Carbohydrate Polymers 251 (2021) 117036

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Injectable hydrogel derived from chitosan with tunable mechanical

properties via hybrid-crosslinking system
Jeong Wook Seo a, Su Ryon Shin b, Min-Young Lee c, Jae Min Cha d, Kyung Hyun Min e,
Sang Cheon Lee f, Seon Young Shin g, Hojae Bae a, *
a
Department of Stem Cell and Regenerative Biotechnology, KU Convergence Science and Technology Institute, Konkuk University, Seoul, 05029, Republic of Korea
b
Division of Engineering in Medicine, Department of Medicine, Harvard Medical School, Brigham and Women’s Hospital, Cambridge, MA 02139, USA
c
Smart Healthcare Research Institute, Biomedical Engineering Research Center, Samsung Medical Center, 81, Irwon-ro, Gangnam-gu, Seoul, 06351, Republic of Korea
d
Department of Mechatronics, College of Engineering, Incheon National University, Incheon, 22012, Republic of Korea
e
Department of Pharmacy, School of Pharmacy, Jeonbuk National University, Jeonju, Jeonbuk 54896, Republic of Korea
f
Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul, 02447, Republic of Korea
g
Department of Internal Medicine, The Catholic University of Korea, Seoul, 02841, Republic of Korea

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

Keywords: Thermo-sensitive injectable hydrogels that spontaneously react to physiological temperature have been widely
Chitosan studied to be used in biomedical fields. However, several challenges on their unstable structures with large-sized
Hydroxybutyl methacrylation pores and low mechanical strength under physiological conditions must be addressed to enable their practical
Injectable hydrogel
applications. We synthesized the hydroxybutyl methacrylated chitosan (HBC-MA) hydrogel that possesses both
Hybrid-crosslinking
Microstructure
thermo-sensitive and photo-crosslinkable properties. The HBC-MA showed effective sol-gel transition under
physiological temperature as well as a sensitive photo-crosslinkable property with visible light capable of skin
penetration. The co-nonsolvency property and thermo-sensitivity of HBC-MA prevented unintended loss of the
hydrogel graft after being subcutaneously injected in mice. Subsequently applied visible light on the skin beneath
which the hydrogel was injected significantly improved the mechanical strength and stability of the graft. The
injectable HBC-MA hydrogel developed in this study can be applicable to a wide range of biomedical fields such
as drug delivery system and tissue engineering.

1. Introduction physiological conditions, limiting its potential application (Cho et al.,

Hydrogels have been used in various biomedical applications
because of their advantageous properties with high water content (Wang
et al., 2010), biocompatibility (Wei et al., 2015), and three-dimensional
network structure (Qi et al., 2010; Zhu, Harris, & Zhang, 2016).
Thermo-sensitive hydrogels can spontaneously provoke a sol-gel tran­
sition in response to physiological temperature without additional
chemical reactions and have been considered a useful biomaterial for
drug delivery system, regenerative medicine, and tissue engineering
(Chen et al., 2016; Fu et al., 2012). The controlled sol-gel transition of
the hydrogels allows minimally invasive injection via syringes or cath­
eters even to the irregularly shaped sites (Liu et al., 2017; Shibata et al.,
2010). However, structural variation in thermo-sensitive hydrogels can
result in low mechanical strength and instability of the gel under
2016; Potta, Chun, & Song, 2010). For example, owing to such charac­
teristics of thermo-sensitive hydrogels, an unintended early ‘burst’
release of drugs could be caused when applied to drug delivery systems.
(Bae, Okano, & Kim, 1989; Dong et al., 2012; Mukae, Bae, Okano, &
Kim, 1990). On the other hand, photo-crosslinkable hydrogels retain
different aspects in the injectability features, such that they show su­
perior mechanical strength and stability to the once they have been
photo-polymerized. However, if the graft was injected as
photo-polymerized, they are less versatile in the shapes of graft, and
when injected as a prepolymer solution before polymerization, any
leakages of the sample cannot be avoided, which would lead to a sig­
nificant loss of graft.
Chitosan is a biomaterial that has shown the following beneficial
abilities: inhibition of infection (Lin, Lin, & Chen, 2009),

* Corresponding author at: KU Convergence Science and Technology Institute, Department of Stem Cell and Regenerative Biotechnology, Konkuk University,
Hwayang-dong, Gwangjin-gu, Seoul, 05029, Republic of Korea.
E-mail address: hojaebae@konkuk.ac.kr (H. Bae).

