Composites Science and Technology 208 (2021) 108763

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Composites Science and Technology

journal homepage: http://www.elsevier.com/locate/compscitech

3D printed gellan gum/graphene oxide scaffold for tumor therapy and

bone reconstruction
Shanshan Zhu, Lingyun Yao, Cile Pan, Jinhuan Tian, Lihua Li, Binghong Luo, Changren Zhou,
Lu Lu *
Department of Material Science and Engineering, College of Chemistry and Materials, Jinan University, Guangzhou, 510632, China

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

Keywords: Compared with traditional tissue engineering scaffold processing methods, 3D printing technology has obvious
3D printing advantages in achieving high structural complexity, flexibility and specific patient needs. Printing ink materials
Gellan gum with both high printability and biocompatibility remains a major challenge. Herein, a hydrogel bio-ink with
Graphene oxide
excellent thixotropy and recovery properties was prepared by combining gellan gum (GG) and graphene oxide
Curcumin
Bone tumor therapy
(GO) for extrusion 3D printing. The GG/GO scaffold constructed by 3D printing has a regular and connected
porous structure, which maintains high fidelity with the model. Cell proliferation and attachment in the printed
3D curcumin-loaded scaffold revealed that the bifunctional GG/GO/Cur scaffold could inhibit human osteo­
sarcoma cell line (MG-63) growth significantly and induce tumor cell death effectively in vitro. Simultaneously,
the scaffold could support the attachment and proliferation of mouse osteoblast cell (MC3T3). Therefore, the
functional GG/GO/Cur scaffold is expected to be used to repair the bone defects induced by surgery and kill the
possibly residual tumor cells at the same time to achieve the purpose of tumor therapy and bone reconstruction.

1. Introduction applied to fabricate poly(vinyl alcohol)/β-tricalcium phosphate com­

Bone cancer and its subtypes are considered to be the most common
primary bone malignancies [1]. Current treatment criteria for bone
cancer include preoperative or postoperative chemotherapy and sur­
gery. However, inhibiting tumor regeneration and repairing bone de­
fects in the meantime after surgery is a major challenge [2,3]. In bone
tissue engineering, biomaterial scaffolds can provide the better physical
support and biocompatible environment for tissue regeneration in vivo,
so they have potential application in repairing postoperative bone de­
fects [2,4,5]. Traditional scaffold manufacturing methods, such as gas
foaming [6], freezing-drying [7], fiber adhesion, particle/salt leaching
[8], emulsification [9], phase separation/transformation, etc., cannot
control the precise structure of the scaffold. Therefore, it is difficult to
accurately mimic the extracellular microenvironment and meet the re­
quirements of personalized customization [10,11]. 3D printing tech­
nology provides an ideal solution for manufacturing complex fine
structures and individual customization.
3D printing technology has been widely used in the biomedical field
due to its fast, accurate and controllable manufacturing process [12,13].
For instance, the fused deposition modeling has been successfully
posite scaffold [14] and polycaprolactone/hydroxyapatite composite
scaffold [15] for bone tissue engineering. Although some progress has
been made in 3D printing inks, the development of 3D bio-inks with
appropriate mechanical properties, biocompatibility and excellent pro­
cessing properties continues to be a pressing issue [16,17].
Hydrogels are the best choice for 3D bioprinting ink. They have the
structure similar to natural extracellular matrix, which makes it easy to
load living cells and create 3D tissue models in vitro [18,19]. In addition,
hydrogels provide convenient means for regulating the mechanical
strength and biochemical signals of the cells’ microenvironment [20].
Relevant studies have shown that the mechanical properties of hydrogel
scaffolds can be regulated by varying the crosslinking density, thereby
affecting the proliferation, survival and migration of the embedded cells,
and inducing the differentiation of loaded stem cells into specific line­
ages [21–23]. Furthermore, hydrogels can be chemically modified and
drug-loaded to present cell attachment sites and functionalization,
which are critical to tumor cell death, endothelial migration and normal
cell growth [23–26].
Gellan gum (GG) is an anionic microbial polysaccharide composed of
a tetra-saccharide repeating unit of L-rhamnose, D-glucuronic acid and

* Corresponding author.
E-mail address: tlulu@jnu.edu.cn (L. Lu).

https://doi.org/10.1016/j.compscitech.2021.108763
Received 8 December 2020; Received in revised form 10 March 2021; Accepted 11 March 2021
Available online 16 March 2021
0266-3538/© 2021 Elsevier Ltd. All rights reserved.
S. Zhu et al. Composites Science and Technology 208 (2021) 108763

Table 1
Overview of evaluated bio-inks with different GG/GO concentrations (Green parts means the com­
ponents that suitable for printing).

