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Reversible physical crosslinking strategy with optimal temperature for 3D

bioprinting of human chondrocyte-laden gelatin methacryloyl bioink

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DOI: 10.1177/0885328218805864

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Biomaterials Processing

Journal of Biomaterials Applications

2018, Vol. 33(5) 609–618
Reversible physical crosslinking strategy ! The Author(s) 2018
Article reuse guidelines:
with optimal temperature for 3D sagepub.com/journals-permissions
DOI: 10.1177/0885328218805864
bioprinting of human chondrocyte-laden journals.sagepub.com/home/jba

gelatin methacryloyl bioink

Yawei Gu1, Lei Zhang1, Xiaoyu Du2, Ziwen Fan1, Long Wang1,
Weiyan Sun1, Yu Cheng3, Yufang Zhu2 and Chang Chen1

Abstract
Gelatin methacryloyl is a promising material in tissue engineering and has been widely studied in three-dimensional
bioprinting. Although gelatin methacryloyl possesses excellent biocompatibility and tunable mechanical properties, its
poor printability/processability has hindered its further applications. In this study, we report a reversible physical
crosslinking strategy for precise deposition of human chondrocyte-laden gelatin methacryloyl bioink at low concentra-
tion without any sacrificial material by using extrusive three-dimensional bioprinting. The precise printing temperature
was determined by the rheological properties of gelatin methacryloyl with temperature. Ten percent (w/v) gelatin
methacryloyl was chosen as the printing formula due to highest biocompatibility in three-dimensional cell cultures in
gelatin methacryloyl hydrogel disks. Primary human chondrocyte-laden 10% (w/v) gelatin methacryloyl was successfully
printed without any construct deformation or collapse and was permanently crosslinked by ultraviolet light. The printed
gelatin methacryloyl hydrogel constructs remained stable in long-term culture. Chondrocyte viability and proliferation
that were printed under this optimal temperature were better than that of chondrocytes printed under lower temper-
atures and were similar to that of chondrocytes in the non-printed gelatin methacryloyl hydrogels. The results indicate
that with this strategy, 10% (w/v) gelatin methacryloyl bioink presented excellent printability and printing resolution with
high cell viability, which appears to be suitable for printing primary human chondrocytes in cartilage biofabrication and
can be extensively applied in tissue engineering of other organs or in other biomedical fields.

Keywords
Gelatin methacryloyl, three-dimensional bioprinting, bioink, printability, reversible crosslinking

