Bioactive Materials 15 (2022) 185–193

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Bioactive Materials
journal homepage: www.keaipublishing.com/en/journals/bioactive-materials

Stem cell-laden hydrogel bioink for generation of high resolution and

fidelity engineered tissues with complex geometries
Oju Jeon a, Yu Bin Lee a, Sang Jin Lee a, Nazilya Guliyeva a, Joanna Lee b, Eben Alsberg a, c, d, e, *
a
Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, IL, 60607, USA
b
Department of Chemistry, University of Illinois at Chicago, Chicago, IL, 60607, USA
c
Department of Orthopaedics, University of Illinois at Chicago, Chicago, IL, 60607, USA
d
Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, 60607, USA
e
Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, Chicago, IL, 60607, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Recently, 3D bioprinting has been explored as a promising technology for biomedical applications with the
3D bioprinting potential to create complex structures with precise features. Cell encapsulated hydrogels composed of materials
Hydrogel bioink such as gelatin, collagen, hyaluronic acid, alginate and polyethylene glycol have been widely used as bioinks for
Biomaterial
3D bioprinting. However, since most hydrogel-based bioinks may not allow rapid stabilization immediately after
High resolution and fidelity
3D bioprinting, achieving high resolution and fidelity to the intended architecture is a common challenge in 3D
bioprinting of hydrogels. In this study, we have utilized shear-thinning and self-healing ionically crosslinked
oxidized and methacrylated alginates (OMAs) as a bioink, which can be rapidly gelled by its self-healing property
after bioprinting and further stabilized via secondary crosslinking. It was successfully demonstrated that stem
cell-laden calcium-crosslinked OMA hydrogels can be bioprinted into complicated 3D tissue structures with both
high resolution and fidelity. Additional photocrosslinking enables long-term culture of 3D bioprinted constructs
for formation of functional tissue by differentiation of encapsulated human mesenchymal stem cells.

1. Introduction structures at high resolution [11]. Cell encapsulated hydrogels

composed of materials such as gelatin, collagen, hyaluronic acid, algi­
Hydrogels are three-dimensional (3D) networks of hydrophilic nate and polyethylene glycol have been used extensively as bioinks for
polymers that can hold a large amount of water in their swollen state [1, 3D printing [11–13]. Since most hydrogel-based bioinks do not allow
2], and over the past few decades, they have been widely used in the rapid stabilization immediately after extrusion printing, achieving high
tissue engineering and regenerative medicine fields due to their excel­ resolution and fidelity and self-supporting tissue constructs remains a
lent biocompatibility [3,4] and capacity to be engineered to mimic as­ significant challenge in 3D hydrogel bioprinting [14,15]. Thus, there is a
pects of the native extracellular matrix (ECM) of tissues. A variety of critical need to develop new hydrogel-based bioinks that can be applied
hydrogels, endowed with special characteristics, such as tunable phys­ to directly print structures of clinically relevant size-scale with high
ical and biochemical properties, stimuli responsiveness, printing resolution and fidelity, tunable mechanical strength and
multiple-network crosslinking and ultra-toughness, have been adopted degradation rates, and high cell viability [16].
with innovative strategies, like photolithography, interpenetrating To overcome this limited capacity for rapid mechanical stabilization
polymer network formation, coacervation and microgel assembly, for of hydrogel-based bioinks, multi-material, interpenetrating network,
biomedical engineering applications [5–9]. However, they are limited in nanocomposite, supramolecular and shear-thinning material-based
their ability to be used to create precisely controlled complex structures bioinks [16,17] and supporting medium systems composed of microgel
and intricate 3D microarchitectures replicating those in tissues and or­ slurries, cell aggregates and shear-thinning hydrogels [14,18–21] have
gans [10]. Recently, 3D bioprinting has been explored as a promising been developed to improve hydrogel printability and mechanical sup­
technology for creating engineered tissue constructs with complex port for 3D printed structures. Among these, shear-thinning hydrogels

Peer review under responsibility of KeAi Communications Co., Ltd.