https://doi.org/10.1016/j.carbpol.2020.117036
Received 19 February 2020; Received in revised form 19 August 2020; Accepted 31 August 2020
Available online 6 September 2020
0144-8617/© 2020 Elsevier Ltd. All rights reserved.
J.W. Seo et al.
anti-inflammation (Azuma, Osaki, Minami, & Okamoto, 2015),
biocompatibility, and biodegradability (Rinaudo, 2006). In addition, it
consists of many reactive functional hydroxyl and amino/acetamido
groups with ease of diverse chemical modifications. In the previous re­
ports, hydroxybutylation of chitosan resulted in an efficient sol-gel
transition under physiological temperatures by influencing hydrogen
bonds between hydrophilic and hydrophobic groups (Bin, Changzheng,
Chunlin, Qisheng, & Dajun, 2010). Meanwhile, methacrylation of chi­
tosan has been frequently used to make photo-crosslinkable hydrogels
with photo-initiators to enable photo-polymerization.
The aim of this study is to develop a novel injectable, stable, robust,
chitosan-based hydrogel using photo-crosslinkable and thermo-sensitive
chemical strategies, to enable robust & conformable gel administration
to different shaped injection sites. Our hypothesis is that HBC-MA ob­
tained by conjugating hydroxybutylation and methacrylation groups to
the side chains of chitosan will form a hybrid-crosslinked hydrogel
under physiological conditions in the presence of a photo-initiator. Our
investigation uses the crosslinking strategies of thermal, photo, and
hybrid-crosslinking of HBC-MA. Cytotoxicity and the physicochemical
properties including degradation, de-swelling are investigated. Inject­
ability of the novel hydrogel is investigated using a mouse model.
2. Materials and methods
2.1. Materials
Chitosan (50~190 kDa; 20~300 cP, 1 wt. % in 1% acetic acid;
75~85 % deacetylated), methacrylic anhydride (MA), 1,2-epoxybutane,
eosin Y (dye content~99 %), triethanolamine (TEOA), 1-vinyl-2-pyrro­
lidinone (NVP, contains sodium hydroxide as an inhibitor, >99 %),
2,4,6-trinitrobenzene sulfonic acid solution (TNBS), and glycine were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum
(FBS), penicillin streptomycin (P/S), high glucose Dulbecco’s Modified
Eagle’s Medium (DMEM), Dulbecco’s Phosphate Buffered Saline
(DPBS), and trypsin-EDTA solutions were purchased from WelGene
(Daegu, Gyeongbuk, Korea). All other chemical agents used in this paper
were analytical grade.
2.2. Preparation of HBC-MA solution and hydrogel
The HBC-MA was synthesized through two processes of hydrox­
ybutylation and methacrylation of chitosan. To start, 1 g of chitosan was
completely dissolved in 60 ml of 0.1 M Acetic acid followed by pH
adjustment to 6 using 5 M NaOH and addition of 20 ml of 1,2-epoxybu­
tane (99 %). Then the mixture was reacted at 55 ◦ C for 24 h. After the
reaction was complete, washing was performed using a centrifuge to
remove any remaining 1,2-epoxybutane. Finally, hydroxybutyl chitosan
(HBC) solution was lyophilized for 4 days (Tsukamoto, Akagi, Shima, &
Akashi, 2017).
To make HBC-MA, 1 g of the recovered HBC was dissolved in 60 ml of
distilled water overnight followed by the addition of methacrylic an­
hydride (94 %) at a rate of 1 ml/min until 15 % (v/v) was reached, and
was allowed to react for 4 h (pH 7). The mixture was dialyzed against
distilled water for 1 week using 12− 14 kDa cut-off dialysis tubing to
remove residual methacrylic anhydride, and finally the purified HBC-
MA polymer was lyophilized (Yoon et al., 2016).
For preparation of HBC-MA solution, lyophilized HBC-MA was
completely dissolved in distilled water to a 3 % (w/v) concentration and
then 0.01 mM Eosin Y which is a Food and Drug Administration (FDA)-
approved photoinitiator responding to visible light (450− 550 nm), 0.1
% (v/v) TEOA as a co-initiator, and 37 nM NVP as a co-monomer were
dissolved. Based on a previously reported study, we refer to a compo­
sition with high biocompatibility of photo-initiators (Bahney et al.,
2011; Noshadi et al., 2017). The HBC-MA solution was then adjusted to
pH 7.4.
To perform photo-crosslinking, a polydimethylsiloxane (PDMS)
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Carbohydrate Polymers 251 (2021) 117036
mold with a diameter of 8 mm and a height of 2.5 mm was filled with
HBC-MA solution and then Omnicure S2000 (Lumen Dynamics; Mis­
sissauga, ON, Canada) equipped with a visible light filter (450− 550 nm)
was used. The luminous intensity was 10 mW/cm2, and each sample was
exposed to visible light for 10, 20, and 30 s at room temperature (27 ◦ C).
For hybrid-crosslinking, the samples were incubated at 37 ◦ C in PBS.
1
2.3. H nuclear magnetic resonance (NMR) spectroscopy
1
H NMR spectroscopy was performed to determine the degree of
substitution (DS) of HBC-MA. Chitosan, HBC, and HBC-MA were dis­
solved in 0.25 % DCl at 1% (w/v) and analyzed using the 500 MHz FT-
NMR spectrometer (Varian; Palo Alto, California, USA).
2.4. Fourier transform infrared spectroscopy (FTIR)
The chemical structures of chitosan, HBC, and HBC-MA at room
temperature were analyzed using an FTIR - 4100 (Jasco; Tokyo, Japan).
Two (2) mg of samples (chitosan, HBC, and HBC-MA) was mixed with
100 mg potassium bromide, and pellets were prepared for the analysis
(Shigemasa, Matsuura, Sashiwa, & Saimoto, 1996).
2.5. Scanning electron microscopy (SEM)
The HBC-MA hydrogels were prepared under three conditions
(thermal-crosslinking, photo-crosslinking, and hybrid-crosslinking). The
prepared hydrogel was carefully sealed in a 50 ml conical tube, then
immersed in liquid N2 for 10 min and frozen. After unsealing, lyophili­
zation was performed at − 80 ◦ C for 24 h. All samples were cut with a
sharp knife to provide a clear view of the interior. Samples were then
secured on a stub via carbon tapes and coated with platinum. Cross-
sectional morphologies were imaged with an SU-8010 Scanning Elec­
tron Microscope (Hitachi; Tokyo, Japan). The pore size was confirmed
by measuring all lyophilized pores in the SEM image at 300x
magnification.
2.6. Reversible characterization of hybrid-crosslinked hydrogel
The 2,4,6-Trinitrobenzenesulfonic acid (TNBS) assay was applied to
measure glycine mixed in the water evacuated during thermal cross­
linking (Zhang & Chu, 2004). Glycine was dissolved at a concentration
of 2 mg/mL in a 3 % (w/v) HBC-MA solution. Glycine-loaded hydrogel
was then immersed in PBS (pH 7.4) for 24 h in the 37 ◦ C incubator. The
hydrogel was then carefully transferred to PBS at 20 ◦ C and 37 ◦ C for
intervals of 30 min. To measure evacuated glycine, 0.25 ml of 0.01 %
(w/v) TNBS solution was added to 0.5 ml of each sample solution. The
samples were then incubated at 37 ◦ C for 2 h. Absorbance was measured
at 335 nm using a UV/vis spectrophotometer (Optizen POP; Seoul,
Korea).
2.7. Rheological analysis
The temperature-dependent rheological properties of the HBC-MA
hydrogel were measured using the MCR302 Rheometer (Anton Paar
Ltd.; Graz, Austria) with parallel plate geometry (50 mm in diameter).
The temperature was increased at a rate of 1 ◦ C/min between 4 and 50
◦
C. Small amplitude y (0.02) and low frequency x (1.00 rad/s) were
applied for temperature sweep measurements of the HBC-MA solution.
2.8. Mechanical testing
A CT3 Texture analyzer (Brookfield; Toronto, ON, Canada) with a
4500 g load cell (Brookfield; Toronto, ON, Canada) in compression
mode was used to measure the compressive strength of photo-
crosslinked HBC-MA hydrogel. Two types of hydrogel (photo-cross­
linked and hybrid-crosslinked hydrogel) were prepared with three types
J.W. Seo et al.
of visible light exposure time (10, 20, and 30 s) to determine the dif­
ference in strength. Each sample was exposed to visible light (as
described in Section 2.2) at room temperature. Photo-crosslinked
hydrogel samples were stabilized in PBS at RT for 15 min so that no
further crosslinking occurred. Hybrid-crosslinked hydrogel samples
were then reacted at 37 ◦ C for 15 min for further thermal-crosslinking. A
probe 12.7 mm in diameter was used to compress with a trigger load of
0.05 N at a test speed of 0.05 mm/s. The compressive modulus was
determined as the slope of the linear region corresponding to 5~15 %
strain (Nichol et al., 2010).
2.9. Swelling test
The HBC-MA solution was pipetted into a PDMS mold. The sample
was exposed to visible light (as described in the Section 2.2) at room
temperature. Single group samples were prepared that were exposed to
visible light for 20 s. The photo-crosslinked hydrogels were stored at 37
◦
C. The swollen weight (St) was measured at the predetermined time
intervals in the PBS-containing samples, and the dry weight (Dt) was
measured after lyophilization. Finally, swelling ratio (%) was calculated
as follows:
Swelling ratio (%) = St / Dt × 100
2.10. Hydrogel degradation
Samples for degradation test were prepared by the method described
in Section 2.2. Degradation tests were performed using lysozyme
(40,000 U/mg, Sigma, USA). The samples were placed in PBS containing
lysozyme at concentrations of 0.5 mg/mL. Control samples were placed
in PBS without lysozyme. The hydrogels were then carefully stored at 20
◦
C and 37 ◦ C for a defined time course. To keep the enzyme active, the
PBS containing lysozyme was changed daily. After 1, 3, 5, 7, 14, and 21
d, the samples were thoroughly washed with deionized water to remove
any remaining PBS and lysozyme, and the water remaining on the sur­
face was removed by blotting with a kimwipe. Then, each hydrogel