Introduction
In thoracic surgery, reconstruction of a long tracheal
defect has always been a challenge as it is difficult to
find a well-functioning tracheal substitute. Although
traditional inorganic tubular scaffold had excellent
mechanical properties, its biocompatibility was poor,
which could not provide the basis for the occurrence
of normal biological functions.1,2 Since the trachea is a
multi-layered organ, organogenesis indicates that both
epithelial and cartilage integrity depend on each
other.3,4 Therefore, cartilage reconstruction is as
important as the epithelial regeneration.5 In recent
years, articular cartilage regeneration has undergone
considerable progress. Abundant novel materials and
methods have been established and used. Moreover,
the development and use of three-dimensional (3D)
printing has also made great advances. The emergence
of 3D bioprinting in recent years has especially made
the biofabrication procedure more precise, and printing
of cell-laden materials is now realizable.6 Among the
1
2
Tongji University Affiliated Shanghai Pulmonary Hospital, Shanghai, China
University of Shanghai for Science and Technology, Shanghai,
Shanghai China
3
The Institute for Biomedical Engineering & Nano Science, Tongji
University School of Medicine, Shanghai, Shanghai China
Corresponding author:
Chang Chen, Tongji University Affiliated Shanghai Pulmonary Hospital,
507 Zhengmin Road, Shanghai 200433, China.
Email: chenthoracic@163.com
610
newly developed materials, gelatin methacryloyl
(GelMA) is one of the most promising scaffolding
materials due to its excellent biocompatibility and pho-
topolymerization features. Gelatin is denatured colla-
gen that retains natural cell binding motifs such as Arg-
Gly-Asp (RGD) sequences and matrix metalloprotei-
nase sensitive degradation sites.7,8 After modification
with functionalized groups of methacryloyl, GelMA
can be photopolymerized into a stable hydrogel at
physiological temperature.
As a printing bioink, GelMA should have good
printability. One of the simplest methods that can
improve printability is to increase GelMA concentra-
tion. However, high concentration will lead to highly
crosslinked networks inside the hydrogel, which will
constrain cell proliferation and migration,9,10 nutrient
absorption or byproduct exchange,11 thus negatively
influencing subsequent biological behaviors such as dif-
ferentiation and secretion.12,13 In previous studies, sac-
rificial material like hyaluronic acid14 or gellan gum15
were used to increase viscosity, but the additional
effects of these materials on the encapsulated cells
should be further investigated. Removal of sacrificial
material would also influence the hydrogel construct’s
mechanical properties. Another ultraviolet (UV) pre-
crosslinking strategy was also applied in order to
enhance the GelMA printability,16 while prolonged
UV light exposure could cause more damage to cells.
GelMA still retains the temperature-sensitive fea-
tures of unmodified gelatin when it is not photopoly-
merized. Each concentration of GelMA has a certain
gelation temperature; below this temperature, GelMA
solution starts to gel, forming a physically crosslinked
hydrogel. This crosslinking state will be greater as the
temperature further decreases until fully crosslinked
networks are formed. In contrast, this hydrogel
becomes a solution when the temperature is above
the gelation temperature. By taking advantage of this
reversible physically crosslinked feature, we demon-
strated an optimized strategy in printing chondrocyte-
laden GelMA bioink with high cell viability.
Materials and methods
GelMA synthesis
GelMA was synthesized as previously described.17,18
Briefly, gelatin powder (type A, Vetec, V900863,