* Corresponding author. Department of Biomedical Engineering, University of Illinois at Chicago, Chicago, IL, 60607, USA.
E-mail address: ealsberg@uic.edu (E. Alsberg).

https://doi.org/10.1016/j.bioactmat.2021.11.025
Received 16 September 2021; Received in revised form 15 November 2021; Accepted 16 November 2021
Available online 22 December 2021
2452-199X/© 2021 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
O. Jeon et al. Bioactive Materials 15 (2022) 185–193

are often favored since they undergo a substantial reduction in viscosity supported by Cura software (Ultimaker) which is an open source 3D
under increasing shear stress [22]. Some shear-thinning hydrogels printer host software. The design for the syringe pump extruder and
exhibit viscous flow under shear and self-healing upon removal of the image files were downloaded as stereolithography (STL) files from
applied shear stress [23]. However, many of these shear-thinning bio­ the National Institutes of Health 3D Print Exchange (http://3dprint.
inks rely on nonspecific interactions between macromers or the devel­ nih.gov) under an open-source license.
opment of bioinks by modifications enabling long-range interactions
between specific binding molecules on macromers, resulting in pro­ 2.3. Ca-crosslinked OMA bioinks
longed self-recovery times following shear-thinning after extrusion
through a needle [24,25]. This may limit their suitability as bioinks OMAs (2 w/v %) were dissolved in DMEM (Sigma) with a photo­
because they may collapse due to instability before self-healing, which initiator [2-Hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone,
results in the poor resolution and fidelity of 3D printed structures [26]. 0.05 w/v %, Sigma] at pH 7.4. OMA (1 ml) solutions were loaded into a
To achieve rapid self-healing of hydrogels, supramolecular hydrogels 1-ml syringe. 40 μl of calcium sulfate slurries (CaSO4⋅2H2O) at con­
have been developed as shear-thinning and self-healing hydrogel bio­ centrations of 0, 52.5, 78.8, 91.9, 105.0, and 210.0 mg/ml were added
inks, which show rapid self-healing time, based on guest-host in­ into another 1-ml syringe. After the two syringes were connected
teractions [27]. Though due to steric hindrance effects of long macromer together with a female-female luer lock coupler (Value Plastics), the
chains, the guest-host complexation can be retarded because the host two solutions were mixed back and forth 40 times. The Ca-crosslinked
molecules cannot efficiently interact with the guest molecules [28,29]. OMA solution was further mixed back and forth 10 times every 10 min
Moreover, non-covalent crosslinking of the guest-host molecules endows for 30 min. Ca-crosslinked OMA bioink was loaded into a 2.5-ml sy­
the hydrogels with poor mechanical properties [30]. ringe (Gastight Syringe, Hamilton Company) with a 0.5-inch (length)
22 G stainless steel needle (McMaster-Carr). The syringe was then
2. Experimental section mounted onto the syringe pump extruder on the modified Printrbot 3D
printer, the pneumatic extrusion system on a commercial extrusion-
2.1. Synthesis of OMA based 3D bioprinter (Biobot Basic, Advanced Solutions Life Sciences)
or the syringe pump printhead on a commercial syringe pump-based
The dual-crosslinkable oxidized and methacrylated alginate (OMA) 3D bioprinter (Bio X™, Cellink). The OMA bioinks were printed
was prepared by the oxidation and methacrylation of alginates [21,31]. using Cura software for the modified Printrbot 3D printer, TSIM®
Briefly, sodium alginate (10 g, Protanal LF120 M [high viscosity (251 software for the Biobot Basic printer, or the in-house software in the
mPa S at 1 % water) or low viscosity (157 mPa⋅S at 1 % water), FMC Bio X™ printer.
Biopolymer] was dissolved in ultrapure deionized water (diH2O, 900 For the cell-laden OMA bioinks, human bone marrow-derived
ml) overnight. Sodium periodate (0.1, 0.2 or 0.5 g, Sigma) was dissolved mesenchymal stem cells (hMSCs) were isolated from bone marrow
in 100 ml diH2O, added to the alginate solution under stirring in the aspirates obtained from the posterior iliac crest of a healthy twenty
dark at room temperature (RT) and then allowed to react for 24 h. To eight-year old male donor under a protocol approved by the Univer­
synthesize OMA, 2-morpholinoethanesulfonic acid (MES, 19.52 g, sity Hospitals of Cleveland Institutional Review Board. The aspirates
Sigma) and NaCl (17.53 g) were directly added to an oxidized alginate were washed with growth medium comprised of DMEM-LG (Sigma)
(OA) solution (1 L), and the pH was adjusted to 6.5 with 5 N NaOH. with 10 % prescreened fetal bovine serum (FBS, Gibco). Mononuclear
N-hydroxysuccinimide (NHS, 2.12 g; Sigma) and 1-ethyl-3-(3-dimethy­ cells were isolated by centrifugation in a Percoll (Sigma) density
laminopropyl)-carbodiimide hydrochloride (EDC, 7.00 g; Sigma) gradient and the isolated cells were plated at 1.8 × 105 cells/cm2 in
(molar ratio of NHS:EDC = 1:2) were added to the mixture to activate 20 DMEM-LG containing 10 % FBS and 1 % penicillin/streptomycin (P/S,
% of the carboxylic acid groups of the alginate. After 5 min, 2-amino­ Thermo Fisher Scientific) in an incubator at 37 ◦ C and 5 % CO2. After 4
ethyl methacrylate (AEMA, 3.04 g, Polysciences) (molar ratio of NHS: days of incubation, non-adherent cells were removed and adherent
EDC:AEMA = 1:2:1) was added to the product, and the reaction was cell were maintained in DMEM-LG containing 10 % FBS, 1 % P/S and
maintained in the dark at RT for 24 h. The reaction mixture was 10 ng/ml fibroblast growth factor-2 (FGF-2, R&D) with media
precipitated with the addition of acetone in excess, dried in a fume hood, changes every 3 days. After 14 days of culture, the cells were passaged
and rehydrated to a 1 w/v % solution in diH2O for further purification. at a density of 5 × 103 cells/cm2, cultured for an additional 14 days,
The OMA was purified by dialysis against diH2O (MWCO 3500, Spec­ and then stored in cryopreservation media in liquid nitrogen until use.
trum Laboratories Inc.) for 3 days, treated with activated charcoal (5 To encapsulate hMSCs into OMA bioink, hMSCs were expanded in
g/L, 100 mesh, Oakwood Chemical) for 30 min, filtered (0.22 μm filter) growth media consisting of DMEM-LG with 10 % FBS (Sigma), 1 % P/S
and lyophilized. To determine the levels of alginate oxidation and and 10 ng/ml FGF-2. To prepare the cell-laden OMA bioinks, hMSCs
methacrylation, the OMAs were dissolved in deuterium oxide (D2O, (passage 3) were harvested with trypsin/EDTA (Thermo Fisher) and
Sigma) at 2 w/v %, and 1H NMR spectra were recorded on a Varian concentrated by centrifugation at 300×g for 5 min. Following aspi­
Unity-300 (300 MHz) NMR spectrometer (Varian Inc.) using 3-(trime­ ration of the supernatant, pelleted hMSCs were suspened in OMA
thylsilyl)propionic acid-d4 sodium salt (0.05 w/v %, Sigma) as an in­ solution (5 × 106 cells/ml), and then the OMA solution with sus­
ternal standard (Fig. S20 and Table S1). pended cells was crosslinked as described above.

