International Journal of Biological Macromolecules 158 (2020) 452–460

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Advanced gellan gum-based glycol chitosan hydrogel for cartilage tissue

engineering biomaterial
Sumi Lee 1, Joo Hee Choi 1, Ain Park, Mina Rim, Jina Youn, Wonchan Lee, Jeong Eun Song, Gilson Khang ⁎
Department of BIN Convergence Technology, Department of Polymer Nano Science & Technology Research Center, Jeonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-
do, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Gellan gum (GG), a nature-derived polysaccharide, is one of the materials widely used in cartilage tissue engi-
Received 17 January 2020 neering (TE). Glycol chitosan (GC), a derivative of chitosan, is a water-soluble natural polymer that has excellent
Received in revised form 24 February 2020 biocompatibility and biodegradability as well as cell adhesion. Herein, GG was physically blended with GC to en-
Accepted 18 April 2020
hance the mechanical properties and microenvironment of the GG to apply in cartilage TE. The study was con-
Available online 23 April 2020
ducted with a hydrogel model which is similar to the extracellular matrix (ECM) of cartilage tissue. The
Keywords:
physicochemical studies were carried out with morphological study, swelling ratio, weight loss, and sol fraction.
Hydrogel The mechanical characterization was conducted with compression test and rheological study to confirm avail-
Gellan gum ability in cartilage TE material. Furthermore, in vitro studies such as morphology investigation, viability assay,
Glycol chitosan GAG content, qRT-PCR, and histological study were performed to verify biocompatibility and chondrogenesis
Cartilage of the material. The mechanical and biological properties improved with a proper amount of GC. Overall results
Chondrocyte verify the potential of the material and can be further used for the cartilage TE.
Regeneration © 2020 Published by Elsevier B.V.

1. Introduction

Gellan gum hydrogel (GG) exhibited cell suitability for various cell
types and allowed cell encapsulation in many studies. The GG forms a
random coil at high temperatures, but at low temperatures it forms a
double helix which leads to physical crosslinking [1,2]. Furthermore, ad-
dition of cations such as Ca2+, Mg2+, Na+, and K+ or controlling the
concentration of GG allows tunable mechanical performance [3]. How-
ever, despite its merits in tissue engineering application, GG fails to
meet the conditions of cell delivery system due to the disadvantages
such low mechanical property and lack of cell binding sites for cells
growth and migration. These drawbacks may lead to a major cell
death, low proliferation, hinder growth and differentiation [4].
Many studies have been conducted to solve these problems. The GG
backbone was modified chemically with biological factor or GG itself to
decrease the gelation temperature, enhance the microenvironment, and
include adhesive sites [5,6]. Another approach to improve mechanical,
physiological, and biological properties of the GG is synthesizing with
different types of polymers [7,8]. Many strategies using chemical modi-
fications have been proposed, but they have the disadvantage that they
can be time consuming and highly toxic in vivo [9]. Therefore, a number
of methods have recently been proposed to enhance the characters of
hydrogels by using a simple method.
In this study, composite matrix of GG and glycol chitosan (GC) was
manufactured to evaluate the effect of the composite in chondrogenesis.
Chitosan is widely investigated as a suitable composite for bio-
applications [10,11]. However, its solubility in water is poor and re-
quires aqueous solution containing acid which lead to the protonation
of amino groups (R-NH+ 3 ) [12]. This may induce toxicity in vitro and
in vivo [13]. Therefore, chitosan was modified into new properties by
modifying the backbone. Among many chitosan derivatives, GC is
water-soluble, biocompatible, biodegradable, and easily available on
the market [14]. Furthermore, the presence of –NH2 can increase the
cell adhesion which may enhance the microenvironment of the matrix
[15,16]. The composite hydrogel of GG and GC (G/C) was characterized
chemically and mechanically and evaluated in vitro to confirm its appli-
cability in cartilage tissue engineering.
2. Materials and methods