300 g Bloom) was dissolved into Dulbecco’s
phosphate-buffered saline ([DPBS], Hyclone) in 10%
(w/w) at 60 C, until fully dissolved. Methacryloyl
anhydride ([MA], Macklin, M813089) was added to
the gelatin solution at 0.5 ml/min and reacted at 55 C
for 3 h. After being diluted five times with DPBS, the
mixed solution was transferred into a 12–14 kDa
Journal of Biomaterials Applications 33(5)
dialysis tube and dialyzed against deionized water for
seven days in order to remove unreacted MA and reac-
tion byproducts. The dialyzed GelMA solution was
then centrifuged at 3300 r/min for 10 min, and sedi-
ments were removed. The clarified GelMA solution
was stored at –80 C for at least 12 h (until it reached
solid state) before 48 h of lyophilization. The lyophi-
lized GelMA was stored at 4 C before use.
Hydrogel precursor preparation
Lyophilized GelMA (w/v) and 0.1% (w/v) 2-hydroxy-
1–(4-(hydroxyethoxy) phenyl)-2-methyl-1-propanone
(Irgacure 2959, Macklin) was dissolved in Dubelcco’s
modified Eagle’s medium: Nutrient Mixture F-12
(DMEM/F12, Hyclone) at 60 C for 4 h until complete
dissolution as stock solution. The stock solution was
filtered through 0.45 lm filters (Merck Millipore) for
sterilization before use. The volume of DMEM/F-12
was 90% of final volume, and the rest 10% was fetal
bovine serum (FBS, Gibco) and cells. FBS was used to
resuspend cells.
Degree of functionalization
The degree of functionalization (DoF) of GelMA was
determined by proton nuclear magnetic resonance (1H
NMR) after each synthesis as previously described.19
Briefly, GelMA was dissolved at 10 mg/ml in D2O at
50 C until fully dissolved. 1H NMR spectra was
recorded at room temperature at a frequency of
400 MHz with 32 scans and a recycle delay of 30 s by
using an NMR spectrometer (Ascend 600, Bruker,
Germany). DoF was determined by comparing the
intensity of the amine groups’ regions with the intensity
of double-bond regions.
Rheological characterization of GelMA bioink
Rheological measurements were performed with a rota-
tional rheometer (MCR302, Anton Paar, Germany)
operating in the oscillatory mode with a strain of
0.1% and a frequency of 1 Hz. In rheological tests,
G0 was defined as the storage modulus and G00 was
defined as the loss modulus. GelMA stock solutions
with different concentration and cell-laden GelMA
mixture were loaded to the test plate, and temperature
ramps of 1 Cmin1 were applied from 50 C to 0 C.
Both G0 and G00 were recorded constantly during
the tests.
Mechanical testing
To determine the effects of GelMA concentration on
the mechanical properties of UV crosslinked hydrogel,
the GelMA precursor at various concentrations was
Gu et al.
pipetted into a round mold with 10 mm diameter and 2
mm height. The GelMA precursor was then exposed to
365 nm UV light for 90 s to form stable hydrogel disks
and stored in PBS overnight in order to reach equilib-
rium swelling. Samples (n ¼ 3 in each group) were
placed on the flat horizontal testing platform and
unconfined uniaxial compression was performed on
samples with a displacement rate of 1 mm/min
(GT-TCS2000, Gotech, Dongguan, China). The com-
pression modulus was calculated as the slope of the
initial linear stage of the stress–strain curve in the
0%–10% strain range.
Cell culture
Primary human chondrocytes were isolated from
resected human bronchial tissues of patients undergo-
ing pulmonary surgery at Shanghai Pulmonary
Hospital. This study was approved by the Ethics
Committee of Shanghai Pulmonary Hospital (Ethical
Code: K17-013). Epithelium and other tissues were
carefully removed from bronchial tissues, and the
remaining cartilage rings were minced and digested
with 0.2% collagenase type II (17101015, Gibco) over-
night at 37 C. The tissue digest was filtered through a
70-lm cell strainer (Falcon) to remove tissue residue,
and the filtered cell suspension was then centrifuged for
5 min at 300g. Pelleted cells were then washed in PBS,
cultured in DMEM/F-12 with 10% FBS, 100 uni-
tsml1 penicillin and 100 lgml1 streptomycin
(Gibco) at 37 C in 5% CO2. The culture medium was
changed every two days, and chondrocytes were pas-
saged with 0.25% trypsin (Trypsin-EDTA, Gibco)
until 90% confluency in the monolayer was reached,
and passage 1 to 3 was used. The primary chondrocytes
were identified by immunofluorescent staining with col-
lagen type II. For 3D cultures, digested chondrocytes
were suspended with GelMA stock solution
(as described in Hydrogel precursor preparation) and
FBS. The chondrocyte-laden GelMA solution was
then pipetted into a square mold with side length
10 mm and height 2 mm. The chondrocyte-laden
GelMA mixture was then exposed to UV light
(k ¼ 365 nm, E ¼ 170 mW/cm–2, h ¼ 5 cm, UVATA,
portable cordless UV LED pen) for 90 s in order to
achieve fully crosslinked hydrogel disks and then
immersed in culture medium.
Bioprinting of cell-laden constructs and culture
The GelMA bioink was preheated at 37 C, and FBS
(10%, v/v) was added to the GelMA precursor before
use. The primary human chondrocytes were detached
with 0.25% trypsin and suspended in the preheated
(37 C) GelMA bioink precursor to form homogenously
611
distributed cell suspension. The cell-laden GelMA
hydrogel bioink precursor was loaded into the syringe