GO
0wt% 0.3wt% 0.6wt% 0.9wt% 1.2wt%
GG
1.2wt% GG1.2GO0 GG1.2GO0.3 GG1.2GO0.6 GG1.2GO0.9 GG1.2GO1.2

1.5wt% GG1.5GO0 GG1.5GO0.3 GG1.5GO0.6 GG1.5GO0.9 GG1.5GO1.2

1.8wt% GG1.8GO0 GG1.8GO0.3 GG1.8GO0.6 GG1.8GO0.9 GG1.8 1.2

two D-glucose subunits. As a bio-ink, it has many advantages over other
hydrogels including shear thinning behavior, high gelling efficiency at
physiological temperature and reasonable production cost [27,28].
However, the brittleness of GG hydrogel limit its application in stress
environment [29]. In this study, we introduced graphene oxide (GO)
into GG hydrogel to improve the mechanical properties of the system,
and developed a high-fidelity GG/GO bio-ink for extrusion 3D printing.
GO has good mechanical properties and many oxygen-containing func­
tional groups, which is conducive to its dispersion and modification
[30]. Nanohybrid films with the structural features of natural nacre
based on the self-assembly of GG-GO has been reported, and proved that
there are combined interactions, including coordination bonding, ionic
bonding, and hydrogen bonding among the constituents [31]. Subse­
quently, curcumin was loaded on the 3D printed GG/GO scaffold to
prepare the functional bone repair scaffold (GG/GO/Cur) for tumor
therapy and bone reconstruction.
min in an ice-water bath. After the mixture had been stirred for 20 min at
35 ◦ C, deionized water (300 mL) was slowly added and heated to 80 ◦ C
for 15 min H2O2 (100 mL of 30 wt % aqueous solution) and deionized
water (1000 mL) were then added, and the mixture was stirred for 2 h at
room temperature. After standing for 12 h, discarding the supernatant,
the resulting mixture was washed repeatedly with hydrochloric acid
solution (1 mol/L) and centrifuged for 3–4 times, and then fully cleaned
with deionized water. Finally, the mixture was dialyzed against deion­
ized water for a week until the pH was neutral. After freeze drying, GO
was obtained.
GG (Bio-Reagent, Sigma-Aldrich) was dissolved in deionized water at
80 ◦ C. Graphene oxide (GO) was dispersed in deionized water by
ultrasonication for 0.5 h to acquire GO dispersion. Bio-inks were pre­
pared by mixing GG and GO aqueous solutions with different concen­
trations (Table 1) at 37 ◦ C.

2. Materials and methods 2.2. Rheological characterization

2.1. Bio-inks preparation
GO was prepared from high purity flake graphite (300 meshes,
99.99% purity, Nanjing Xianfeng Nano Materials Technology Co., Ltd,
China) according to the modified Hummers’ method as previously re­
ported [32,33]. Briefly, flake graphite (6 g) and H2SO4 (120 mL) were
mixed in a flask at 0 ◦ C. Then KMnO4 (15 g) was slowly added over 15
Rheological tests were performed on an AR-G2 rheometer (TA In­
struments, USA) equipped with a 1.03◦ aluminum cone plate geometry
(40 mm diameter) at 37 ◦ C. Shear-thinning and recovery properties of
the bio-inks were investigated in both dynamic and static modes.
Oscillatory strain sweep test and time sweep test conducted with alter­
nating low/high strains of 200/0.1% every 120 s were performed at a
fixed frequency (1 Hz). The static shear sweep test with a linearly
ramped shear rate from 1 × 10− 3 to 1 × 103 s− 1 and the shear recovery

Fig. 1. Scheme showing the fabrication process of GG/GO and GG/GO/Cur scaffolds.