⁎ Corresponding author. 2.1. Preparation of G/C hydrogels

E-mail addresses: sumilee@jbnu.ac.kr (S. Lee), zooheechoi@jbnu.ac.kr (J.H. Choi),
ainpark@jbnu.ac.kr (A. Park), miiing95@jbnu.ac.kr (M. Rim), yjina@jbnu.ac.kr (J. Youn),
lwc95@jbnu.ac.kr (W. Lee), songje@jbnu.ac.kr (J.E. Song), gskhang@jbnu.ac.kr (G. Khang). Low-acyl gellan gum (GG, Gelzan™, Sigma-Aldrich, St. Louis, MO,
1
First co-authors. USA) was used in this experiment. First, 1% (w/v) GG was dissolved in

https://doi.org/10.1016/j.ijbiomac.2020.04.135
0141-8130/© 2020 Published by Elsevier B.V.
 S. Lee et al. / International Journal of Biological Macromolecules 158 (2020) 452–460
90 °C deionized water (DW) to fabricate homogeneous GG hydrogel so-
lution and CaCl2 was added to the solution. The temperature was
lowered to 60 °C and the glycol chitosan (GC, Sigma-Aldrich, USA)
was added to the solution at the amount of 0%, 0.05%, 0.075%, 0.1%,
and 0.2% (w/v); GG, G/C0.05, G/C0.075, G/C0.1, and G/C0.2, respectively.
The solutions were stirred until the GC was completely dissolved. The
homogeneous solutions were poured into petri dish and left at room
temperature for gelation. The fabricated hydrogels were punched with
a biopsy punch (Kai Medical, Honolulu, HI, USA) to make 3 mm height
and 6 mm diameter cylindrical disk and were used for further studies.
2.2. Characterization
2.2.1. FT-IR analysis
The chemical structures of GG, GC, and G/C hydrogel were character-
ized by a Fourier transform infrared spectroscopy (FTIR, Perkin Elmer,
USA) at the wavelength range of 400–4000 cm−1. The samples were
measured in a dried state.
2.2.2. Swelling analysis
The specimens were prepared with the method of Section 2.1. Initial
weight of the hydrogel was measured (Mf) and then placed in a 24-well
plate. A phosphate-buffered saline (PBS, pH 7.4, Gibco, Big Cabin, OK,
USA) was added and stored in a 37 °C incubator until at a specific time
point. At a certain point, the PBS was removed and the residual solution
was removed using a filter paper. The weight of the samples was re-
corded (mw) and the samples were rinsed with a DW 3 times. The
hydrogels were dried in a 60 °C oven for 3 h and the weight was mea-
sured (mf). The swelling ratio and the weight loss were calculated
using the Eqs. (1) and (2) respectively [17,18]. The study was evaluated
on 1, 7, 14, 21, and 28 days. PBS was replaced every 3 days.
Swelling ratio ð%Þ ¼ ðmw −m f Þ=m f  100
Weight loss ð%Þ ¼ ðmi −m f Þ=mi  100
2.2.3. Sol fraction
The weight of freeze-dried hydrogels was measured (mi). The mea-
sured samples were immersed in a DW (good solvent) and agitated on a
shaker for 1 h. The DW was removed and the hydrogels were freeze-
dried again. The weight of the samples was recorded (mf). The sol frac-
tion was calculated by following Eq. (3) [19].
Sol fraction ð%Þ ¼ ðmi −m f Þ=mi  100%
2.2.4. Compressive strength
The compression test was carried out under an unconfined condi-
tion. The prepared cylindrical shape hydrogels were placed on a cylin-
der plate of a Texture Analyzer (FTC, Sterting, Virginia, USA). The
hydrogels were compressed at the speed of 2 mm/min and load cell of
10 N. Before characterization, the height, and diameter of the samples
were measured using a caliper (Mitutoyo, South Korea) to calculate
the accurate compressive strength.
2.2.5. Rheological evaluation
The rheological character and gelation temperature of the hydrogels
were characterized with a viscometer (AMETEK Brookfield, Middleboro,
MA, USA). The water circulation bath (VCB-07, JONGRO Industrial Co.,
Ltd., South Korea) was preheated and the 5 mL of a premade hydrogel
solution was transferred to the viscometer. The temperature was grad-
ually lowered until the circulation bath reached 18 °C. The router speed
was set at 0.5 rpm and cone and plate spindle (SC-32 spindle, AMETEK
Brookfield, USA) were used for this study.
453
2.2.6. SEM analysis
The porous structure of the hydrogels was studied by Bio-LV scan-
ning electron microscope (Bio-LV SEM, Hitachi, SN-3000 Hitachi,
Japan). The hydrogels were frozen in a deep-freezer (−60 °C). The spec-
imens were cross-sectioned and gold sputtered using a vacuum sputter
(SC500k, Emscope, UK) for the evaluation.
2.3. In vitro study
2.3.1. Animal experiment
All the animal experiments were performed following the guidelines
and approval of Chonbuk National University Animal Care Committee,
Jeonju, Republic of Korea (CBNU 2016-50).
2.3.2. Isolation and culture of chondrocytes from rabbit model
All materials were autoclaved before the experiment. Chondrocytes
were isolated from a 4 weeks old female New Zealand white rabbits
(Hanil laboratory animal center, Wanju, Korea) following the lab
protocol [20]. Briefly, flesh was removed from the extracted legs and
washed with a sterilized PBS to eliminate residual blood and flesh. A car-
tilage tissue was peeled off using a blade and digested it in a 0.02% col-
lagenase A (Roche, Germany) for overnight. Separated cells were
centrifuged in a 1200 rpm at 4 °C for 3 min. The centrifuged
chondrocytes were resuspended in a Dulbecco's Modified Eagle Me-
dium Nutrient Mixture F-12 (DMEM /F12, Gibco, Big Cabin, OK, USA)
supplemented with 10% of Fetal Bovine Serum (FBS, Gibco, USA) and
1% of Penicillin/Streptomycin (PS, Gibco, USA) and cultured in cell cul-