cartridge of a 3D bioprinter (Bioplotter, EnvisionTec,
GmbH, Gladbeck, Germany). A 27-G needle (inner
diameter 210 lm) was used as a printing nozzle.
A reversible physical crosslinking strategy was applied
in bioprinting. Cartridge and the platform temperatures
were set at temperatures ranging from 19 C to 18 C.
The cartridge was then maintained for 30 min at each
temperature after loading bioink in order to convert the
bioink into a partial gel. For the control temperature,
cartridge and platform temperatures were set at 15 C.
The printed cell-laden hydrogel constructs consisted of a
four-layer quadrate pattern of 10 mm in side length with
a strand-to-strand distance of either 600 or 800 lm and
crossing angle at 90 . All of the samples were printed at
a speed of 4.5 mms–1 with extrusive pressure between
0.8 and 1.2 bar, and the displacement between two
layers was set at 180 lm. After printing was finished,
the whole construct was exposed to UV light for 90 s
to achieve permanent fully-crosslinked hydrogel con-
structs. The 3D printed constructs were cultured at
37 C in 5% CO2, and culture medium was changed
every two days. Additionally, a hollow cylinder with
inner diameter of 10 mm was also printed to further
examine printability. During the printing procedure,
the construct was exposed to UV light for 2 min after
every two layers had finished. The printing procedure
continued until the whole construct had started
to collapse.
Cell proliferation assay and viability assay
Cell counting kit-8 (CCK-8, Dojindo) was used to
assess the proliferation of human chondrocytes both
in a 2D monolayer dish culture and in 3D constructs.
Samples (n ¼ 3 in each group) were incubated in fresh
culture medium with 10% (v/v) CCK-8 solution at
37 C for 3 h and 4.5 h for 2D and 3D cultures, respec-
tively. One-hundred microliters of the supernatant was
transferred into a 96-well plate in order to read the
optical density (OD) value at 450 nm wavelength
using a microplate reader (Varioskan Flash
Multimode Reader, Thermo Scientific). The prolifera-
tion assay was performed over a time course. A fluo-
rescent live/dead staining kit was used to determine cell
viability in the 3D cell-laden constructs according to
the manufacturer’s instructions. Briefly, samples were
gently washed in PBS three times. One micromolar
Calcein-AM (Sigma-Aldrich, 17783) and 2 lm propi-
dium iodide (PI, Sigma-Aldrich, P4170) per ML were
used to stain live cells and dead cells (green and red,
respectively) for 30 min in a cell incubator. A fluores-
cence imaging system (EVOS FL imaging system,
AMF4300, Life Technologies) was used for
612
observation and image acquisition. Cell viability was
counted by Image-J software and calculated by divid-
ing the total number of cells by green (live) stained
cells. Four random fields were counted for each sample.
Statistical analysis
Unless specifically stated, all statistical analyses were
performed using GraphPad Prism 7.0 software
(California, USA). All data were shown as mean
 standard deviation. A one-way analysis of variance
(ANOVA) was used for data with one influence factor,
and a two-way ANOVA was used for data with two
independent influence factors.
Results
DoF of GelMA
All GelMA were synthesized using the same MA con-
centration (10 M). The actual percentage of the func-
tional MA groups grafted to gelatin molecules was
verified using 1HMR after each batch of GelMA was
synthesized. The percentage of modified MA groups
was determined by comparing the integrated intensity
of all amine groups’ regions, representing the gelatin
concentrations, with the intensity of the double-bond
5.63
5.58
5.34
Hb Ha
60.0
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5
f1 (ppm)
Figure 1. 1H NMR spectra of GelMA. Inset shows the areas of interest between 5.3 and 5.6 ppm. The peaks Ha and Hb (5.34 and
5.58 ppm, respectively) show the methacryloyl groups incorporated into the gelatin via double bonds formation. 1H NMR: proton
nuclear magnetic resonance; GelMA: gelatin methacryloyl.
Journal of Biomaterials Applications 33(5)
regions, representing modified lysine groups20
(Figure 1). The DoF of GelMA varied from 85% to
94% (89.25  4.83).
Mechanical properties
To determine the concentration effects of GelMA on
mechanical properties of formed hydrogels, a uniaxial
compression was performed on samples at GelMA con-
centrations of 10%, 15% and 20% with 0.1% (w/v)
Irgacure2959. The slope of the stress–strain curve
increased with increasing GelMA concentrations. The
compressive modulus was calculated from the slope of
the initial linear part of the stress–strain curve in the
0%–10% strain range. Generally, hydrogel stiffness
increased as the concentration of GelMA increased.
The compressive modulus of 10%, 15% and 20%
GelMA was 13.85  2.00 kPa, 70.42  5.57 kPa and
186.95  14.57 kPa, respectively (Figure 2).
3D cell encapsulation at different GelMA
concentrations
To investigate the biocompatibility of different concen-
trations of GelMA hydrogel, the proliferation of chon-
drocytes was tested in patterned GelMA hydrogel
disks. Encapsulated human chondrocytes viability
700
600
500
400
300
200
100
0
1.00
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 –0.5
Gu et al. 613