2.2. Modification of the Printrbot 3D printer 2.4. Rheological properties of OMA bioinks

The 3D printer with a syringe-based extruder was prepared as Dynamic rheological examination of the OMA bioinks was per­
previously reported. Briefly, the thermoplastic extruder assembly was formed to evaluate their mechanical properties and shear-thinning,
removed from the 3D printer (Printrbot®) and replaced with a custom- shear-yielding and self-healing behavior with a Kinexus ultra +
built syringe pump extruder (Fig. S21). The custom syringe pump rheometer (Malvern Panalytical). In oscillatory mode, a parallel plate
extruder was designed to enable the use of the NEMA-17 stepper motor (25 mm diameter) geometry measuring system was employed, and the
from the original thermoplastic extruder-based printer and mounting gap was set to 1 mm. After each OMA bioink was placed between the
of it directly in place of the original extruder on the x-axis carriage. The plates, all the tests were started at 25 ± 0.1 ◦ C, and the plate temperature
syringe pump extruder was printed with polylactic acid using the was maintained at 25 ◦ C. To determine the shear-thinning and shear-
thermoplastic extruder on the Printrbot® before its removal. By using yielding behaviors of the OMA bioinks, viscosity change was
the same stepper motor, the syringe pump extruder was natively measured as a function of shear rate and shear stress, respectively.