ture dishes (Eppendorf, Hamburg, Germany) under standard conditions
(5% CO2 and 37 °C). The media was changed every 3 days and primary
passage 2 of chondrocytes were used for this study.
2.3.3. Cell culture in the hydrogels
ð1Þ All materials were sterilized with autoclave and under ultraviolet
(UV) lamp before the experiment. The hydrogels were fabricated fol-
ð2Þ lowing the 2.1. Preparation of G/C hydrogels method. The manufactured
solutions were filtered through a 0.45 μm filter (Sigma-Aldrich,
Germany). The trypsinized chondrocytes (2 × 106 cells/mL) were care-
fully mixed with the hydrogels at the temperature before the gelation
occur (35–37 °C). The co-cultured solutions were cast into a petri dish
and left at RT for gelation. The stable hydrogels were punched into
3 mm height and 6 mm diameter size with a biopsy punch. Each hydro-
gel was transferred to a 24-well plate and cultured in a 1 mL of DMEM/
F12 cell culture media. The media was changed every 3 days.
2.3.4. Cell viability assay
ð3Þ Cell viability study was conducted with MTT (3-[4,-dimethylthiazol-
2-yl]-2,5-diphenylterazolium bromide; thiazolyl blue, Amresco, TX,
USA) assay. The evaluation was carried out on 3, 7, 14, and 21 days of
culture. The existing medium was replaced with a fresh medium and
added MTT solution (5 mg/mL in PBS) to make a 10% reagent. The
treated samples were stored in a humidified 5% CO2 and 37 °C environ-
ment for 3 h. The supernatant was removed and the samples were ho-
mogenized with dimethyl sulfoxide (DMSO, Samchun chemical, South
Korea) using a glass tissue grinder (WHEATON, USA) dissolve formazan
crystal. The solutions were transferred into a 96-well plate and mea-
sured under an absorbance of 570 nm with a microplate reader (Syn-
ergy MX, Biotek, Vernusky, VT, USA).
2.3.5. Live/dead assay
A live/dead cell imaging kit (Invitrogen, Carlsbad, CA, USA) was used
in this study according to the kit protocol. Briefly, the hydrogels were
washed with PBS and sliced into half. The samples were transferred to
a coverglass bottom dish (SPL, Lifescience, Korea). A mixed reagent
which contains calcein AM (green) and ethidium homodimer (red)
were added on top of the specimens. The treated samples were incu-
bated under standard conditions (5% CO2 and 37 °C). The image was
454 S. Lee et al. / International Journal of Biological Macromolecules 158 (2020) 452–460
Fig. 1. FT-IR analysis of GG, GC and G/C hydrogel in the wavelength range of
400–4000 cm−1.
taken under a super resolution confocal laser scanning microscope (LSM
880 with Airyscan, Carl Zeiss, Germany) using a Z-stack method. The
fluorescence intensity was measured by using an image J program [21].
2.3.6. Morphology observation
The cell encapsulated hydrogels were cultured for 21 days. The spec-
imens were washed with PBS 3 times and fixed with 2.5% glutaralde-
hyde (Sigma-Aldrich, USA) for 24 h in 4 °C. The fixed samples were
stored in −60 °C deep-freezer for 24 h and were lyophilized for 48 h
to remove water. The SEM image of the prepared samples was observed
by following Section 2.2.6.
Fig. 2. (A) Swelling ratio (%) (B) weight loss (%) and (C) sol fraction (%) of the hydrogels (values are mean ± SD, n = 3, P b 0.05 (*), P b 0.01 (**), P b 0.001 (***)).
2.3.7. Histological analysis
The specimens were cultured for 21 days and washed. At specific
time point, the samples were washed with PBS 3 times and fixed with
a 4% formaldehyde (Sigma-Aldrich, USA). The specimens were frozen
with a CryoMatrix (Thermo Fisher Scientific, USA) in a liquid nitrogen.
The prepared samples were sectioned in a 10 μm thickness using a
cryomicrotome (Thermo Fisher Scientific, USA). Hematoxylin & Eosin
(H&E) and Safranin-O staining were carried out according to the stan-
dard histological methods. All the reagents were bought from Sigma-
Aldrich except for hematoxylin which was obtained from Fisher Scien-
tific, Hampton, USA.
2.3.8. GAG quantitative analysis
Glycosaminoglycan (GAG) analysis was performed at specific time
points. The samples were rinsed with PBS and were lyophilized. The
weight of the samples was recorded and papain solution was added
and treated for 16 h in a 60 °C oven to perform enzymatic digestion.
The papain digestion solution was prepared by dissolving 125 μg/mL
of papain (Sigma-Aldrich, USA) and 10 mM L-cystein (Sigma-Aldrich,
USA) into a premade PBE buffer (pH 6.5) which constituted of
100 mM Na2HPO4 (SHOWA, Japan) and 10 mM EDTA (Sigma-Aldrich,
USA). GAG content was characterized with a 1,9-dimethylmethylene
blue (DMMB, Sigma-Aldrich, USA) dye-binding assay with a chondroi-
tin sulfate from bovine trachea (Sigma-Aldrich, USA) as a standard
[22]. The biosynthetic activity of the cells was assessed by normalizing
the GAG quantification to the double-stranded DNA (dsDNA) content
which was studied by a Qunati-iT PicoGreen reagent (Life Technologies,
Carlsbad, CA, USA) according to the product protocol. Standard curves
were prepared at concentrations ranging from 0 to 2 μg/mL. The mea-
surement was conducted with a microplate reader at a wavelength for
GAG content (525 nm) and the fluorescence intensities at an excitation
wavelength of 485/20 nm and an emission wavelength of 528/20 nm for
the dsDNA quantification.
 S. Lee et al. / International Journal of Biological Macromolecules 158 (2020) 452–460 455