(a) (b)
600
10%GelMA 250
500

Compression modulus (kPa)

15%GelMA ****
20%GelMA 200
400
Stress (kPa)

300 150

200 ****
100

100
50

0
0
–0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 10% 15% 20%
Strain GelMA concentration

Figure 2. Mechanical properties of different GelMA hydrogel concentration. Stress–strain curves (a) and compression modulus
(b) for GelMA hydrogel at 10%, 15% and 20% (w/v) concentration (****p < 0.0001).
GelMA: gelatin methacryloyl.

(a) (b) A B
10000
cell-laden GelMA G′
0.6 cell-laden GelMA G″
1000 non-cell GelMA G′
10%GelMA non-cell GelMA G″
15%GelMA 100
Storage modulus G′ (Pa)
Loss modulus G″ (Pa)

20%GelMA
0.4 10
Control
OD value

0.2 0.1

0.01

0.0 1E-3
Day 1 Day 7 Day 28 Day 56 0 10 20 30 40 50
Culture time Temperature (°C)

Figure 3. OD value of the primary human chondrocyte proliferation in non-printed GelMA hydrogels disks at concentrations of 10%
(w/v), 15% (w/v) and 20% (w/v) (a). Rheological properties of 10% (w/v) GelMA (b). Dashed lines A and B indicate the optimal printing
temperature of cell-laden and non-cell GelMA bioink (18 C and 21 C, respectively) under which G0 was highest, while G0 and G00
were the same order of magnitude.
OD: optical density; GelMA: gelatin methacryloyl.