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Oscillatory frequency sweep (0.1–10 Hz at 1 % shear strain) tests were chondrogenic media was changed every other day. After 4 weeks of
performed to measure storage moduli (G′ ) and loss modulis (G′′ ). culture, 3D printed hMSC constructs were harvested, fixed in 10 %
Oscillatory strain sweep (0.10–100 % shear strain at 1 Hz) tests were neutral buffered formalin overnight at 4 ◦ C. Whole intact 3D printed ears
performed to determine the G’/G” crossover. To demonstrate the self- were stained with Toluidine blue O. Fixed chondrogenically differenti­
healing properties of the OMA bioinks, cyclic deformation tests were ated and control 3D printed cuboids (3 × 3 × 1 mm) were embedded in
performed at 100 % shear strain with recovery at 1 % shear strain, each paraffin, sectioned at a thickness of 10 μm, stained with Toluidine blue
for 1 min at 1 Hz. O, and then imaged using a microscope (TMS-F, Nikon) equipped with a
digital camera (Coolpix 995, Nikon). To measure GAG production,
2.5. 3D printing of OMA bioinks chondrogenically differentiated 3D printed cuboids (3 × 3 × 1 mm)
were homogenized at 35000 rpm for 60 s using a TH homogenizer (Omni
OMA or hMSC-laden OMA bioinks were loaded into syringes as International) in buffer (1 mL, pH 6.5) containing papain (25 μg m/l,
described above, connected to 0.5-inch (length) 22 G stainless steel Sigma), L-cysteine (2 × 10− 3 M, Sigma), sodium phosphate (50 × 10− 3
needles and mounted on the Printrbot 3D printer. The tip of each needle M, Thermo Fisher Scientific) and EDTA (2 × 10− 3 M, Thermo Fisher
was positioned at the center and 0.1 mm from the bottom of the glass Scientific) and then digested at 65 ◦ C overnight. GAG content was
dish (Cellink), and the print instructions were sent to the printer using quantified by a dimethylmethylene blue assay and DNA content was
Cura software. For 3D printing of patterned structures, bioink-loaded measured using the PicoGreen® assay. GAG content was normalized to
syringes were connected to a 0.5-inch (length) 22 G stainless needles DNA content.
and mounted onto the commercial extrusion-based 3D bioprinter (Bio­
bot Basic) or the commercial syringe pump-based 3D bioprinter (Bio 2.9. Statistical analysis
X™, Cellink), and the bioink was printed using the software indicated
earlier for these printers. Quantitative data were expressed as mean ± standard deviation.
Statistical analysis was performed by one-way analysis of variance
2.6. Analysis of 3D printed structures (ANOVA) with the Tukey significant difference post hoc test using Prism
software (GraphPad). A value of p < 0.05 was considered statically
Linear filaments of the OMA bioink were printed in the OMA significant.
microgel supporting baths with 22, 25 and 27 G needles using the
BioX™ printer, photocrosslinked under UV light a 20 mW/cm2 for 1 min 3. Results and discussion
and then filaments were imaged using a fluorescence microscope (TMS-
F, Nikon) equipped with a digital camera (Coolpix 995, Nikon). Di­ Here, ionically crosslinked oxidized and methacrylated alginate
ameters of the 3D OMA filaments were measured at least 400 times using (OMA) has been utilized as a bioink, which can be further stabilized after
10 different printed filaments for each group using ImageJ (National bioprinting via secondary crosslinking (Fig. 1 and Fig. S1). The OMA
Institutes of Health). bioink exhibits shear-thinning and rapid self-healing properties (Fig. 2,
To assess the quality and accuracy of the 3D printing of OMA bioinks, Fig. S2 and Fig. S7), and are expected to be applicable to 3D bioprinting
cuboids [10 × 10 × 5 mm, Fig. 3(c)] were printed with 20 and 22 G systems with high resolution and fidelity. While OMA bioinks could be
needles. The dimensions were then measured using a caliper. The directly extruded through the printing needle as continuous fibers via