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S. Zhu et al.
Fig. 2. Rheological properties of the GG/GO bio-inks. The shear rate sweep test (a) and oscillation strain sweep test (b) of sample GG1.5GO1.2. Three step shear-
thinning and recovery characterization under the static (c) and oscillation (d) mode. In (b) and (d), the solid points represent storage modulus (G′ ) and the hol­
low points represent loss modulus (G′′ ).
test including low (0.01 s− 1)-high (300 s− 1)-low (0.01 s− 1) shear rate 3
steps switching test were measured with duration 120 s, 30 s and 120 s,
respectively.
2.3. Printability experiments
Based on extrusion bioprinting, an integrated 3D printing system
developed by our lab controlled with Repetier software (Repetier-Host
1.6.2, Germany) was used for all printing tests. The printability exper­
iments were proceeded using a 22-gauge needle as the nozzle under the
same printing conditions as follows: nozzle temperature 37 ◦ C, substrate
kept at 25 ◦ C, bioprinting speed 10 mm s− 1.
The shape of the ink at the printing nozzle was observed and the
filament length from the nozzle tip to the separating location was
measured. Furthermore, three typical structures, including the lattice
matrix, pentagram and gradient spacing structure were designed using
creo™ elements/pro™ 5.0 software (PTC, USA) for printability evalu­
ation. After printing, the morphological features of these structures were
investigated and imaged with a stereomicroscope (Discovery.V20, Zeiss,
Germany).
2.4. Fabrication of GG/GO and GG/GO/Cur scaffolds
According to printability experiments, the selected GG/GO ink was
used to print 3D scaffolds with rectilinear and honeycomb infill patterns.
The as-printed scaffolds were then soaked in CaCl2 solution (8 × 10− 3
mol/L) for 10 min. The GG/GO scaffolds were obtained after frozen in
liquid nitrogen and subsequently freeze-dried for 24 h.
The curcumin (98.0% pure, Sigma-Aldrich) powder was dissolved in
75% alcohol solution and prepared into 1 mg/mL curcumin solution.
Subsequently, the GG/GO scaffolds were soaked in curcumin solution
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Composites Science and Technology 208 (2021) 108763
for 2 h at 75 ◦ C to prepare the GG/GO/Cur scaffolds. Fig. 1 shows a
schematic of the scaffold preparation process, including in vitro cell
culture.
2.5. Characterization of scaffolds
Scanning electron microscope (SEM) images were taken on a mi­
croscope (SEM, XL30 FESEM FEG, PHILLPS) at an accelerating voltage
of 20 kV. Before imaging, the samples were sputtered with gold. Cross-
sectional SEM image was observed through the fracture of scaffolds in
liquid nitrogen.
The compression test was conducted on cylinder samples (12 mm in
diameter and 10 mm in initial height) at a rate of 2 mm/min by using a
mechanical testing machine (AG-I, SHIMADZU, Japan) equipped with a
200 N load cell.
The chemical structure of scaffolds was analyzed using Fourier
transform infrared spectra (FTIR, Bruker EQUINOX 55 FTIR, Germany)
with a scanning range from 4000 to 500 cm− 1.
2.6. Curcumin loading and release from the GG/GO scaffolds
Curcumin release from GG/GO/Cur scaffolds was measured at a
wavelength of 428 nm by using an ultraviolet spectrophotometry de­
tector (Shimadzu, Japan). Phosphate buffered saline (PBS, pH 7.4) was
prepared with 30% V/V alcohol and then was used to prepare 5, 10, 15,
20 and 25 μg/mL curcumin standard solution to obtain the standard
curve. The relationship between concentration (C, mg/mL) and absor­
bance (Abs, a.u.) can be described by eqs. (2–1):
C = 10.8601 × Abs − 0.0016 (2–1)
GG/GO scaffolds were immersed in 1 mg/mL curcumin solution
S. Zhu et al.
(with 75% V/V alcohol) at 75 ◦ C for 10–360 min, and samples were
taken out every 10 min. The loading efficiency of curcumin can be
calculated via eqs. (2–2):
(Ws − Wt ) × 1000
∅=
W0
where φ (μg/mg) is the mass ratio of loaded curcumin on GG/GO, Ws is
the initial amount of curcumin, Wt is the total curcumin amount in the
supernatant after loading, and W0 is the amount of GG/GO used for
binding.
Curcumin release from GG/GO/Cur scaffolds with rectilinear and
honeycomb infill patterns divided into two equal aliquots was measured.
The scaffolds were immersed in 5 mL PBS (containing 30% ethanol) with