was highest at 5% GelMA on the first day. However, chondrocytes proliferated most slowly in 20%
5% GelMA would collapse over the time it is in culture GelMA hydrogel, even after 56 days of culture, and
and partially melt after 10 days, which caused cell loss obvious proliferation could hardly be observed
during medium change. This may be caused by insuf- (Figure 3(a)). This result was in agreement with previ-
ficient crosslinked structures inside the hydrogel result- ous studies.9,21
ing from low GelMA concentration. The results
showed that cell proliferation could be observed at all Choice of optimal printing temperature
GelMA hydrogel concentrations. In 10% GelMA based on rheological properties of different
hydrogel, the optical density value of the proliferation
assay doubled on the 56th day. Human chondrocytes
GelMA concentrations
also showed good proliferation in 15% GelMA hydro- Under the best printing temperature, GelMA should be
gel but was slower than in 10% GelMA. Human viscous enough (but not too viscous) to form a
614
hydrogel cylinder when it was extruded through the
printing nozzle, because too high viscosity would gen-
erate high shear stress. To determine this printing tem-
perature, we first measured the gelation temperature of
each GelMA concentration. The gelation temperature
is defined as the intersection of G0 and G00 curves. We
continuously recorded both the G0 and G00 from 50 C
to 0 C in different GelMA concentrations (5%, 10%,
15% and 20%). At 5% GelMA, gelation could not or
barely be observed most of the time (data not shown).
The gelation temperatures of 10%, 15% and 20% con-
centration of non-cell GelMA were 23 C, 27 C and
31 C, respectively. GelMA started to gel at the gelation
temperature but was still too weak to form regular
hydrogel cylinders. The printing temperatures were
21 C–22 C, 25 C–26 C and 28 C–29 C, according to
the rheological property curves, under which G0 was
the highest when G00 was the same order of magnitude
(dashed lines, supplemental Figure S1). G00 reached a
plateau sooner upon additional cooling, while G0 sig-
nificantly increased. This significant increase in G0
caused the GelMA solution to steadily become a gel,
which produced a GelMA solution viscosity that was
too high and caused twisted filaments and more fre-
quent clogging during printing. This highly viscous
GelMA bioink state was not suitable for printing.
According to the biocompatibility test of various
GelMA concentrations, cell-laden 10% (w/v) GelMA
bioink was further tested, and a temperature range
between 18 C and 19 C was determined as the printing
temperature (Figure 3(b)). Cell addition influenced the
GelMA bioink’s rheological properties to some
extent,22,23 making the temperature shift to the right,
which was 3 C lower than the non-cell 10% (w/v)
GelMA bioink (Figure 3(b)).
30
28
Temperature (°C)
26
24
(a)
22
20
18
16
10*
GelMA concentration (%, w/v)
Figure 4. Phase diagram of printability at various GelMA concentration under temperatures from 16 C to 31 C. The asterisk (*)
indicates cell-laden GelMA bioink. Black dots in yellow boxes indicate optimal printing state, square frames in brown boxed indicate
unprintable and triangle frames in grey boxes indicate impaired printability. Dashed boxes show the crosslinking status of GelMA
bioink: partially crosslinked (a), non-crosslinked (b) and heavily crosslinked (c).
GelMA: gelatin methacryloyl.
Journal of Biomaterials Applications 33(5)
3D bioprinting human chondrocyte-laden
GelMA bioink
Based on biocompatibility and rheological results, 10%
GelMA was chosen as a bioink formula. For each tem-
perature, the bioink was held in the cartridge in order
to achieve a temperature-balanced state. Both cartridge
and platform temperatures were first set at 19 C since
19 C was the upper boundary of the theoretical print-
ing temperature range and was closer to physiological
temperature. At 19 C, the GelMA bioink could be
extruded in form of irregular filaments with some
drips, but hardly form continuous threads through
the nozzle. The cartridge and platform temperature
were then lowered until they reached 18 C (the lower
boundary of theoretical printing temperature range),
and GelMA bioink was extruded in the form of a con-
fluent filament. Printed constructs maintained fidelity
without undergoing any severe fluctuations. Otherwise,
when the temperature <18 C, the viscosity of the
GelMA bioink increased, resulting in filaments twist-
ing, discontinuity of bioink or nozzle block (such as at
10 C) (Figure 4).
Although the theoretical printing temperature was
between 18 C and 19 C, in the experiments, it was
observed that the best printability occurred only
when the temperature was set at 18 C. Even when the
temperature increased slightly (to 19 C), the GelMA
bioink could barely form regular filaments, so that
the printability was relatively poor. Therefore, the
printing temperature was restricted. Either the incre-
ment or reduction of temperature was responsible for
impairment of the GelMA bioink printability. Finally,
the four-layer chondrocyte-laden GelMA hydrogel
constructs were successfully printed without any col-
lapse or deformation. In the exploratory printing,
(b)
(c)
10 15 20
Gu et al.
a 15-layer hollow cylinder was also successfully printed