measured dimensions were compared to the original dimensions speci­ their shear-thinning properties, the 3D printed OMA constructs could be
fied in the 3D models. The fidelity (%) was calculated as Lengthmeasured/ stabilized and maintained through rapid self-healing properties. We
Lengthoriginal × 100 (N = 3). have successfully demonstrated that stem cell-laden calcium-crosslinked
OMA hydrogels can be bioprinted into complicated 3D tissue structures
2.7. Cytotoxicity of 3D printing process with high resolution and fidelity. Additional photocrosslinking enables
long-term culture of 3D bioprinted constructs for formation of functional
Viability of hMSCs in the photocrosslinked 3D printed constructs was tissue by differentiation of encapsulated human mesenchymal stem cells
investigated using Live/Dead staining comprised of fluorescein diac­ (hMSCs).
etate (FDA, Sigma) and ethidium bromide (EB, Fisher Scientific). The 3D bioprinting is an important tool for the development of complex
staining solution was freshly prepared by mixing 1 ml FDA solution (1.5 structures of tissues and organs for tissue engineering and regenerative
mg/ml in dimethyl sulfoxide, Sigma) and 0.5 ml EB solution (1 mg/ml in medicine applications [2]. Since the hydrogel-based bioinks for 3D
PBS, Thermo Fisher Scientific) with 0.3 ml PBS (pH 8). After photo­ bioprinting are extruded via syringe through a narrow printing nozzle,
crosslinking of 3D printed constructs (5 mm diameter and 1 mm height) they must possess a balance between a high viscosity for rapid gelation
in a 24-well tissue culture plate under UV light at 20 mW/cm2 for 1 min, after extrusion and low shear stress for cytocompatibility, which is
1 ml growth media was added into each well. 20 μl of staining solution difficult to achieve at the same time in a biomaterial [32]. Since the
was added into each well and incubated for 3–5 min at room tempera­ shear-thinning and self-healing characteristics and low shear yield stress
ture, and then stained hMSC-hydrogel constructs were imaged using a could enhance the 3D printing capacity of bioinks [22], OMA bioinks
fluorescence microscope (ECLIPSE TE 300) equipped with a digital were evaluated to determine whether they exhibit these properties
camera (MU1402-NI05, AmScope). before utilizing them for 3D bioprinting. As the oxidation and meth­
acrylation degrees of OMA can affect the physical properties of OMAs (e.
2.8. Chondrogenesis g., mechanical and rheological properties and swelling and degradation
profiles), a variety of OMA bioinks were evaluated (i.e., 1 and 2 %
After 3D printing of the bioinks, the 3D printed constructs were theoretical oxidation and 5, 10, and 20 % theoretical methacrylation).
further stabilized by photocrosslinking under UV at 20 mW/cm2 for 1 The ionically crosslinked OMA bioinks exhibited shear-thinning
min. After photocrosslinking, 3D printed constructs were transferred behavior, characterized by the decrease in viscosity with increasing
into 100 ml spinner flasks (Bellco Glass Inc., Vineland, NJ) containing shear rate [Fig. 2(A)], while the uncrosslinked OMA solution (OMA-0)
80 ml of chondrogenic differentiation media [1 % ITS + Premix, 100 nM exhibited typical liquid behavior, indicating significantly lower and
dexamethasone, 37.5 μg/ml L-ascorbic acid-2-phosphate, 1 mM sodium variable viscosities over the shear rate screen (Fig. S2(A)). Additionally,
pyruvate, 100 μM nonessential amino acids, and 10 ng/ml TGF-β1 in HG- all of the OMA bioinks exhibited low shear yield stress [Fig. 2(B) and
DMEM] or growth media as a control. The spinner flasks were placed in Fig. S2(B), <5 Pa], which was determined using shear stress sweeps,
a humidified incubator at 37 ◦ C with 5 % CO2 and stirred at 40 rpm. The indicating that only a small stress is required to allow the OMA bioinks