pH 7.4 and pH 5.4 respectively, and incubated in a shaking incubator at
37 ◦ C. The cumulative release efficiency of curcumin could be obtained
from eqs. (2-3):
CS × V
Ψ= × 100%
WL
where Ψ is the cumulative percentage of drug release, Cs is the calcu­
lated curcumin concentration in the buffer solution, V is the remaining
volume of the liquid in the whole system, and WL is the initial amount of
curcumin on GG/GO calculated after the loading process.
2.7. In vitro osteoblast and osteosarcoma cell culture study
Mouse osteoblast cell (MC3T3) cultured in α-minimum essential
medium (α-MEM, Gibco) and human osteosarcoma cell line (MG-63)
cultured in high-glucose Dulbecco’s Minimal Eagle Medium (DMEM,
Gibco) were purchased from American Tissue Culture Collection (ATCC,
USA). The two mediums were supplemented with 10% fetal bovine
serum (FBS, Gibco) and 1% penicillin/streptomycin (P/S, Gibco).
Briefly, sterilized GG/GO and GG/GO/Cur scaffolds were placed in 24
well plates. Cells were seeded on the scaffold surface at a density of 1 ×
104 cells/sample and then cultured in an incubator at 37 ◦ C under an
atmosphere of 5% CO2.
The viability of cells seeded on scaffolds was evaluated by cell
counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) and
Fig. 3. Printability of GG/GO bio-inks. Optical images of printed lattice matrix structures (a), pentagram structures (b) and gradient spacing structures (c). The
printing fidelity for the GG/GO bio-inks (d, e, f).
Composites Science and Technology 208 (2021) 108763
Live/Dead cell assay kit (Life Technologies) according to the manufac­
turer’s instructions.
2.8. Statistics
(2-2)
All the in vitro experiments data results were shown as the means ±
standard deviation (SD) for n = 5. Statistical analyses were performed
using SPSS. One-way ANOVA and Bonferroni post-tests were used for
statistical analyses. p values ≤ 0.05 were considered as statistically
significant.
3. Results
3.1. Rheological behavior of GG/GO bio-inks
The rheological behavior of bio-inks significantly affects the 3D
printing process and its products. For extrusion-based printing, the bio-
ink should exhibit a relatively low viscosity under the high shear stress
(2–3)
to be extruded, and be able to recover quickly after extrusion, enabling a
self-supporting structure for printing. In addition, the bio-ink must be
stable and homogeneous to prevent clogging in the print nozzle [34].
Fig. 2 displays the rheological properties of the GG/GO bio-inks. The
viscosity of the bio-ink decreased with the increasing shear rate,
showing typical pseudoplastic fluid properties (Fig. 2a). As the shear
rate increased, the disentanglement rate between GG macromolecules
increased, and the physical cross-linking points through non-covalent
interaction between GG and GO sheet were destroyed under the action
of shear stress, therefore the viscosity of the system gradually decreased.
During the dynamic strain sweep test (Fig. 2 b), the bio-ink showed a
gel-sol transition. Under 10− 2-101% small strain, the bio-ink exhibited
the solid gel like behavior (G′ > G′′ ). As the strain increased above 20%,
the sample showed liquid like behavior (G′′ > G′ ). This transformation
indicates that the structure of the system has changed under stress. GG
macromolecules have a typical double helix structure and associate with
GO through dynamic interactions, such as van der Waals forces and
hydrogen bonds. Due to the reversibility of dynamic bonding structure,
GG/GO hydrogel exhibited shear-thinning and gel-sol transition
behavior. This property ensures that the bio-ink can be extruded from
print nozzle easily.
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S. Zhu et al.
Fig. 4. The morphology of lyophilized 3D printed GG/GO hydrogel scaffolds. (a–e) Optical images of the scaffolds. SEM images of the surface (f–o) and cross section
(p–t) of the scaffolds. GG1.5GO1.2-H represents the honeycomb scaffold printed by sample GG1.5GO1.2.
In order to simulate the forming state of bio-ink immediately after
extrusion, static shear recovery and oscillation time sweep tests were
performed. The shear rate during printing can be calculated by the flow
rate and needle diameter [35]. In our printing process, the shear rate
was about 320 s− 1 and the shear rate of 300 s− 1 was set in the step
shear-thinning test. As shown in Fig. 2(c) and (d), the gel-sol transition