(Figure 5).
Cell viability and proliferation
The viability of human chondrocytes after printing
under 18 C reached 96.13%  1.72% on day 1, which
was similar to the cell viability in non-printing GelMA
hydrogel disks (97.07%  1.11%). Cell viability in both
printed constructs (18 C) and non-printed disks was
always maintained at >90% within 28 days despite sta-
tistical significance between these two groups through-
out the culture time (Day 7, p < 0.01; Day 28, p < 0.05).
Cell viability in the constructs on Day 1 printed
under 15 C was at the level of 87.67%  2.48% and
continuously decreased to 81.63%  1.55% by Day
28 (Figure 6). Human chondrocyte viability in all
three groups showed a decline after seven days.
This might be due to damage caused by UV light
exposure and photoinitiator-induced toxicity.24 For
printing groups, shear stress generated from extrusive
printing, lower temperatures and holding time would
bring more cell injuries compared to non-printing
group.25,26 In optimal printing (18 C) and non-
printing groups, human chondrocytes proliferated
during the subsequent culture period, and cell viability
presented an incremental increase. However, chondro-
cytes could hardly recover when printed under a lower
temperature (15 C) and thus presented a sustained
decline in cell viability. Live/dead chondrocyte staining
in three groups on days 1, 7 and 28 is presented in
Figure 7, and red arrows indicate dead cells. Dead
cells were obviously more in the 15 C-printed group
than in the other two groups. The results indicated
that additional lowering of the temperature and
higher shear stress caused more injuries, leading to sus-
tained chondrocyte apoptosis.
Figure 5. Gross view of a printed quadrate construct (a), and the scale bar is 4 mm. Microscopic view of printed quadrate construct
(b), and the scale bar is 400 lm. Gross view of a 15-layer hollow cylinder (c), and the scale bar is 2 mm.
615
Discussion
GelMA is a versatile biomaterial that is widely used for
various biomedical applications because of its excellent
biological properties and tunable mechanical charac-
teristics.18 In recent years, GelMA has gained special
attention in 3D cultures and regenerative medicine.21
As an ideal nature-derived culture medium, GelMA
hydrogels present good performance with respect to
cell compatibility and for allowing cell adhesion, prolif-
eration and migration. The tunable mechanical proper-
ties based on concentration and crosslinking density also
offer more possibilities in simulation of physical features
of different tissues.27 Compared to other hydrogels,
these advantages make GelMA hydrogel an optimal
alternative scaffold material for biofabrication.
However, technology such as extrusive 3D bioprint-
ing has requirements for printed materials as printabil-
ity is the basic property in this advanced fabrication
technique.28 Only with processability, complicated
tissue structures with high resolution could be built.
Nevertheless, the superior biocompatibility of GelMA
could only be present in low concentrations (not higher
than 10% w/v),9 which was also verified in this study.
**** ****
**** **** ****
**** ** *
100
15°C printing
Viability of chondrocytes
18°C printing
non-printing
50
0
Day1 Day7 Day28
Figure 6. Viability of primary human chondrocytes in printed
and non-printed GelMA hydrogel constructs. (*p < 0.05,
**p < 0.01, ****p < 0.0001).
GelMA: gelatin methacryloyl.
616
Day 1
Figure 7. Calcein-AM (green)/propidium iodide (red) staining of primary human chondrocytes in GelMA hydrogel constructs printed
at 15 (a, b, c), at 18 C (d, e, f) and non-printed GelMA hydrogel disks (g, h, i). Red arrows indicate dead cells. The scale bar is 500 lm.
GelMA: gelatin methacryloyl.
In 3D cultures with GelMA hydrogel, primary chon-
drocytes proliferated best in 10% (w/v) GelMA hydro-
gel; however, as the concentration increased, the
proliferation rate decreased. This was because high
GelMA concentrations could form denser crosslinked
networks inside the hydrogel, which constrained encap-
sulated cells’ growth and migration. Stiffness also
increased with increment in GelMA concentration. It
has been found that cells tend to exhibit more natural
biological behaviors when cultured in a microenviron-
ment that is similar to in-vivo conditions.29,30 Not only
the concentration of GelMA but also the concentration
of photoinitiator and UV exposure time influence the
stiffness and density of the crosslinked networks inside
hydrogel.24 Based on this fact, only GelMA at lower
concentrations could be more ideal in biofabrication,
but it also presented a dilemma because GelMA at low
concentration could not be handled in printing under
physiological temperature, while higher concentrations
would impair GelMA’s advantages in biocompatibility.
Therefore, various strategies were explored to improve
Journal of Biomaterials Applications 33(5)
Day 7 Day 28
GelMA bioink printability. Sacrificial materials like
gelatin,31 gellan gum15 and sodium alginate32 were
added to GelMA, making the mixture temporarily vis-
cous enough to be extruded. Although this strategy
allowed the possibility for printing with low GelMA’s
concentration, additional effects from sacrificial mate-
rials could not be ignored. On one hand, these sacrifi-