sample was lyophilized and the dry weight (Dt) was recorded. Four
samples were measured for freeze-dry weight immediately after prepa­
ration and the mean weight (D0) was recorded. Gel remaining (%) was
calculated as follows:
Mass loss (%) = (D0 − Dt) / D0 × 100
2.11. In vitro hydrogel crosslinking test
The HBC-MA solution at 20 ◦ C was added into deionized water at 37
◦
C in a petri dish using a 20 G needle syringe. Compression force of the
syringe was adjusted manually (Fig. S1A and B, Supporting Informa­
tion). The sample was exposed to visible light (as described in Section
2.2). The distance between the sample and the light source was main­
tained at 2 cm (Fig. S1C, Supporting Information).
2.12. Live/Dead fluorescence assay
To investigate the cytotoxic effect of the HBC-MA, cell viability was
investigated using a calcein AM/ethidium homodimer Live/Dead assay
kit (Invitrogen, Carlsbad, CA). NIH3T3 fibroblast cells were grown and
maintained in Dulbecco’s Modified Eagle Medium (DMEM; Welgene,
Daegu, Korea) containing 10 % fetal bovine serum (FBS). The cells were
cultured in an incubator supplied with 5 % CO2 at 37 ◦ C. The culture
medium was changed daily. Cells were seeded in 48-well plates at 103
cells per well in 700 μl DMEM (10 % FBS, 1 % penicillin) supplemented
with 0.1, 1, and 10 % (v/v) sterilized HBC-MA solution, and incubated at
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Carbohydrate Polymers 251 (2021) 117036
37 ◦ C in 5 % CO2 atmosphere. After 6, 12, and 24 h incubation, 700 μl of
the staining solution was added according to the manufacturer’s pro­
tocol, and cells were imaged using fluorescence ECLIPSE Ts2 microscope
(Nikon; Tokyo, Japan). The imaged cells were counted using ImageJ
1.50a software (National Institutes of Health, Bethesda, Maryland). The
ratio of live cells to the total number of cells was used as a metric of cell
viability.
2.13. Cell counting kit-8 (CCK-8) assay
To further investigate the cell proliferation and cytotoxicity of the
HBC-MA, cell viability was investigated using a Cell Counting Kit-8
(CCK-8; Dojindo, Kumamoto, Japan). HBC-MA was prepared using the
same method (as described in the Section 2.13). Cells were seeded in 96-
well plates at 1000 cells per well in 100 μl of DMEM (10 % FBS, 1 %
penicillin) supplemented with 0.1, 1, and 10 % (v/v) sterilized HBC-MA
solution, and incubated at 37 ◦ C in 5 % CO2 atmosphere. After 24, 48,
and 72 h incubation, the CCK-8 assay was performed according to the
manufacturer’s protocol. After incubation at 37 ◦ C for 2 h, the absor­
bance of the samples was measured at 480 nm with a Microplate
Spectrophotometer (Biotek Instruments, Vernon Winston, USA).
2.14. Subcutaneous injection study of HBC-MA hydrogels in mice
All experimental procedures were approved by the Institutional
Animal Care and Use Committee (IACUC) of Konkuk University (KU-
20035) and these mice were handled in accordance with the guidelines
approved by the IACUC of Konkuk University. The hydroxybutyl
methacrylated chitosan (HBC-MA) solution was injected into mice
without incision, and experiments were conducted to confirm that
thermal- and photo-crosslinked stable hydrogels could be formed.
Additionally, it was intended to determine the biodegradation tendency
of hydrogels according to the control of photo-crosslinking in vivo. HBC-
MA solution was sterilized using a PES syringe filter (0.22 μm pore size;
Millipore; Bedford, Massachusetts, USA). Athymic nude mice (20~30 g,
8 weeks old, male) were purchased from Orient Bio (Seongnam,
Kyunggido, Korea). Four mice per group (0 s, 10 s, 20 s, and 30 s) were
used and then the implanted constructs were retrived at 1, 3, and 5 days.
HBC-MA solution without visible-light exposure (0 s) was used as con­
trol. The dorsal skin of each mice was divided into 4 types according to
the light exposure time. Total of twelve mice were weighed and intra­
peritoneally injected using alfaxan 24 mg/kg and xylazine 10 mg/kg.
After sterilizing the injection site with 70 % (v/v) ethanol (99 %), HBC-
MA solution was injected into the subcutaneous dorsal through the
needle. All injection procedures were performed identically. To main­
tain a constant injection volume, a syringe pump (Legato 101; KD Sci­
entific, Holliston, MA, USA) was run for 10 s at an injection rate of 0.6
ml/min for all the mice to inject 100 μl of 3 % (w/v) HBC-MA solution.
Photo-crosslinking was induced by exposing the injection site to visible
light using the Omnicure S2000 (Lumen Dynamics, Canada), which is
equipped with a visible light filter (450~550 nm), for a defined time
period (0, 10, 20, and 30 s). Other than at the injection site, the light was
completely blocked. The light intensity was 10 mW/cm2. For safety
reasons, the light guide was always at least 3 cm away from the mice.
After the visible-light exposure step, the mice were placed in the mouse
cage. After 1, 3, 5 d, mice were intraperitoneally injected using alfaxan
and xylazine, and then the dorsal subcutaneous skin was resected using
surgical instruments. The obtained dorsal skin samples were fixed in 4%
formaldehyde and then hydrogel samples were carefully isolated. The
separated hydrogel was weighed immediately. Then each hydrogel
sample was lyophilized and the dry weight (Dt) was recorded. Four
hydrogel samples made with 100 μl HBC-MA solution were measured for
freeze-dry weight immediately after preparation and the mean weight
(D0) was recorded. Mass loss (%) was calculated as follows:
Mass loss (%) = (D0 − Dt) / D0 × 100
J.W. Seo et al.
Fig. 1. (A) Chemical structure of HBC-MA. Hydroxybutylation substituted for pristine chitosan is shown in blue, and methacrylation is shown in red. (B) 1H NMR of
pristine chitosan (black) versus HBC (blue), HBC-MA (red) to confirm chemical modification. (C) FTIR spectra of chitosan, HBC, and HBC-MA. (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version of this article).
2.15. Statistical analysis
To evaluate statistical significance, an ordinary one- and two-way
analysis of variance (ANOVA) followed by Tukey test was performed.
Data are mean ± standard deviation (SD) and means were compared
using unpaired Student’s t-tests. A p-value<0.05 was considered to
indicate statistical significance. All analyses were performed using
GraphPad Prism 8.0.2 (GraphPad Software; La Jolla, CA).
3. Results and discussion
3.1. Synthesis and characterization of HBC-MA conjugate
3.1.1. 1H Nuclear magnetic resonance spectroscopy
Chitosan was reacted with epoxybutane to obtain HBC through a
substitution reaction of epoxybutane to hydroxy and amine group under
neutral condition. Then, HBC-MA was obtained through reaction be­
tween unsubstituted amine group of HBC and methacrylic anhydride.
New signals at 0.92 and 1.5 ppm (peak 1~2, Fig. 1A and B) were
observed due to the substitution of methyl group of the hydroxybutyl.
The degree of hydroxybutylation was calculated based on the ratio of the
integrated area of the peaks of chitosan moiety between 3.5 and 4.0 ppm
(peaks 5~9, Fig. 1A and B) to that of the CH2 peaks of the hydroxybutyl
group at 0.92 and 1.5 ppm. The degree of hydroxybutylation of chitosan
was found to be 73.64 % by 1H NMR using the above method. Peaks at
5.7 and 6.11 ppm (peak 3, Fig. 1A and B) are methylene peaks that occur
as covalent bonds in methacryloyl groups are replaced by meth­
acrylamide. The methyl protons of the methacryloyl group in
4
Carbohydrate Polymers 251 (2021) 117036
methacrylic anhydride bound to HBC-MA were labeled at 1.8 ppm (peak
4). This demonstrated that HBC with the addition of hydroxybutyl
groups, a modification of chitosan, resulted in the successful addition of
methacrylamide to a proton of -NH2. The peaks from 3.5 to 4.0 ppm
(peaks 5~9, Fig. 1A and B) corresponded to the non-anomeric protons
(Yang et al., 2012). The degree of methacrylation was calculated based
on the ratio of the integrated area of the peaks of chitosan moiety be­
tween 3.5 and 4.0 ppm (peaks 5~9, Fig. 1A and B) to that of the CH2
peaks of the methacrylate group at 5.7 and 6.11 ppm (peak 3, Fig. 1A
and B). Since methacrylic anhydride bonds mainly to reactive amine
groups on the chitosan backbone, the degree of methacrylation of chi­
tosan was found to be 24.8 % by 1H NMR using the above method
(Valmikinathan et al., 2012).
3.1.2. Fourier transform infrared spectroscopy (FTIR)
The synthesis of HBC-MA was also confirmed through FTIR com­
parison. In the FTIR of chitosan, a broad band in the region of
3247~3398 cm− 1 corresponded to the overlapping of the vibrations of
− OH and -NH. The bands at 1024 and 1067 cm− 1 presented the char­
acteristic band of 6− OH in chitosan. A peak of 1153 cm− 1 is typically
attributed to asymmetric C–O–C bridge. The absorption bands at 2911
and 2873 cm− 1 can be presented to C–H symmetric and asymmetric
stretching (Fernandes Queiroz, Melo, Sabry, Sassaki, & Rocha, 2015). In
the FTIR of HBC, the band at 1020~1060 cm− 1, which mainly represents
6− OH, is found to be less than that of the FTIR of chitosan spectra. The
new peaks at 2944~2892 cm− 1 and 1585 cm− 1 indicate bending and
C–H stretching of the newly formed CH3 groups due to the introduction
of 1,2-epoxybutane. It was confirmed that the hydroxybutyl group was
mainly substituted with 6− OH (Wang et al., 2013). In the FTIR of
HBC-MA, a peak rise for HBC-MA at 1614 cm− 1 associated with the NH2
J.W. Seo et al. Carbohydrate Polymers 251 (2021) 117036