Fig. 3. Morphological analysis under SEM.

2.3.9. Quantitative real-time polymerase chain reaction (qRT-PCR) 2.4. Statistics

A quantitative real-time polymerase chain reaction (qRT-PCR) was
carried out to investigate gene expression of chondrocytes cultured in
the hydrogels. On 21 days of culture, hydrogels were washed 3 times
with PBS and cells were frozen in −60 °C deep freezer for overnight.
Cell lysis was conducted by homogenizing the cell encapsulated
hydrogels with a Trizol (Invitrogen, USA) in the glass tissue grinder.
The solution was transferred to a 1.5 mL Eppendorf tube (EP tube). A
chloroform (SAMCHUN chemicals, South Korea) was included and the
solution was shaken well to mix and centrifuged at 12,000 rpm in 4 °C
for 15 min. The supernatant was carefully transferred to another EP
tube and mixed with isopropanol (Sigma-Aldrich, USA) to sink ex-
tracted mRNA. The samples were stored in 4 °C overnight and centri-
fuged at 12,000 rpm in 4 °C for 5 min. The supernatant was removed
and washed with a 75% ethanol. The samples were centrifuged at
7500 rpm in 4 °C for 10 min and the supernatant was removed. Ultra-
Pure DNase/RNase-free distilled water (Invitrogen, USA) was added to
the extracted samples. The concentration of mRNA was determined
using a Biospectrophotometer (Eppendorf, USA). cDNA was synthesized
by using TOPscriptTM RT DryMIX (dT18 plus) (Enzynomics, South
Korea). qRT-PCR was performed by StepOnePlus Real-Time PCR system
(Applied Biosystems, USA) using a SYBR Green Master Mix (Applied
Biosystems, USA). Aggrecan (AGG), Collagen type 2 (COL2), and SOX9
were applied for the study and GAPDH were used as a housekeeping
gene.
All the numerical results are presented by the mean ± standard de-
viation (SD). The GraphPad Prism 5.0 software (GraphPad Software, La
Jolla, CA, USA) was utilized to perform the statistical analysis. The stud-
ies were analyzed employing one-way analysis of variance (one-way
ANOVA test), and the differences were considered significant at
P b 0.05(*), P b 0.01(**), and P b 0.001(***).
3. Result and discussion
3.1. Characterization
3.1.1. FT-IR analysis
The chemical composition of GG, GC, and interaction among GG and
were evaluated with FT-IR analysis (Fig. 1). The specific peak of GG was
observed at 3300 cm−1 a stretching band of –OH groups, at 2914 cm−1
which corresponds to stretching vibrations of the –CH groups, at
1618 cm−1 due to the presence of C_O stretching vibration from car-
bonyl groups, and at 1030 cm−1 which is caused by –COC stretching.
The FTIR spectrum of GC showed characteristic peaks associated with
C_O stretching amide I, –NH bending of primary amines, and amide
II. The blended network exhibited shifted frequency band at
3375 cm−1, 2943 cm−1, 1619 cm−1, and 1041 cm−1 [23]. This phenom-
enon appeared due to the electrostatic interaction of carboxylate groups