under high shear rate or large strain and the reassembly of the network
after removing the shear rate or strain applied to the hydrogels were
almost instantaneous. The concentration of GG and GO directly affected
the viscosity of the bio-ink, and GO was the main influencing factor. The
introduction of GO provides physical cross-linking points between GG
macromolecular chains. With the increase of GO content, the viscosity
and storage modulus of bio-inks increased significantly. Under the low
shear rate, the viscosity of sample GG1.5GO1.2 reached 105 Pa S and
could recover 85% of the original viscosity within 5 s after shear thin­
ning. The lower the GO content in the sample, the longer the recovery
time. The recovery time of the sample GG1.5GO0 without GO was 20 s,
and the viscosity was only around 103 Pa S, which cannot maintain the
line shape well after extrusion. Therefore, it is not suitable for extruded
3D printing to form a stable self-supporting structure. The addition of
GO increased the mechanical strength and recovery ability after
shearing of the hydrogel. All samples could recover more than 90% of
the original storage modulus within 5 s after the stress was released
(Fig. 2d). Once the GG/GO hydrogel is extruded, it quickly recovers its
original high storage modulus, so it can form self-supporting structure in
the z-axis direction without being affected by gravity and maintaining a
stable structure. These results indicate that GG/GO bio-ink is printable
and can be used for extrusion 3D printing potentially.
3.2. Printability characterization
Filament extrusion test can evaluate the printability of bio-ink easily
and quickly [28]. As shown in Fig. S1 (a), due to the relatively low
viscosity, the samples GG1.5GO0, GG1.5GO0.3, GG1.5GO0.6 and
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Composites Science and Technology 208 (2021) 108763
GG1.2GO0.9 could not form uniform filaments but in the shape of droplets
after extrusion. The viscosity of the sample is higher, the filament will be
longer, more uniform with a gel-like status after extrusion. The average
filament length of sample GG1.2GO1.2, GG1.5GO0.9, GG1.5GO1.2, and
GG1.8GO0.6 reached 32.7, 64.4, 82.2 and 67.6 mm (Fig. S1b), respec­
tively. According to our previous experiments on hydrogel printability,
the appropriate average filament length should be greater than 20 μm
[28]. Therefore, sample GG1.2GO1.2, GG1.5GO0.9, GG1.5GO1.2 and
GG1.8GO0.6 would be further tested for subsequent printing performance
evaluation.
Three typical 3D models (lattice matrix, pentagram and gradient
spacing structure) were designed and selected to further evaluate the
printing fidelity of the bio-ink samples. The models of these three
structures and the actual printed schematic diagrams are illustrated in
Fig. S2 and the experimental results are shown in Fig. 3. For the lattice
matrix structure, all samples could keep the lattice matrix structure well.
However, due to the time dependence of the physical cross-linking point
and the influence of gravity, the stacked filaments of sample GG1.2GO1.2
and GG1.8GO0.6 fused together and spread around, so the shape of the
meshes changed from a square to a circle and unable to maintain self-
supporting property in the Z-axis direction. The sample GG1.5GO1.2
has suitable viscosity, stable shear thinning and recovery behavior. The
extruded fiber has self-supporting ability in the Z-axis direction, thus
forming a complete and clear lattice matrix structure, with the optimal
Fa reaching 94.52%. Compared to single straight-line or circle structure,
it is always difficult to maintain the set angle and shape when suddenly
changing printing direction. To test the angle fidelity of bio-inks, we
selected pentagram as print model and measured sharp and blunt angles
with Image-J (Fig. S2 (c) and (d)), and calculated the deviation rates of
sharp and blunt angles. Among them, the sample GG1.5GO1.2 performed
best with a sharp angle deviation of 22.1% and a blunt angle deviation of
6.9%. In the gradient spacing structure, the straight-line were printed
back and forth by changing printing direction 180◦ and decreasing the
distance between the filaments. The worse the self-supporting
S. Zhu et al. Composites Science and Technology 208 (2021) 108763