Fig. 2. Reversible physical properties of HBC-MA hydrogel. (A) Schema to describe the network formation of thermal-crosslinked HBC-MA hydrogel. Interactions
between hydrophobic chains occur with deswelling of H2O at physiological temperatures. (B) Schema to describe a hydrogel network; the methacrylic group of HBC-
MA was crosslinked using visible light irradiation in the presence of a photo-initiator at 20 ◦ C. (C) Schema to describe Hybrid-crosslinked HBC-MA hydrogel network
with both thermal-crosslinking and photo-crosslinking at 37 ◦ C. This was accompanied by a deswelling of H2O and showed reversible characteristics with respect to
temperature. (D~F) SEM images of micropore structure according to different crosslinking methods: thermal, photo, and hybrid-crosslinking. The magnifications of
SEM images are x100 and x300. (G~I) Pore distribution frequency obtained from SEM images of hydrogels prepared by different crosslinking methods: thermal,
photo, and hybrid-crosslinking. (J) Average pore size according to cross-linking method: thermal, photo, and hybrid-crosslinking. * p < 0.05, *** p < 0.001 vs.
hybrid-crosslinking group. Data are represented in means ± SD, n = 3. (K) Absorbance graph measured using TNBSA for glycine deswelling by reversible hybrid-
crosslinking. Data are represented in means ± SD, n = 3. A photograph of the water deswelling is attached.