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Fig. 1. Schematic illustration of photocrosslinkable calcium-crosslinked OMA bioink. The 3D bioprinted OMA bioink could be further photocured to produce a
chemically and mechanically stable biomimetic 3D bioprinted construct.

to flow. Frequency sweep tests of the OMA bioinks showed significantly 1OX20MA; 1 and 20 % theoretical oxidation and methacrylation,
higher G′ than G′′ , indicating that the OMA bioinks were mechanically respectively), which had the lowest amount of calcium ions and the
stable [Fig. 2(C–F) and Fig. S3(A-D)], while the uncrosslinked OMA crossover point of G’ and G” at the lowest shear strain [Fig. 2(H)]
solutions show typical viscoelastic liquid behavior (Fig. S4). In oscilla­ amongst the compositions which printed at high fidelity, was used for
tory strain sweep tests, G′′ of the OMA bioinks surpassed G′ at approx­ subsequent experiments.
imately 25 % strain [Fig. 2(G-J) and Fig. S5(A-D)], indicating the phase To mimic native tissue architecture using an extrusion-based 3D
change from solid-like to liquid-like, which is an important property of bioprinting, it is important to evaluate the resolution of bioinks. Since
hydrogel materials for injectability and/or printability through a the diameter of a printing nozzle has a significant effect on the resolution
printing nozzle. In contrast, the uncrosslinked OMA solutions show of printed constructs [34], the effect of the printing needle diameter on
typical viscoelastic liquid behavior [Fig. S6(A-B)]. When investigated bioprinting resolution was investigated. The robustness of
under the cyclic strain sweeps by alternating low (1 %) and high (100 %) high-resolution printing was determined by measuring the diameter of
strains, the OMA bioinks went from solid-like to liquid-like behavior in printed OMA filaments with various printing needle gauges from 22 to
response to strain [Fig. 2(K–N) and Fig. S7(A-D)]. Furthermore, the re­ 27 G [Fig. 3(A)]. As the inner diameter (ID) of the printing needles
sponses of shear moduli to high strain and recoveries at low strain were decreased from 413 μm (22 G) to 210 μm (27 G), the diameters of
rapid and repeatable. The combination of shear-thinning, low printed OMA filaments decreased from 361 μm to 223 μm [Fig. 3(B)],
shear-yielding and self-healing properties allows for the rapid transition which were 88–106 % of the inner diameters of the needles, respec­
from solid-like to liquid-like behavior [33]. Moreover, the rapid recov­ tively, and confirmed the capability of high-resolution printing. This
ery of mechanical properties after removal of shear stress, as occurs after trend was further confirmed with OMA bioinks prepared with low vis­
the deposition of the OMA bioinks, enables their stabilization immedi­ cosity alginate. 3D printed OMA filaments with 22 G needle exhibited
ately after extrusion. These properties make the OMA bioinks higher solution compared to 3D printed filaments with 20 G needle
well-suited for injection and extrusion-based 3D bioprinting. These (Fig. S14).
characteristics were further confirmed with various OMA bioinks pre­ One of the primary goals of 3D bioprinting is to print complex ge­
pare with low viscosity alginate (Figs. S8–S12). OMA-20, OMA-25 and ometries. However, current hydrogel-based bioinks lack the mechanical
OMA-40 bioinks with different amounts of calcium exhibited similar strength as well as printability to print macroscale constructs with high
high fidelity to the printed structure of a cuboid, while the OMA-15 fidelity [33]. Although high resolution of hydrogel bioink filaments have
bioink exhibited the lowest fidelity of the 3D printed structure [Fig. 2 been reported in some studies [26], there are still limitations with
(O) and Fig. S13]. Therefore, the OMA-20 bioink (high viscosity respect to achieving high shape fidelity of 3D printed structures. To