Fig. 4. (A) Compressive strength and (B) rheological characteristics (values are mean ± SD, n = 3, P b 0.05 (*), P b 0.01 (**), P b 0.001 (***)).
456 S. Lee et al. / International Journal of Biological Macromolecules 158 (2020) 452–460
Fig. 5. MTT assay exhibited in optical density values (values are mean ± SD, n = 3).
and amide groups in GG and GC lead to shifting in the specific peak of
GG peak in the G/C hydrogel [24]. Besides the characteristic peaks corre-
sponding to GG and GC, a new absorption peak did not appear, implying
that the network is formed with the physical interaction.
3.1.2. Physicochemical characterization of GC/GG
The swelling and degradation kinetics were carried out for 28 days.
The swelling rate of the hydrogel is important because it is related to
nutrient uptake and in vivo fluid retention in cell culture media
in vitro [19]. The swelling ratio of the hydrogels showed gradual in-
crease as the time passed which may be due to the increased
Fig. 6. (A) LSM image of chondrocytes encapsulated in the hydrogels analyzed with a Z-stack mode and (B) relative intensity of LSM images (scale bar = 50 μm) (values are mean ± SD,
n = 3, P b 0.05 (*), P b 0.01 (**), P b 0.001 (***)).
hydrophilicity of glycol group in the GC (Fig. 2A). The weight loss of
the hydrogels also showed the similar aspect as the swelling kinetic
(Fig. 2B). This may be due to difference in the osmotic pressure with
the physiological aqueous solution. The difference in an osmotic pres-
sure leads hydrogels to absorb water and swell and reach equilibrium
state. However, as the matrix degrade, the elastic pressure decreases
and cause more swollen state [25]. The initial state of all the specimens
showed fast degradation which may be due to the release of free GG and
GC chains [20]. The swelling and weight loss rate did not show signifi-
cant differences between each specimen until 14 days of measurement.
However, after 14 days, GG and G/C0.05 showed a progressive decrease
in the rate of degradation, while the rest of the groups continued to in-
crease. This may be due to the lower polymers that are involved in the
matrix by the inhibition of crosslinking from GC incorporation as
shown in the sol fraction result (Fig. 2C) [26]. The morphological analy-
sis of all the hydrogels showed continuous porous structures which re-
sult from ice crystal formation from freeze-drying step [27]. The pore
size increased as the GC content and displayed lamellar porous struc-
tures (Fig. 3). It is assumed that the entanglement between the two net-
works allowed interconnected porous structures.
3.1.3. Mechanical study
The compressive strength and viscosity of the fabricated materials
were characterized to investigate influence of GC. The compressive
strength of the hydrogel is an essential property for cartilage tissue engi-
neering materials in order to sustain the external force [28]. The compres-
sive modulus increased as the GC was included in the GG (Fig. 4A). The
viscosity of the hydrogels displayed higher level when GC was added in
the matrix (Fig. 4B). This may be due to the higher interaction between
two matrices [29]. The gelation temperature was measured according to
the temperature change. It is important for gelation to occur at proper
 S. Lee et al. / International Journal of Biological Macromolecules 158 (2020) 452–460
Fig. 7. SEM images of chondrocytes encapsulated in the hydrogels.
temperature which is not too high or low which may affect the survival of
the cells [30]. Also, it is important for hydrogel to solidify after cells are
uniformly suspended in the hydrogels [31,32]. Each hydrogel showed ge-
lation temperature at 32.1 °C, 32.2 °C, 33.6 °C, 33.7 °C, and 34.2 °C in GG,
G/C0.05, G/C0.075, G/C0.1, and G/C0.2, respectively. The gelation temper-
ature was not significantly affected of inclusion of GC which verifies the
applicability in cell culturing material [33].
3.2. In vitro study
3.2.1. Biocompatibility
The biocompatibility is an important factor to consider in choosing
tissue engineering materials. The MTT assay which confirms the viabil-
ity of cells by metabolic activity showed that all the groups showed sim-