band was observed. Newly formed amide 3, 2, and 1 band absorbances
corresponded at 1306, 1542, and 1710 cm− 1. This confirms that the
additional substitution from HBC to HBC-MA was successfully conju­
gated (Fig. 1B).
3.2. Preparation and characterization of HBC-MA hydrogel
3.2.1. Morphological analysis
The effect of cross-linking methods on the morphology of HBC-MA
hydrogels was investigated by SEM. Scanning electron microscopy

5
J.W. Seo et al. Carbohydrate Polymers 251 (2021) 117036

Fig. 3. Rheological analysis and mechanical characterization. (A) Temperature dependent storage modulus (G’) and loss modulus (G’’) of HBC-MA aqueous solution.
In this process, temperature is increased from 4 to 50 ◦ C (heating rate: 1 ◦ C; frequency: 1 rad/s). (B) The temperature-gelation point at which G’ intersects G’’ is
shown. (C) Mechanical properties of photo-crosslinked and hybrid-crosslinking HBC-MA with various visible light exposure times. Compressive modulus was
compared with the time of exposure to visible light. *** p < 0.001 vs. photo-crosslinking 10 s; ### p < 0.001 vs. hybrid-crosslinking; +++ p < 0.001 between the
indicated group. Data are represented in means ± SD, n = 8. (D) A representative stress-strain curve of photo-crosslinking and hybrid-crosslinking HBC-MA hydrogel
with visible light exposure time.

micrographs clearly show the difference in pore size of the lyophilized
hydrogel according to the crosslinking mechanism. HBC-MA solution
forms hydrophobic interaction domains by binding hydrophobic chains
surrounded by water molecules at 37 ◦ C. This process is accompanied by
an evacuation phenomenon in which water molecules are deswelled
(Zhang, Xu, Cheng, & Zhuo, 2008). The thermal crosslinked hydrogel
forms a porous matrix, but the micropores are relatively larger in
diameter (Fig. 2D). When the HBC-MA solution is exposed to visible
light, photo-initiators bind between methacrylamide chains to form
crosslinking networks (Kuwajima, Yoshida, & Hayashi, 1976). When the
degree of methacrylation of chitosan was 24.8 % and exposed to short
time visible light for 10 s, the photo-crosslinking bridge was shorter than
the chitosan backbone, resulting in an elongated micropore (Fig. 2E). If
more methacrylamide is introduced or exposed to light for a longer time,
it is expected to form a denser matrix. Hydrogels formed by
photo-crosslinking are responsive to temperature and can form tighter
and more consistent micropores in response to temperature (37 ◦ C)
(Fig. 2F). There was a difference in distribution of pore size depending
on the crosslinking method used. The micropore size of the
thermal-crosslinked samples showed an irregular distribution (Fig. 2G),
whereas the micropore size of the photo-crosslinked sample was uni­
formly distributed (Fig. 2H). A smaller pore size and more regular
structure than the pore structure of thermal-crosslinking hydrogel was
observed. In the hybrid-crosslinked sample, the pore distribution was
more constant than the photo-crosslinked sample (Fig. 2I). This more
compact and regular pore structure provides additional mechanical
strength stability. The average pore sizes for lyophilized temperature,
photo, and hybrid-crosslinking hydrogel were 69.5 ± 5.5, 27.9 ± 3.5,
and 18.0 ± 1.9 μm, respectively (Fig. 2J).
3.2.2. Reversible characteristics of HBC-MA hydrogel
The temperature reversible characteristics of hybrid-crosslinking
hydrogels were investigated by applying the internal water molecule
evacuation phenomenon (Zhang, Xu, Cheng, & Zhuo, 2008). The reac­
tion of HBC-MA with temperature occurs through
hydrophobic-interaction. As the temperature rises, crosslinking occurs
between hydrophobic chains. This process is accompanied by an evac­
uation phenomenon in which water molecules are released. The
photo-crosslinked HBC-MA hydrogel gradually became opaque due to
the hydrophobic-interaction that occurred during hybrid-crosslinking at
37 ◦ C, and gradually decreased in volume as water releasing occurred
(Videos 1, Supporting Information). Conversely, the hybrid-crosslinked
hydrogel showed reversible characteristics in response to low temper­
ature (20 ◦ C). When the temperature is lowered, crosslinking between
the hydrophobic chains is released. The opaque HBC-MA hydrogel
became transparent enough to see the text on the back and the volume
increased again by swelling the surrounding water quickly (Videos 2,
Supporting Information). As a representative material of hydrogel,
PEG-DMA, which has low reactivity to temperature, was used as a
control. If the photo-crosslinked HBC-MA hydrogel can form additional
crosslinking according to temperature, it is assumed that the evacuation
phenomenon will occur, and the hydrogel will release the
glycine-dissolved water. In this case, it is possible to know whether the
thermal crosslinking of HBC-MA occurred when the glycine concentra­
tion of PBS in which the hydrogel remained when the crosslinking
occurred, and the crosslinking was released by temperature was
measured. When glycine-loaded photo-crosslinked HBC-MA hydrogel
was transferred to PBS at 20 ◦ C and 37 ◦ C at 30-minute intervals, the
absorbance was measured after reacting the released glycine with

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J.W. Seo et al. Carbohydrate Polymers 251 (2021) 117036

Fig. 4. Swelling and degradation characteristics. (A) Swelling of photo-crosslinked HBC-MA hydrogel from 0 to 36 h at 37 ◦ C in PBS. Photographs were taken before
and after the deswelling. Data are represented in means ± SD, n = 3. (B) Degradation of hybrid-crosslinked HBC-MA hydrogel from 0 to 21 d at 37 ◦ C. Two groups
(PBS and 0.5 mg/mL lysozyme) with three visible light exposure conditions (10, 20, and 30 s) were tested according to the lysozyme concentration. Data are
represented in means ± SD, n = 3. (C) Degradation of photo-crosslinked and hybrid-crosslinked HBC-MA hydrogel from 0 to 21 d. Two groups (20 and 30 ◦ C) with
three visible light exposure conditions (10, 20, and 30 s) were tested according to the temperature condition. Data are represented in means ± SD, n = 3.