Fig. 5. Mechanical properties of lyophilized 3D printed GG/GO hydrogel scaffolds. (a) Compressive stress-strain curves and (b) Young’s modulus in the linear stress-
strain range from 0 to 10%. GG1.5GO1.2-H represents the honeycomb scaffold printed by sample GG1.5GO1.2.

performance of the sample, the easier the filament fusion occurs, and the
lower the printing accuracy and fidelity that can be achieved. The
spacing fidelity of sample GG1.5GO1.2 reached 89.6% with the printable
minimum accuracy spacing of 0.25 mm. These experiments evaluated
the ability of bio-inks to print high-fidelity and complex 3D structure
from three aspects: area, angle and gradient pitch. The results demon­
strated that sample GG1.5GO1.2 had the best printability.
3.3. Characterization of the 3D printed scaffold
During the printing process, GG/GO bio-ink was extruded from the
syringe, and then the lattice matrix and honeycomb scaffolds were
formed. In order to prevent the scaffolds from shrinking under capillary
pressure when they were dried in air, we soaked them in a CaCl2 solution
to promote the formation of a double helix structure of GG, then freeze-
dried to obtain porous scaffolds [36–38]. As shown in Fig. 4(a–e), all the
printed scaffolds maintained structural integrity and accuracy. Both the
printed lattice matrix and honeycomb structure showed interconnected
porous structure with pore sizes ranging from a few millimeters to
hundreds of microns (Fig. 4(f–t)). The mesh shape of all the lattice
matrix scaffolds was consistent with the printability experiment results
before freeze-dried. The width of the extruded filaments was larger than
the inner diameter of the needle (0.22 mm), which was mainly due to the
expansion effect of the macromolecules and the deformation of the ink
after extrusion. For sample GG1.2GO1.2 and GG1.8GO0.6, at the inter­
section of the two filaments, the stacked structure in the Z-axis direction
could not be clearly distinguished. Sample GG1.5GO1.2 had the highest
fidelity structure and clear lines. The mesh hole size was uniform, about
500 μm. The cells loaded on the scaffold can migrate from the surface to
the interior of the scaffold and grow longitudinally during the cell cul­
ture process [39]. Furthermore, the cross-section image of the sample
GG1.5GO1.2 showed that the lattice matrix had a more regular and