cial materials should be non-toxic to encapsulated cells,
but on the other hand, they are supposed to degrade
or be carefully removed under controlled condi-
tions.11,33,34 However, the removal of sacrificial materi-
als might influence constructs’ physical features,
including, for example, the mechanical strength, micro-
pore size and other factors. When taking these factors
into consideration, the fabricated structure design and
bioink formation should be adjusted to fit this modifi-
cation. Another pre-polymerized strategy reported by
Bertassoni et al.16 also made GelMA bioink printable
at low GelMA concentrations. Nevertheless, this
strategy demanded a glass capillary nozzle or other
non-opaque nozzles in order to permit the UV light to
Gu et al.
pass through it. The UV light exposure was supposed to
be homogeneously distributed, so that the GelMA pre-
cursor could form a homogeneous hydrogel. Moreover,
whether two-step UV light exposure would generate
more cell injuries still needs further investigation.
This study demonstrated a reversible physical cross-
linking strategy for printing pure GelMA bioink. In this
approach, neither sacrificial materials nor special equip-
ments were required, which is adapted to common extru-
sive bioprinters. Another advantage of this strategy is
that the first step of physical crosslinking is reversible,
which will not have much effect on the crosslinked net-
work density or the mechanical properties when it
increases to physiological temperature. The printing tem-
perature was determined by the GelMA bioink’s rheolog-
ical properties. By investigating storage modulus and loss
modulus under various temperature, the printing temper-
ature could be restricted to a small range, which mini-
mized the temperature effects. It is notable that the
printing temperature was not the gel formation point
(intersection of the G00 and G0 curves) but consisted of
a temperature that could be determined by specific data
(see Methods and materials). Under this temperature,
both the G0 and G00 were of the same order of magnitude.
Theoretically, the printing temperature should be a
range, but in our study, we used the lower boundaries
of the printing temperature range (18 C for cell-laden
GelMA bioink) as higher temperatures did not guarantee
continuous printing and the printed GelMA bioink
tended to transfer into solution in a very short period
of time. This optimal temperature guarantees printability
with lowest shear stress during the printing procedure. In
addition, high cell viability proved that cell injuries
caused by this temperature was subtle. We also tried
printing GelMA bioink under the intersection of the G00
and G0 curves (data not shown) as in some studies, this
temperature was suggested to be proper printing temper-
ature. However, GelMA bioink could not form a viscous-
enough hydrogel to be printed under these conditions.
This was because the GelMA precursor started to gel at
this temperature, but the crosslinked network was not
dense enough to form filament when passing through
the nozzle. Additionally, we also successfully printed
GelMA bioink under lower temperature (15 C) and the
printed constructs had good fidelity as well. However,
lower temperature would further increase the bioink’s
viscosity, thus generating higher shear stress that could
cause cell injuries.25 What’s more, lower temperatures
also produced more cold injuries to cells,26,35 with
longer holding time, and caused irreversible cell
damage. Cooling would make the viscosity of the
GelMA hydrogel too high, which would lead to twisted
filaments, discontinuity of extrusion or frequent clogging.
In our study, we used primary chondrocytes as the
seed cells. Beside chondrocytes, mesenchymal stem cells
617
(MSCs) and cartilage-derived progenitor cells (CDPCs)
were also used in cartilage regeneration and were
proven to also have compatibility with GelMA hydro-
gel.36–38 Despite mature application and stability, pri-
mary chondrocytes presented little pluripotency and
poorer secretion function compared to MSCs or
CDPCs. As the strategy in this study improved cell
viability of chondrocytes, it could be extensively used
in printing MSCs or CDPCs, which needs to be fur-
ther verified.
Conclusion
In summary, this study presents a reversible physical
crosslinking strategy for extrusive bioprinting of cell-
laden low-concentration GelMA bioink. The results
emphasized precise printing temperature based on the
rheological properties of GelMA bioink. Under opti-
mal printing temperature, the printability of pure
GelMA bioink had obviously improved, and primary
human chondrocytes could be printed with less cell
injury and high cell viability retention after long-term
culture. Furthermore, the results demonstrated that
with this strategy a 15-layered hollow cylinder con-
struct could be successfully printed.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of
this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This study was supported by the National Natural Science
Foundation of China (NSFC, Nos. 81770091, 81570014).
Supplemental material
Supplemental material for this article is available online.
ORCID iD
Chang Chen http://orcid.org/0000-0002-9981-3110
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