TNBSA. Hydrogel reswelled the water at 20 ◦ C and released the water
containing glycine at 37 ◦ C. The results showed that glycine was
released even after repeated reversible temperature changes. This con­
firms that hybrid-crosslinking and photo-crosslinking can be reversibly
switched on and off by changing the temperature. In addition, the
shorter photo-crosslinking time (10 s) resulted in unexpected glycine
loss, even at 20 ◦ C. Perhaps the loss of glycine occurs as the pore, where
the hybrid-crosslinking hydrogel condenses, is released again (Fig. 2K).
It was confirmed that the temperature-reactive crosslinking was a
reversible reaction and was accompanied by a swelling and deswelling
phenomenon. Through this data, the photo-crosslinked hydrogel can be
a hybrid-crosslinked hydrogel at physiological temperature, and the
hybrid-crosslinked hydrogel can be converted into photo-crosslinked
hydrogel in response to low temperature (20 ◦ C). This characteristic
can be expected to be applied to induce stimuli-drug release by inten­
tionally creating low temperature environment in the hydrogel during
drug release through continuous deswelling via physiological tempera­
ture in the body.
3.2.3. Rheological analysis
Rheology analysis was performed to characterize the sol-gel transi­
tion of HBC-MA solution as a function of temperature. As shown in
Fig. 3A and B, the loss modulus (G”) is higher than the storage modulus
(G’) at the beginning of the measurement at 4 ◦ C, but both show very
low coefficients. As the temperature rises, the sol-gel transition takes
place, and the storage modulus also rises slightly (Fig. 3A). However,
depending on the increase in temperature, storage modulus (G’) and loss
modulus (G”) showed different rates of rising. The gelation point was
defined as the temperature at which the storage modulus (G’) and loss
modulus (G’’) intersect. The intersection of the storage modulus and loss
modulus of the HBC-MA solution was 27.3 ◦ C, confirming that this point
is the temperature-gelation point (Fig. 3B). As the temperature rises past
the gelation point, the storage modulus rises at a faster rate. In this
experiment, the gelation temperature of 3 % (w/v) HBC-MA solution
was 27.3 ◦ C. It was confirmed that thermal-crosslinking could be
exhibited at physiological temperature.
3.2.4. Mechanical analysis
The reactivity of the HBC-MA solution was considerably high, so that
photo-crosslinking occurred in a short time. When exposed to visible
light, photo-crosslinking between the methacrylamide group and the
photo-initiator resulted in the formation of a tighter pores as the visible
light exposure time increased. When exposed to visible light for 10 s,
compressive modulus of the hydrogel sample was 15.1 ± 1.7 kPa. It was
confirmed once again that hydrogels could be formed even after expo­
sure to visible light for a short time (10 s). The 20 and 30 s groups
showed a statistically high difference compared to the 10 s group. The
20 and 30 s groups were 34.1 ± 2.9 kPa (*** p < 0.001) and 42.0 ± 4.1
kPa (*** p < 0.001), respectively. In the case of hybrid-crosslinking, the
compressive modulus of 10 s (hybrid) was 25.7 ± 2.7 kPa (+++ p <
0.001), which is 41.5 % higher than 10 s exposure only to visible light.
This was a statistically significant difference. Due to thermal cross­
linking, additional pore formation affected compressive modulus. The
20 and 30 s (hybrid) groups did not show statistically significant dif­
ferences when compared to the 20 and 30 s groups. The 20 and 30 s
(hybrid) groups were 37.4 ± 2.2 kPa and 45.4 ± 2.8 kPa, respectively.
Increasing the degree of photo-crosslinking by increasing the visible
light exposure time decreases the mobility of the polymer, thus limiting
the hydrophilic-hydrophobic interactions that occurred as the temper­
ature rises, which led to no significant difference in modulus (Fig. 3C).
Differences between photo-crosslinking and hybrid-crosslinking groups
were also observed in the strain-stress curve. As the visible light expo­
sure time increased, the initial strain (~ 40 %) showed high stiffness for
each group. In strain later point, yield points appeared at lower strains
and yield stresses tended to decrease. As a result, both groups had
stronger intensities with increased exposure to visible light and had

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J.W. Seo et al. Carbohydrate Polymers 251 (2021) 117036

Fig. 5. Cell viability analysis by Live/Dead assay. (A) Image of NIH3T3 fibroblasts stained for 6 to 24 h by calcein-AM (green) / ethidium homodimer (red) Live/
Dead assay in media containing various concentrations of HBC-MA prepolymer solution concentration (0, 0.1, 1, and 10 %). The scale bars represent 100 μm and
magnification is x100. (B) NIH3T3 viability (%) according to HBC-MA concentration from Live/Dead image analysis. There was no statistically significant difference.
Data are represented in means ± SD, n = 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

lower elastic regions. But when comparing the two groups at the same
visible light exposure time, a unique difference was observed. At 10 s,
hybrid-crosslinking samples with additional thermal crosslinking
compared to photo-crosslinking only had a lower yield stress value that
differed by more than 40 kPa at lower or similar strain points. At 20 s,
the strain point of hybrid-crosslinking was reduced compared to photo-
crosslinking, but the difference gap was not significant compared to 10 s.
However, the addition of thermal crosslinking increased the deforma­
tion rate. At 30 s, the strain of hybrid-crosslinking increased by about 10
% compared to photo-crosslinking. But in contrast to the previous re­
sults, it was found that the fracture stress showed only minor change
(Fig. 3D). The 10 s (hybrid) sample with the largest change in thermal
properties showed a decreased fracture stress as the compressive
modulus increased due to thermal crosslinking. On the other hand, the
30 s (hybrid) sample, which had no significant difference in compressive
modulus due to the change of thermal properties, showed a large change
in deformation as strain increased to the highest value.
3.2.5. Swelling test
The swelling of the hydrogel network is important in applications as
it affects mechanical properties and solute diffusion (Peppas, Hilt,
Khademhosseini, & Langer, 2006). As has already been reported, in
conventional polymer scaffolds, the gel expands due to the interaction
between the polymer and solvent depending on the pore size (Chen,
Park, & Park, 1999). But in the case of HBC-MA polymer, hydrophobic
interaction could occur due to the temperature (37 ◦ C) even after
photo-crosslinking. The interaction of hydrophobic residues with hy­
drophilic residues results in condensation and release of water (Zhang &
Chu, 2004). During formation of the polymer network by temperature,
the ratio between the swollen hydrogel and the dry hydrogel was
reduced. When the occurrence of additional crosslinking due to tem­
perature reached saturation, the swelling ratio of the hydrogel showed
the lowest ratio. The lowest swelling ratio was observed at 4–6 h (2 h).
Hydrogel converted to hybrid-crosslinking after saturated thermal
crosslinking showed an increase in the swelling curve from 6 to 12 h.
After 12 h, the hybrid-crosslinked hydrogel a maintained equilibrium
swelling ratio (Fig. 4A). This data showed that temperature-sensitive
crosslinking is possible even after photopolymerization once again,
and deswelling can be continuously maintained in a physiological
temperature environment such as 37 ◦ C.
3.2.6. Degradation test
Chitosan is known to degrade glycoside bonds by lysozyme (Pan­
gburn, Trescony, & Heller, 1982). The lysozyme mainly contributes to
the initial degradation rate because it binds to the hexamer bond site
with 3–4 acetylated units of chitosan (Pangburn et al., 1982). To confirm
the difference in the biodegradation pattern of HBC-MA hydrogel ac­
cording to the light exposure time, experiments were conducted under
three light exposure time conditions (10, 20, and 30 s). First, to further
investigate the degradation patterns of HBC-MA, lysozyme (40,000