Fig. 6. (a) The ability of curcumin loaded on GG/GO scaffolds. (b and c) Curcumin released from the GG/GO/Cur scaffolds in phosphate buffer at pH = 7.4 and pH =
5.4 during the (b) first 6 h and (c) whole process. (d) Curcumin released from the GG/GO/Cur-H scaffolds in phosphate buffer at pH = 5.4.

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Fig. 7. Viability of osteoblast cells cultured on the 3D printed scaffolds (R stands for the scaffold with linear lattice filling pattern, H stands for the scaffold with
honeycomb filling pattern). (a) CCK-8 osteoblast cell viability assay (n = 3) after 1, 3 and 5 days of culture (*p < 0.05), (b) Results of live/dead fluorescent stained
osteoblast cells cultured on the scaffolds after 1, 3 and 5days. Viable cells were fluorescent green, while non-viable cells were fluorescent red.

obvious interconnected structure between the layers compared with the
honeycomb structure, which indicated that the specific surface area of
the lattice matrix scaffold was higher and it had the potentially advan­
tages in drug delivery systems. SEM observation results also proved that
GO was uniformly dispersed in GG, and there was no obvious GO
agglomeration in the scaffolds.
Compression tests were conducted to measure the stress-strain
curves of GG/GO scaffolds. Fig. 5 shows the compressive stress-strain
curves and the compressive modulus in the linear stress-strain range
from 0 to 10% of the 3D printed GG/GO scaffolds. Compression tests
revealed that the compression modulus of the GG/GO scaffolds was
greater than 0.15 kPa, and the Young’s modulus reached more than 10
kPa. Although the mechanical strength of the GG/GO hydrogel is not
comparable to that of load-bearing bone, it can meet the requirements of
filling and supporting the damaged tissue [40]. Compared with
high-strength inorganic and polyester materials [41,42], GG/GO
hydrogel has better 3D bioprinting performance and cell compatibility
for its viscoelasticity similar to natural extracellular matrix. The addi­
tion of GO significantly improved the mechanical properties of the
system. The compressive stress and Young’s modulus became higher as
the content of GO increased. In addition, the infill pattern of the scaffold
is also an important factor affecting its mechanical properties. Sample
GG1.5GO1.2-H (honeycomb infill pattern) had a higher Young’s modulus,
but the compressive stress of under 60% strain was much lower than that
of sample GG1.5GO1.2 (rectilinear infill pattern). Under the same filling
density, the honeycomb infill pattern had a higher degree of tightness
and a higher Young’s modulus under compressive stress. On the other
hand, the SEM observation results showed that the internal pores of the
honeycomb scaffold were large and distributed unevenly (Fig. 4t),
resulting in low compression strength under high compressive strain.
The GG1.5GO1.2 scaffold had optimal mechanical properties, which
could maintain the long-term stability of the printed structure, thereby
providing mechanical support and nutrient transmission for cell and
tissue culture process.
Fig. S3 shows the FTIR spectra of GG, GO, Cur, GG/GO and GG/GO/
Cur scaffolds. Ca2+ can not only cross-link with the carboxyl group on
the GG macromolecular chain, but also provide the ionic interaction
with GO [43]. Therefore, the interaction between GG and GO should
include coordination bonding, ionic bonding, and hydrogen bonding
interactions. When curcumin was loaded on GG/GO scaffold, the
carbonyl vibrational peak disappeared, the carboxylic acid group and
epoxy bond were significantly reduced. Moreover, the characteristic
peak of curcumin could still be observed, which was significantly
reduced at 1626 cm− 1, indicating that curcumin partially reduced GO

