus (G') and the viscousmodulus (G'').

2.3.4. Printability of hybrid bioinksA designed syringe holder was printed in polylactic acid (PLA) using

the thermoplastic extruder on a FlashForge Creator Pro (FlashForge,China). The original thermoplastic extruder from the x-axis carriagewas then removed and replaced with the custom-made syringe holder.The modified FlashForge Creator Pro's operation software was un-altered, except for manual settings such as nozzle diameter, filamentdiameter, and extrusion temperature. Liver mimetic honeycomb models(2.4 mm spacing, 0.48 mm wall thickness, and 1.2 mm wall height asshown in Fig. 1) were designed using Solidworks software and saved asan STL file. This file was then converted to G-code using ReplicatorG.The bioink-loaded syringe was equipped with a 32-gauge disposable tip(EFD Nordson, USA) and was fitted onto the plastic syringe holder.Applied extruding pressure ranged from 5 to 25 psi to control the inkflow rate to match the moving speed of the nozzle at 25 mm s−1. Thebioinks were deposited in 16 layers.

2.4. Morphologies of hydrogels

The structures of hydrogel samples (20/00 and 20/40) were char-acterized using a field emission scanning electron microscope (FE SEM,Zeiss Leo 1530). The samples were first prepared in a custom-madeTeflon mold and immersed in a 1% (w/v) CaCl2 bath. The crosslinkedhydrogels were instantly frozen in liquid nitrogen and lyophilized be-fore being cut into small pieces with a sharp blade. The samples werethen deposited onto an aluminum holder which was placed in a sputtercoater, coating samples with a thin layer of gold. The pore size wasanalyzed using ImageJ software.

2.5. 3D bioprinting of liver-mimetic tissue constructs

2.5.1. Cell cultureFibroblasts were cultured in complete DMEM supplemented with

10% FBS and 100 unit/mL penicillin, 100 µg/mL streptomycin. Humanhepatoma cells were cultured in EMEM with 10% FBS and 100 unit/mLpenicillin, 100 µg/mL streptomycin. Both cell lines were maintained ina 5% CO2 incubator at 37 ℃.

2.5.2. BioprintingFor the construction of liver-mimetic structures, the outer honey-

comb structures were first printed using one syringe with fibroblast-containing bioinks (106 cells/mL) using the aforementioned printingprotocol. The inner cavities with a diameter of 1.5 mm were thenprinted using another syringe with bioink containing human hepatomacells (106 cells/mL). The printed constructs were gelated in 1% (w/v)

Table 1Summary of bioink formulations.

Bioink formulation Alginate (% w/v) CNC (% w/v) Water content (% w/v)

20/00 2 0 9820/10 2 1 9720/20 2 2 9620/30 2 3 9520/40 2 4 9440/00 4 0 9660/00 6 0 94

Y. Wu et al. Bioprinting 9 (2018) 1–6

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Fig. 1. Schematic illustration of the fabrication process.

Fig. 2. (A) TEM image of CNC used in this study. (B) Flow curves of five different bioink formulations, 20/00, 40/00, 60/00, 20/20, and 20/40. (C) Schematic illustration of interactionsbetween alginate (grey) and CNC (green) at static state and under shear stress (τ). At static state (left), alginate and CNC are entangled in the hybrid solution randomly, while under shearstress (right), entangled alginate and CNC align. (D) Elastic modulus (G') and viscous modulus (G'') of three representative bioink formulations 20/00, 60/00, and 20/40 as a function ofoscillatory frequency. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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CaCl2 solution for 10 min and then washed with serum-free DMEM.Next, the constructs were cultured in complete DMEM supplementedwith 5 mM CaCl2 in an incubator. Molded constructs were made ascontrol samples using an extrusion-free method. Briefly, 20/40 bioinkcontaining fibroblasts or hepatoma cells was placed on a sterilized glassslide with two spacers (150 µm thickness) and covered with an ionpermeable membrane to make a slab (26 mm × 26 mm × 0.15 mm),which was then immersed in 1% (w/v) CaCl2 for 10 min and was cul-tured in the same condition as that of the honeycomb constructs.

2.6. Cell viability studies

Viability of naked cells was assessed on day 0 using trypan bluestain and a hemocytometer. A LIVE/DEAD viability/cytotoxicity kit wasutilized to determine the cell viability within the molded and bioprintedconstructs on days 0, 1, and 3. Calcein-AM/ethidium homodimer wasdiluted in serum-free DMEM with ratios of 1: 2000 and 1: 500, re-spectively. The samples were first washed with serum-free DMEM for10 min and then incubated in the staining solution for 2 h. Afterwards,the samples were washed with serum-free medium for another 10 min.A fluorescence microscope (Nikon Eclipse Ti-S) was used to image thelive and dead cells within the constructs. Images from each sample weretaken and analyzed using ImageJ software. Cell viability was calculatedby dividing the number of live cells by the total numbers of cells in theconstructs.