8
J.W. Seo et al. Carbohydrate Polymers 251 (2021) 117036

Fig. 6. Cell viability analysis by cell counting kit-8 (CCK-8) assay. (A) NIH3T3 fibroblasts in media containing various concentrations of HBC-MA (0, 0.1, 1 and 10 %)
from 12 to 72 h by CCK-8 assay. It is a relative graph comparing the measured absorbance with that of the same time course (h) based on the 0 % group. ** p < 0.01,
*** p < 0.001 vs. Same time course (h) in 0% group. Data are represented in means ± SD, n = 7. (B) CCK-8 absorbance graph from 12 to 72 h of 10 % (v/v) HBC-MA
concentration in culture media group. ** p < 0.01, *** p < 0.001 vs. 12 h. Data are represented in means ± SD, n = 7. (C) Image by concentration at 48 h. The scale
bars represent 100 μm and magnification is x100.

U/mg, Sigma-aldrich, USA) was added to PBS at a concentration of 0.5
mg/mL and compared at 37 ◦ C. In the 10 s condition where the most
biodegradation occurred, 47.9 ± 2.4 % degraded, showing the fastest
biodegradation rate in the early stage. In comparison, the biodegrada­
tion rate was relatively slow at 32.2 ± 2.7 % at 30 s condition. At 14 d,
most of 10 s condition degraded, and most the 20 s condition degraded
at 21 d. However, in the 30 s condition, only 76.1 ± 3.2 % was bio­
degraded even at 21 d, and the remaining hydrogel was present. On the
other hand, in the PBS group without lysozyme, the biodegradation
tendency was relatively slow for the initial 7 d. The mass loss ratio did
not exceed 40 % in 10, 20, and 30 s conditions for 21 d. Through this, the
modified chitosan HBC-MA did not inhibit the activity of lysozyme and
was confirmed to be affected by lysozyme (Fig. 4B). Next, the biode­
gradability of HBC-MA hydrogel at low temperature (20 ◦ C) was
confirmed. Unlike the biodegradation tendency shown at 37 ◦ C, no
significant change occurred after 7 d under all conditions. In addition,
no mass loss took place more than 20 % (Fig. 4C). Through this, it was
confirmed that HBC-MA showed tendency to biodegrade at low tem­
perature. It can be anticipated that HBC-MA hydrogel or solution can be
stored for a relatively long time when stored at low temperatures. These
data suggested biodegradability of HBC-MA can be controlled according
to temperature and light exposure time and has the potential to be
optimized and applied according to the specific application.
3.2.7. Visual confirmation of co-nonsolvency effect and crosslinking in vitro
Hydroxybutyl methacrylamide chitosan (HBC-MA) is a polymer in
which both hydrophilic and hydrophobic groups coexist, and when
HBC-MA solution is added to photo-initiator, the HBC-MA solution
shows a co-nonsolvency effect (Wolf & Willms, 1978). We hypothesized
that if a co-nonsolvency effect can be exhibited in an aqueous environ­
ment, crosslinking would be possible. Deionized water was prepared to
minimize the effects of ions and HBC-MA solution (20 ◦ C) was carefully
injected into the 37 ◦ C deionized water using a syringe (Fig. S1A, Sup­
porting Information). As expected, the HBC-MA solution was
successfully crosslinked by the co-nonsolvency effect but was not diluted
upon injection in deionized water. Subsequent thermal-crosslinking by
the temperature took place and was indicated by the alteration of
hydrogel color (opaque pink) (Fig. S1B, Supporting Information).
Sensitivity to temperature was confirmed to operate as expected. In
addition, it was exposed to visible light to confirm its sensitivity to light
(Fig. S1C, Supporting Information). Eosin Y, which was used as a
photo-initiator, is shown as yellow and colorless when exposed to visible
light (Noshadi et al., 2017). The area cross-linked by visible light turned
yellow or colorless, providing visual confirmation of the sensitivity to
light (Fig. S1D, Supporting Information). HBC-MA was not diluted in
PBS as well as deionized water, and it was confirmed that the temper­
ature and photo-crosslinking functioned as behavior of HBC-MA. In
addition, it was confirmed that crosslinking with visible light is possible
after temperature sensing (Videos 3, Supporting Information).
3.3. In vitro cytotoxicity and cell viability test
3.3.1. Live/dead fluorescence assay
To evaluate the effect of HBC-MA and photoinitiator on cell viability,
NIH3T3 cells were cultured in media containing different concentrations
of HBC-MA solution. To compare the cytotoxicity of HBC-MA solution,
we used the NIH3T3 grown in cell-cultured media as a control group. At
the 6, 12 and 24 h time point, cell viability of all groups was higher than
90 %, and therefore statistically significant cytotoxicity compared to
NIH3T3 cells grown without HBC-MA solution was not shown (Fig. 5A
and B).
3.3.2. Cell counting kit-8 (CCK-8) assay
To further evaluate the effect of HBC-MA and photoinitiator on cell
viability and proliferation, NIH3T3 cells were cultured with media
containing different concentrations of HBC-MA solution and subject to
CCK-8 assay at 12, 24, 48, and 72 h. To confirm the cytotoxicity of HBC-
MA, relative cell viability was compared based on the NIH3T3 control

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J.W. Seo et al. Carbohydrate Polymers 251 (2021) 117036

Fig. 7. Subcutaneous injection study of HBC-MA hydrogels. (A) Using a syringe pump, HBC-MA solution was injected into mice at 100 μl. (B) Visible light was
irradiated on the back of respiratory anesthesia mice without incision. The light source was at least 3 cm away from the mice. The light intensity is 10 mW/cm2. The
0 s type was excluded from this process. (C) Confirmation of hydrogel formation after HBC-MA injection 1, 3, and 5 d later. (D) Representative photograph showing
samples after 1, 3, and 5 d for each visible light exposure time after injection. (E) Hydrogel samples were measured for each visible exposure time after injection and a
defined day. Data are represented in means ± SD, n = 4. (F) Degradation of injected HBC-MA hydrogel from 1 to 5 d in mice. Four types (0, 10, 20, and 30 s) were
tested according to visible light exposure time. Data are represented in means ± SD, n = 4.