ilar optical density (Fig. 5). However, live/dead staining of the cells
encapsulated hydrogels showed higher amounts of dead cells in the
GG group while the viability of the cells increased in the GC incorpo-
rated hydrogels (Fig. 6). This may be due to the higher attachment site
from GC and improved mechanical property enhanced the viability of
the cells [16,34].
3.2.2. Morphological and histological observation
Chondrocytes encapsulated hydrogels were incubated for 21 days
and examined under SEM to observe ECM formation, cell morphology
and growth. It is noted that the spherical shape is favorable for chondro-
genesis [35]. All the encapsulated cells displayed roundel shape. How-
ever, while GG showed cells tangled in the matrix rather than being
presented in the pores, higher amounts of cells existing in the porous
structure was detected in hydrogels containing GC (Fig. 7). H&E and
Safranin-O staining were performed on day 21 to evaluate morphology
and expression of cartilaginous matrix. The H&E which stains nucleus
and possible secreted cytoplasm around cells were examined. The
cells were well distributed in the matrix and some of the cells showed
noticeable cytoplasm around the cells (Fig. 8A). The G/C0.075 showed
cytoplasm stretching out of the cells (shown in arrows) which indicate
that the inclusion of GC enhances the microenvironment of the hydro-
gel and has a beneficial effect on the metabolism and growth of the
cells [36,37]. The Safranin-O which stains GAG exhibited higher positive
red staining around the cells in GC loaded hydrogel (shown in arrows)
(Fig. 8B). These results show that the GC has positive effect on improv-
ing microenvironment [38].
457
3.2.3. Biochemical study
GAG is a significant component of cartilaginous ECM [39,40]. The
results of the biosynthesis of chondrocytes encapsulated in
hydrogels showed increasing amount over time (Fig. 9). The GAG
synthesis showed significantly higher level in the G/C0.075 con-
structs when compared to the GG but decreased when higher
amount of GC was incorporated in the GG matrix. Especially on 14
and 21 days, G/C0.075 showed about 2 times higher content com-
pared to the GG. These results show the positive effect of the appro-
priate amount of GC blended GG from the complementary effect of
the two substances which provided enhanced microenvironment to
form GAGs in hydrated macrostructures. Furthermore, the expres-
sion of cartilage-related gene markers such as AGG, COL2, and
SOX9 was confirmed and standardized based on GAPDH, a house
keeping gene. SOX9 is one of the most important transcription fac-
tors that is crucial for chondrocyte differentiation and cartilage for-
mation. COL2 is the main constituent of cartilage tissue and AGG
plays an important role in a cartilage function [41–43]. All the
genes showed higher level in the GC incorporated hydrogels when
compared to the pristine GG (Fig. 10). Especially, G/C0.075 matrix
exhibited a significantly highest amount. As it is specified in the pre-
vious experiments, when appropriate amount of GC was added, the
cell proliferation and growth were active which allowed proper
chondrogenesis by providing the enhanced microenvironment.
4. Conclusion
In this study, GG and GC composite was proposed as an effective ma-
terial for cartilage TE. The GG was manufactured with various amount of
GC with a simple method. GG acted as a platform for the entire support,
while GC improved the mechanical properties of the hydrogel and en-
hanced the microenvironment. The physicochemical and mechanical
properties of the hydrogels were improved as the GC was incorporated.
In vitro results showed higher cell viability and proliferation. The bio-
chemical studies exhibited higher level of GAG synthesis and mRNA ex-
pression of cartilage-specific genes in G/C0.075. The hydrogel was
fabricated with a non-toxic crosslinker in a pH-neutral condition. Also,
material can be improved with the use of stem cells, growth factors,
genes, therapeutic drugs, and biomolecules [44]. Overall, with the ap-
propriate amount of GC in the GG-based hydrogel, G/C hydrogel is ex-
pected to be applicable in cartilage TE.
458 S. Lee et al. / International Journal of Biological Macromolecules 158 (2020) 452–460