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S. Zhu et al.
and was successfully loaded on the surface of GG/GO scaffold through
hydrogen bonding [44].
3.4. Loading and release of curcumin
All experiments to determine the load and release of curcumin were
performed using UV absorption at 428 nm. As shown in Fig. 6 (a), the
longer the soaking time of GG/GO scaffold in curcumin 75% alcohol
solution, the more amount of curcumin was loaded. When the soaking
time reached 120 min, the drug-polymer interactions became satura­
tion, and the loading amount of curcumin on GG/GO was 96.5 μg/mg.
The GG/GO/Cur scaffolds with soaking time of 40 min and 120 min
were selected for release experiment in vitro.
The release of curcumin from GG/GO/Cur scaffolds in different pH
values at 37 ◦ C was investigated, where pH = 7.4 and pH = 5.4 repre­
sented the normal physiological environment and the acidic environ­
ment of tumor cells, respectively [45]. As illustrated in Fig. 6(b) and (c),
in the first 6 h, there was a sudden release of curcumin in all samples,
which accounted for about 14–19% of the load. This was mainly because
a part of the loaded curcumin just adhered to the surface of the scaffolds
would directly diffuse into the medium in the initial release. The release
of curcumin in the scaffold had obvious pH-sensitive properties. Under
the alkaline conditions, the hydroxyl group on the benzene ring of
curcumin acts as oxygen negative ion, which enhances the
electron-giving ability and thus the electrophilic reactivity of the carbon
chain enhanced greatly. With the decrease of pH value, curcumin
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Composites Science and Technology 208 (2021) 108763
Fig. 8. Viability of osteosarcoma cells
cultured on the 3D printed scaffolds (R
stands for the scaffold with linear lattice
filling pattern, H stands for the scaffold
with honeycomb filling pattern). (a)
CCK-8 osteosarcoma cell viability assay
(n = 3) after 1, 3 and 5days of culture
(**p < 0.01, ***p < 0.001, ****p <
0.0001); (b) Results of live/dead fluo­
rescent stained osteosarcoma cells
cultured on the scaffolds after 1, 3 and
5days. Viable cells were fluorescent
green, while non-viable cells were fluo­
rescent red.
produces isomers, and the possibility of nucleophilic reaction on hy­
droxyl carbon increases, resulting in the decomposition of curcumin.
Therefore, the release of curcumin in acidic medium was higher than
that in neutral medium. Among the test samples, GG/GO/Cur120min
scaffold in an acidic environment showed superior release performance.
The release time continued for 14 days with 30% release efficiency. The
low release of curcumin was mainly attributed to the low solubility of
curcumin in the release medium [46]. In addition, different infill pattern
of GG/GO scaffolds also influenced the release of curcumin. As shown in
Fig. 6 (d), under the condition of pH = 5.4, the release efficiency of the
honeycomb-shaped GG/GO/Cur120min-H was 12% in the first 6 h and
22% after 10 days, which was lower than that of the lattice-shaped
GG/GO/Cur120min, mainly due to different specific surface areas of the
scaffolds.
3.5. Cell evaluation in vitro
Fig. 7 (a) shows viability of osteoblast cells cultured on the 3D
printed scaffolds with different filling structures by CCK-8 assay. MC3T3
cells on the scaffolds showed good cell activity and proliferation ability.
There was no significant difference in CCK-8 cell viability from the
scaffold with honeycomb and linear lattice filling patterns. The results of
loading curcumin showed that the number of viable cells in the scaffold
loaded with curcumin was significantly higher than that in the scaffold
without curcumin at 3 and 5 days (p < 0.05), indicating that curcumin
had a positive effect on the proliferation of osteoblasts. MC3T3 cells
S. Zhu et al.
could maintain good adhesion and proliferation ability on the scaffolds.
The results of live/dead fluorescent staining (Fig. 7b) showed that there
were almost no dead cells on the scaffold, and the flattened osteoblast
cells with multiple filopodial prostheses spread well on the scaffolds,
which helped the cells extend and attach to nearby anchor points better.
The results of osteosarcoma MG-63 cells cultured on the scaffold are
shown in Fig. 8. The results demonstrated a significant difference be­
tween the control groups (GG/GO scaffolds) and scaffolds with curcu­
min loaded at all the observed time points. Compared with the control
group, the curcumin-loaded scaffolds reduced cell viability by 50% and
82% on day 1 and day 3, respectively. On day 5, the viability of MG-63
cells on the curcumin-loaded scaffolds decreased by 93%, suggesting
that GG/GO scaffolds loaded with curcumin had a significant function of
eliminating osteosarcoma cells. This is critical for the scaffold located at
the site of bone tumor resection. In terms of cell adhesion and prolif­
eration, MG-63 cells seeded on the scaffolds without curcumin loading
showed rapid growth, and an elongated fibroblast-type cell morphology
with filopodial extension on the surface on the 3rd and 5th days. On the
first and third days, only a few cells survived on the curcumin-loaded
scaffold, and almost no cells survived on the 5th day, which further
proved that GG/GO/Cur scaffolds were effective in killing osteosarcoma
cells. During the process of cell culture in vitro, the scaffold remained
stable in the culture medium. And the ions in the culture medium
contribute to the formation and stability of the double helix structure of
GG. Therefore, the mechanical strength of the scaffold in the body fluid
environment can meet the requirements of supporting the tumor surgery
induced bone defects.
4. Conclusion
Aiming at the high fidelity, good biocompatibility and stability of 3D
bio-ink, GG and GO were combined to develop a new 3D printing ink.
Taking the advantage of the excellent rheological properties of GG, the
introduction of GO effectively improved the mechanical properties of
the system, thereby improving the printing performance of the ink and
the mechanical strength of the construction of the scaffold. At the same
time, good biocompatibility was guaranteed. After evaluation of rheo­
logical properties and printing performance of the system, the optimal
formulation was determined as 1.5 wt% GG and 1.2 wt% GO. The cur­
cumin was loaded onto the 3D printed scaffold by a simple immersion
method, and the application of the functional scaffold as a bone repair
material after bone tumor resection was investigated. The functional
GG/GO/Cur scaffold was pH responsive, and could effectively kill the
tumor cells and suppress the tumor growth in vitro via curcumin release
in the acidic environment of tumor cells. Simultaneously, the scaffolds
could support the attachment and proliferation of osteoblasts. This dual-
functional scaffold that combines tumor therapy with bone regeneration
can provide a promising clinical therapy strategy for the regeneration of
tumor surgery induced bone defects.
Declaration of competing interest
The authors declare that they have no known competing financial
interest or personal relationships that could appeared to influence the
work reported in the paper.
Acknowledgments
This research was supported by the Natural Science Foundation of
Guangdong Province (Grant No. 2018A030313052), the National Nat­
ural Science Foundation of China (Grant Nos. 31872758 and 31771047)
and the National Key Research and Development Project (Grant No.
2019YFD0901905).
9
Composites Science and Technology 208 (2021) 108763
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.compscitech.2021.108763.
Credit author statement
Shanshan Zhu contributed to the investigation, methodology, data
curation, writing original draft. Lingyun Yao assisted in investigation,
methodology, review and editing of the manuscript. Cile Pan and Jin­
huan Tian assisted in investigation, data curation. Lihua Li, Binghong
Luo and Changren Zhou, provided resources, participated in the work of
validation, review and editing of the manuscript. Lu Lu provided re­
sources, and contributed to the conceptualization, supervision as well as
review and editing of the paper.
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