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Fig. 2. Characterization of OMA (1OX20MA) bioinks synthesized from high viscosity alginate. Viscosity measurements of the OMA bioink as a function of (A) shear
rate and (B) shear stress demonstrate its shear-thinning and shear-yielding behaviors, respectively. Frequency sweep tests of (C) OMA-15 [OMA+ 15 μl CaSO4 (1.22
M)], (D) OMA-20 [OMA+ 20 μl CaSO4 (1.22 M)], (E) OMA-25 [OMA+ 25 μl CaSO4 (1.22 M)], and (F) OMA-40 [OMA+ 40 μl CaSO4 (1.22 M)] bioinks indicate that
the OMA bioinks were mechanically stable. Strain sweep tests of (G) OMA-15, (H) OMA-20, (I) OMA-25, and (J) OMA-40 bioinks. G′ and G′′ crossover of the OMA
bioinks as a function of shear strain exhibit their gel-to-sol transition at higher shear strain. Shear moduli changes during dynamic strain tests of (K) OMA-15, (L)
OMA-20, (M) OMA-25, and (N) OMA-40 bioinks with alternating low (1 %) and high (100 %) strains at 1 Hz demonstrate their rapid transitions between solid-like
and liquid-like behavior within seconds, which indicates self-healing or thixotropic properties. (O) Photograph of the 3D printed structures (5 × 5 × 3 mm) using the
OMA bioinks on the BioX printer.

demonstrate the OMA bioinks’ ability to print macroscale shapes with 92–110 % [Fig. 3(D)], indicating the capability of high fidelity, and
high shape fidelity, cuboids (10 × 10 × 5 mm) were printed with 20 (ID there was no significant difference in fidelity achieved with the 20 and
= 603 μm) and 22 G (ID = 413 μm) needles [Fig. 3(C) and Fig. S15], 22 G printing needles. Furthermore, the OMA bioinks exhibited high
measured the dimensions of the 3D printed structures (X, Y and Z axes) fidelity in forming complex and large-scale structures, such as a letter, a
and then calculated the fidelity (%) by comparison with the 3D digital concentric-ring, a two-phase cylinder, a patterned structure, the UIC
images. The quantified fidelities of the 3D printed structures were logo [Fig. 3(E-J)], and an ear [Fig. S16]. Although complex geometries

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Fig. 3. (A) Images of 3D filaments printed in a straight line using the modified Printrbot printer with OMA bioinks synthesized from high viscosity alginate and 22 G
(ID = 410 μm), 25 G (ID = 260 μm), and 27 G (ID = 210 μm) printing needles and (B) their mean diameters. Colored dotted lines indicate the inner diameter of each
respective printing needle. Scale bars indicate 500 μm. *p < 0.05 compared to other groups. Fidelity of the 3D printed structures with various printing needle sizes.
(C) Images of 3D printed structures using the modified Printrbot printer with 22 G and 20 G printing needles, (D) their quantified fidelity, demonstrating high fidelity
of the 3D printed structures. Images of 3D printed structures of (E) a letter “C” formed using the modified Printrbot printer, (F) a concentric-ring fabricated using the
Biobot printer, (G) a two-phase cylinder formed using the BioX printer, (H) a checkerboard-patterned structure fabricated using the Biobot printer (I) the UIC logo
formed using the BioX printer and (J) an ear formed using the modified Printrbot printer. Images of 3D printed overhang geometries of (K) a bowl, (L) a bridge and
(M) a letter “K” using the BioX printer with OMA bioinks synthesized from high viscosity alginate and 22 G printing nozzles. The black scale bars indicate 1 cm.

have been reported in a number of studies using hydrogel bioinks, it is has never been reported before.
currently still very challenging to 3D extrusion print hydrogel bioinks in Since cell-laden OMA bioinks dispensed from a nozzle are exposed to
overhang geometries without additional supporting structures or ma­ shear stress and additionally low-level UV light during photo­
terials due to their mechanical instability. Instantaneous recovery of crosslinking to further stabilize the 3D printed constructs, the cyto­
bioinks’ mechanical strength after extrusion is essential to prevent compability of the bioprinting process was analyzed by measuring cell
collapse of overhang layers [35,36]. Since our OMA bioinks are me­ viability after the printing process and photocrosslinking. High cell
chanically stable and exhibit rapid self-healing (Figs. 2, S1, S3 and S7), viability was observed after printing process and photocrosslinking,
various overhang geometries with self-supporting structural integrity indicating that the macromers, the bioprinting process and the photo­
could be extrusion printed using the OMA bioinks without supporting crosslinking were all cytocompatible [Fig. 4(A-C)].
devices or materials [Fig. 3(K-M) and Fig. S17], which to our knowledge After validating the printability and cytocompability of the OMA