Fig. 8. Histological images of (A) H&E and (B) Safranin-O.

Funding

This research was supported by the Korea Health Technology R&D

Project through the Korea Health Industry Development Institute
(KHIDI), funded by the Ministry of Health & Welfare, Republic of
Korea (HI15C2996) and the International Research & Development
Program of the National Research Foundation of Korea (NRF) funded
by the Ministry of Science, ICT & Future Planning (NRF-
2017K1A3A7A03089427).

Author contributions

We believe that these individuals should be listed as authors be-

Fig. 9. GAG quantitative analysis of chondrocytes encapsulated in the hydrogels (values
are mean ± SD, n = 3).
cause: S. Lee and J.H. Choi designed and conceived the study. S. Lee, J.
Yoon, and W. Lee synthesized and characterized the materials. S. Lee,
J.H. Choi and A. Park performed in vitro study. S. Lee, A. Park, and M.
Rim performed the Histology. S. Lee and J.H. Choi wrote the manuscript.
J.E. and G. contributed reagents and materials for this study.
 S. Lee et al. / International Journal of Biological Macromolecules 158 (2020) 452–460
Declaration of competing interest
We wish to confirm that there are no know conflicts of interest asso-
ciated with this publication and there has been no significant financial
support for this work that could have influenced its outcome.
Acknowledgements
This research was supported by the Korea Health Technology R&D
Project through the Korea Health Industry Development Institute
(KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea
(HI15C2996) and the International Research & Development Program
of the National Research Foundation of Korea (NRF) funded by the Min-
istry of Science, ICT & Future Planning (NRF-2017K1A3A7A03089427).
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Fig. 10. RT-PCR analysis of (A) AGG (B) COL2 and (C) SOX9 normalized by GAPDH (values are mean ± SD, n = 3, P b 0.05 (*), P b 0.01 (**), P b 0.001 (***)).