2.7. Statistical analysis

All data were presented as mean± standard deviation. Differencesbetween samples were determined from the independent t-test andwere considered statistically significant when p

of fibroblast and hepatoma cells in bioprinted constructs on day 0 were71.00% and 67.06%, respectively. No significant differences in cellviability were observed between molded and bioprinted constructs,indicating that the bioprinting process resulted in no observable celldeath and thus is cell-compatible. However, after 3 days, cell viabilitiesof fibroblast and hepatoma cells decreased to 58.91% and 49.51%,respectively (Fig. 5C). The declining viability over time may arise fromthe lack of cell-binding sites in the hydrogel, which limit cell adhesion,viability and proliferation. To circumvent such issue, one potentialsolution is incorporating polymers containing cell-binding sites, such as

gelatin, into the system. There is an ever-growing amount of evidencereported in research literature that gelatin is effective in promoting thecell attachment due to its RGD tripeptide sequences, which are themajor integrin-binding domains present within extracellular matrix(ECM) [33,34]. Our preliminary results (Fig. S4) show that the in-corporation of gelatin in the alginate-CNC hybrid bioink does not in-fluence the viscosity and hence printability of the bioink. Fig. 5D-Eshow the fluorescence images of homogeneously distributed fibroblastsand human hepatoma cells in the bioprinted constructs.

Fig. 3. Optical images of 3D printed constructs using bioink formulations (A) 20/00, (B) 20/10, (C) 20/20, (D) 20/30, and (E) 20/40, respectively. (F) The zoomed-in image of theoutlined area in (E). Scale bars are 5 mm in (A-E) and 500 µm in (F).

Fig. 4. Scanning electron micrographs of freeze-dried (A) alginate (20/00) and (B) alginate/CNC (20/40) hydrogels.

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4. Conclusions

In this work, we developed a hybrid bioink by blending alginatewith CNC. This bioink displayed excellent shear-thinning property,extrudability, and shape fidelity after deposition. The 20/40 (2% algi-nate and 4% CNC) bioink formulation was selected to print a liver-mimetic honeycomb 3D structure containing fibroblast and heptatomacells, and the bioprinting process was found to induce minimal celldeath. Overall, the proposed bioink has good potential for 3D bio-printing.

Acknowledgements

The authors would like to thank Dr. Michael Tam's and Dr. JuewenLiu's group for providing access to the rheometer and fluorescencemicroscope, respectively. Also, we would like to thank Dr. ZengqianShi, Debbie Wu, Yibo Liu, and Jimmy Huang for providing informationon materials characterization and instruments training. This work issupported by a discovery grant from the Natural Sciences andEngineering Research Council (NSERC) of Canada (grant no. RGPIN-2016-04398) to Dr. Tang.

Appendix A. Supporting information

Supplementary data associated with this article can be found in theonline version at http://dx.doi.org/10.1016/j.bprint.2017.12.001.

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Fig. 5. (A) Schematic top-down view of the liver-mimetic engineered tissue constructs. (B) 3D printed constructs with bioink 20/40. Food dyes were used to distinguish fibroblast-ladenbioink (green) from hepatoma cell-laden bioink (purple). (C) Statistical analysis of viabilities (live cells populations/total cell populations) of fibroblast and human hepatoma cells on days0, 1, and 3. (D&E) Representative live/dead fluorescent images of bioprinted (D) fibroblast only and (E) fibroblast plus human hepatoma cells. The dashed lines are the boundaries of thedesigned structure. The cells were stained with Calcein-AM and ethidium homodimer-1 to show live (green) and dead (red) cells. Scale bars are 5 mm in (B) and 250 µm in (D) and (E).(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3D bioprinting of liver-mimetic construct with alginate/cellulose nanocrystal hybrid bioinkIntroductionMaterials and methodsMaterialsBioink preparationRheological properties of the hybrid bioinksDetermination of shear-thinningDetermination of shear rateDeterminations of elastic and viscous moduliPrintability of hybrid bioinks

Morphologies of hydrogels3D bioprinting of liver-mimetic tissue constructsCell cultureBioprinting

Cell viability studiesStatistical analysis

Results and discussionFabrication of molded and bioprinted constructsRheological properties of hybrid bioinksDetermination of shear-thinningDetermination of shear rateDeterminations of elastic and viscous moduliPrintability of hybrid bioninks

Morphological characterizationsCell viability studies

ConclusionsAcknowledgementsSupporting informationReferences