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Fig. 4. Differentiation of 3D bioprinted constructs using the modified Printrbot printer with hMSC-laden OMA bioinks synthesized from high viscosity alginate.
Representative (A) live, (B) dead and (C) merged photomicrographs of photocrosslinked 3D printed constructs at day 0. 3D printed ear using hMSC-laden OMA
bioink (D) before and (E) after photocrosslinking. Chondrogenically differentiated 3D printed ear (F) before and (G) after Toluidine blue O staining. Photomicro­
graphs of Toluidine blue O stained construct sections cultured in (H) chondrogenic (Chondro) and (I) growth media (Control). (J) Quantification of GAG/DNA in the
3D printed constructs. White, red and black scale bars indicate 200 μm, 1 cm and 100 μm, respectively. *p < 0.05 compared to Control. (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)

bioinks, the hMSC-laden constructs were printed [Fig. 4(D)] for long- Numerous natural hydrogel-based cell-laden bioinks have been re­
term culture to investigate the capacity to form cartilage tissue via ported in efforts to biofabricate tissues and organs in the tissue engi­
chondrogenic differentiation of hMSCs while maintaining high shape neering and regenerative medicine fields. However, it’s challenging to
fidelity. Photocrosslinked 3D bioprinted ears [Fig. 4(E)] and cuboids achieve high shape fidelity in 3D printed constructs of clinically relevant
were cultured for 4 weeks in chondrogenic differentiation media, and sizes using hydrogel bioinks due to their aforementioned mechanical
after 4 weeks, chondrogenically differentiated tissue constructs were instability [33,37]. Furthermore, the successful clinical application of
harvested [Fig. 4(F)]. The initial 3D printed structure and high shape cell-laden 3D printed hydrogel constructs depends on the ability to
fidelity were well maintained for long-term chondrogenic culture. Dif­ modulate their biochemical and physical properties to create hierar­
ferentiation down the chondrogenic lineage and resultant formation of chically complex microenvironments that can regulate encapsulated cell
cartilage tissue were confirmed via Toluidine blue O staining; intense behaviors, such as proliferation, differentiation, migration and
purple color was observed throughout the ear constructs [Fig. 4(G)] and apoptosis [38–40]. Therefore, development of bioinks that permit tun­
sectioned cuboid samples [Fig. 4(H)], while there was only slight light ing of these properties is essential to tackle current challenges of
blue color, which is non-positive staining for GAG, on the 3D printed hydrogel-based bioinks and regenerate biomimetic functional tissue
whole ear (Fig. S18) and no positive staining of the sectioned 3D printed constructs [38]. Not only does the bioink system reported here address
cuboid constructs [Fig. 4(I)] cultured in growth media. Lacunae struc­ the problems of fidelity, resolution and mechanical stability, but it is
tures were also observed in sectioned slides of chondrogenically differ­ possible to independently control the OMA biochemical (e.g., cell
entiated constructs [Fig. 4(H)], indicating maturation of cartilage adhesivity) and physical (e.g., mechanical, swelling and degradation
tissues. Successful tissue formation by the 3D printed hMSC-laden OMA profile) properties and bioactive molecule delivery capacity [1,6,
bioinks was further confirmed by quantification of cartilage tissue 41–47]. In this respect, OMA bioinks provide critical unique advantages
glycosaminoglycan (GAG) production in the cuboids [Fig. 4(J) and over the other hydrogel-based bioinks, including the ability to guide the
Fig. S19]. regeneration of tissues of clinically relevant sizes by 3D printing

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O. Jeon et al. Bioactive Materials 15 (2022) 185–193

constructs using bioinks with tissue specific cues to guide the function of org/10.1016/j.bioactmat.2021.11.025.
encapsulated cells. In addition, the OMA bioinks allow controlled
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