group grown on cell-cultured media. In the 0.1 % and 1% HBC-MA
prepolymer solution groups, except for 0.1 % of 24 h, there was no
statistically significant difference in cell viability and proliferation from
the control group. However, at 48 and 72 h, the 10 % group showed a
statistically significant decrease of 70.4 ± 0.7 % (*** p < 0.001) and
64.2 ± 2.4 % (*** p < 0.001), respectively (Fig. 6A). In the same group,
which showed a decrease in cell viability, the absorbance was checked
again to see if proliferation was limited in the cell. Only the 10 % group
was rechecked to see if cell viability was reduced due to limited cell
proliferation (Fig. 6B). Cell proliferation was occurring at all time
points, including the 48 and 72 h samples, which showed a decrease in
cell viability. As a result, the absorbance (480 nm) showed a statistically

10
J.W. Seo et al.
significant increase (*** p < 0.001). These results occurred when HBC-
MA solution reacted with 37 ◦ C of the incubator to form a tiny hydrogel.
Tiny hydrogels in the 10 % group were clouded over the cell, partially
interfering with its nutrient transport. For additional reasons, this also
affected the lack of physical space for cell proliferation in cell-culture
dishes (Fig. 6C). HBC-MA was not toxic to NIH3T3 cells but also did
not provide a space for cells to grow physically due to a lack of cell
adhesion to NIH3T3 while forming hydrogels.
3.4. Subcutaneous injection study of HBC-MA hydrogels in mice
Subcutaneous injection study was performed without an incision to
identify the potential for application as a minimally invasive injectable
hydrogel and biodegradability of the hydrogel in vivo. For the subcu­
taneous injection study, 100 μl of HBC-MA solution was injected using a
syringe pump (Fig. 7A). As expected, the HBC-MA solution retained a
convex shape throughout the experiment at the injection site without
any manipulation due to the viscosity and co-nonsolvency effects of
HBC-MA. There was no change in the convex morphology even after
exposure of visible light (0, 10, 20, and 30 s) at different times for each
type in the dorsal skin of mice without incision (Fig. 7B). After a defined
time course, the mice were sacrificed, and the injected hydrogels were
carefully separated using surgical instruments (Fig. 7C and D). Photo­
polymerization of the HBC-MA solution was successful and temperature
sensitivity was also confirmed by the 0 s types in mice. All samples
obtained from mice were weighed and compared. After day 1, the
weight of the hydrogel obtained from mice was heavier with longer
visible light exposure time to visible light. The 1 d group in the 0 s type
with the lowest weight was 31.4 ± 5.2 mg, and the 1 d group in the 30 s
type with the most weight was 13.4 ± 2.3 mg, which showed significant
difference. The tendency for weight to decrease over time also showed a
significant difference (Fig. 7E). As can be seen in previous experiments,
increasing the degree of photo-crosslinking tended to limit thermal
crosslinking. Conversely, a sample with a low degree of photo-
crosslinking formation initially showed more water release due to
thermal crosslinking, thus showing a low hydrogel weight. However, it
was judged that the degree of water content was not the only factor
causing the change in hydrogel weight. After the hydrogel obtained from
mice was lyophilized to remove all moisture, degradation ratio was
analyzed. Through the in vitro degradation experiment, it was confirmed
that there was a difference in the tendency of degradation of HBC-MA
hydrogel according to the crosslinking density. Samples from early in
vivo time points showed faster biodegradability. The 0 s type with
temperature crosslinking only showed high biodegradability of 61.3 ±
16 % in 1 d group. At 5 d group, most hydrogels were degraded during
the experiment, with mass loss of 92.5 ± 4.1 %. On the other hand, the
30 s type with the longest visible light exposure and additional thermal-
crosslinking due to physiological temperature was 25.2 ± 12.3 % at 1
d group, and 36.5 ± 3.3 % at 5 d group. It showed similar results to the
degradation experiment using lysozyme in vitro. It was confirmed that
there was a difference in degradation of HBC-MA hydrogel according to
the crosslinking density. Unlike in vitro experiments, the degree of
degradation with temperature could not be tested.
4. Conclusion
In this study, we synthesized hydroxybutyl methacrylated chitosan
conjugate and investigated its thermo-sensitive and photo-crosslinkable
characteristics for injectable hydrogel system. The HBC-MA solution
exhibits co-nonsolvency characteristics at neutral pH at 37 ◦ C, so ther­
mal crosslinking can be achieved even under physiological conditions.
The photo-crosslinked HBC-MA hydrogel formed smaller micropore
sizes than the thermo-crosslinked gel, and hybrid-crosslinked HBC-MA
hydrogel significantly improved mechanical strength and structural
stability under physiological conditions. The improved stability is ex­
pected to broaden the application range of thermal-crosslinkable
11
Carbohydrate Polymers 251 (2021) 117036
hydrogels. Despite chemically photo-crosslinking, the hybrid-
crosslinked hydrogel was biodegradable in the presence of lysozyme.
In addition, it was confirmed that degradation optimization according to
temperature and light exposure degree is possible. Slow biodegrad­
ability at 20 ◦ C showed the storage potential of HBC-MA solution or
hydrogel at low temperature. Cytotoxicity and cell viability test using
NIH3T3 cell confirmed the biocompatibility of HBC-MA conjugate. The
subcutaneous injection of the HBC-MA solution into the mice allowed
the formation of thermogel successfully without incision. Further visible
light irradiation from outside the skin greatly increased the stability of
the hydrogel in vivo. The results of the in vivo experiment showed a
similar tendency as the in vitro experiment. It has been demonstrated
that the photo-crosslink density can be adjusted in vivo with visible light
exposure time, and various elements (degradation and mechanical
properties) can be optimized accordingly. Taken together, the biocom­
patible and hybrid-crosslinkable HBC-MA can be considered as an
injectable hydrogel material with high potential in a variety of appli­
cations, including drug delivery system and tissue engineering.
Author contributions
Hojae Bae and Jeong Wook Seo designed the experiments; Jeong
Wook Seo and Seon Young Shin performed the experiments. Hojae Bae
and Jeong Wook Seo arranged the data and wrote the manuscript; Hojae
Bae, Jeong Wook Seo, Su Ryon Shin, Min Young Lee, Jae Min Cha,
Kyung Hyun Min, and Sang Cheon Lee revised the manuscript. All au­
thors significantly contributed to the study and to the interpretation of
the data.
Funding
This article was supported by a 2016 grant from the Konkuk
University.
Declaration of Competing Interest
All the authors declare no financial or commercial conflict of interest
that are directly related to the content of this article.
Acknowledgements
None
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.117036.
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