Radiation-crosslinked scaffolds from gelatin/CM-chitin and gelatin/CM-chitosan hydrogels for adipose-derived stem cell culture
Nuclear Science and Technology, Vol.10, No. 3 (2020), pp. 13-21
Radiation-crosslinked scaffolds from gelatin/CM-chitin and
gelatin/CM-chitosan hydrogels for adipose-derived stem cell culture
Dang Van Phu1,*, Nguyen Quoc Hien1, Le Anh Quoc2, Nguyen Ngoc Duy1, Phan Phuoc Thang1,
Nguyen Thi Ly1, Nguyen Thi Kim Lan1, Tran Le Bao Ha2
1Research and Development Center for Radiation Technology, Vietnam Atomic Energy Institute,
202A, Street 11, Linh Xuan Ward, Thu Duc District, Ho Chi Minh City
2Faculty of Biology and Biotechnology, University of Science, Vietnam National University Ho Chi Minh City,
227-Nguyen Van Cu, Ward 4, District 5, Ho Chi Minh City
(Received 06 October 2020, accepted 30 October 2020)
Abstract: Radiation technology has been providing a useful tool to modify polymers involving the
radiation crosslinking of hydrogels. In the present work, two kinds of hydrogels composed gelatin and
carboxymethyl chitosan (gelatin/CM-chitosan), and carboxymethyl chitin (gelatin/CM-chitin) were
prepared by the radiation crosslinking. The resulting hydrogels were freeze-dried, sterilized under
gamma-ray at a sterilization dose of 25 kGy, and characterized to be utilized as the scaffolds for
culturing adipose-derived stem cells (hADSCs). The physicochemical properties, biodegradability,
and cytotoxicity of these scaffolds were also investigated. The results indicated that both CM-chitosan
and CM-chitin much improved the swelling capacity, porosity, and pore size of the gelatin-derived
scaffolds. The swelling degree of gelatin/CM-chitosan and gelatin/CM-chitin scaffolds was about 7-9
g/g after 6 hrs immerging in PBS, and their porosity was about 70-73% with pore size ranging from
100 to 300 mm. The results also revealed that the compressive modulus of gelatin/CM-chitosan and
gelatin/CM-chitin was 45.6 and 66.4 kPa, respectively, which were suitable for soft tissue engineering.
Both scaffolds were enzymatically biodegradable by collagenase and non-cytotoxic for hADSCs with
an RGR of ~97%. Thus, the resultant scaffolds can be suitably utilized for culturing hADSC in
practice to regenerate soft tissues.
practice, the hydrogel-scaffold materials in
tissue engineering ought to meet several design
criteria to mimic ECM, which is dependent on
the cultured source cells and the targeting
tissues. Alternatively, in the soft tissue-
regeneration technique from the adult
mesenchymal stem cells, a biomaterial scaffold
should be owned to the functional
specifications as follows: the hydrophilic 3D-
microporous structure, porosity >70% and pore
size ~100 – 300 m, swelling degree ~6 – 12
g/g, compressive strength ~1 – 80 kPa,
admissible biodegradability, non-cytotoxicity,
and providing a good platform for cell
adhesion, function, differentiation, and
transplantation [3, 5-8]. In the last couple of
years, the human adipose-derived stem cells
I. INTRODUCTION
Tissue engineering is a quickly enlarging
interdisciplinary field that attempts to provide
solutions for the regeneration of defected or
damaged tissues. Designing of biologically
active scaffolds with optimal characteristics is
one of the most important factors for
successful tissue engineering [1]. Recently,
hydrogel has been acquired substantial
attraction as an immense potential material for
engineered tissue scaffolds due to their unique
compositional and structural similarities to the
natural extracellular matrix (ECM), which is an
indispensable component to ensure the survival,
communication,
proliferation,
and
differentiation of the cells in tissues [2-4]. In
©2020 Vietnam Atomic Energy Society and Vietnam Atomic Energy Institute
RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN…
(hADSCs), one exciting kind of the adult
gelatin on the features of hybrid scaffolds are
still lacking. This work characterizes the
scaffolds derived from gelatin/CM-chitosan
and gelatin/CM-chitin hydrogels obtained by
radiation-crosslinking in combination with
freeze-drying and radiation sterilization for
further applications.
mesenchymal stem cell sources copiously
found in the perivascular area of adipose
tissues, have been examined for application in
regeneration-tissue therapy due to their
abundance, multipotent nature, facile isolation,
and non-controversiality [7, 9]. In terms of
conventional technology, the polymer-based
mesenchymal stem cell culture scaffolds are
hydrophilic hydrogels in the form with a 3D
microporous structure, which were prepared by
chemical crosslinking [3, 8, 9], ionizing
radiation followed by freeze-drying treatment
[2, 10-12]. Among them, the radiation-
crosslinking approach is recognized as a
potential way with some advantages such as no
chemicals to initiate the reaction and the
product sterilized at the same time [1, 13]. The
crosslinking reaction in polymer solution
occurred through the recombination of free
radical macromolecules, which are produced
by the indirect and direct effect of radiation
interaction [10, 12]. The radiation-crosslinked
gelatin is a promising cell culture scaffold due
to its chemical resemblances to the ECM [11].
Despite the advantages, poorly mechanical and
physicochemical properties, and enzymatic
degradation beyond controllability have
restricted its applications and levered the
production of gelatin-based composite
hydrogels [11, 12]. Because of the many
inherent useful bio-properties of CM-chitosan
and CM-chitin, natural polysaccharide
derivatives of modified chitosan/chitin, it has
been considered as the leading materials to
make hydrogels for biomedical applications.
So far, several novel hybrid scaffolds have
been developed from a combination of
gelatin/CM-chitosan [2, 11, 12] as well as
gelatin/CM-chitin [4, 5] for the adult
mesenchymal stem cell tissue engineering.
However, studies on the effect of CM-
chitosan and CM-chitin in mixture with
II. MATERIALS AND METHODS
A. Materials
Three polymers including gelatin (CAS
9000-70-8, type A, source from porcine skin
with bloom ~300, an average molecular weight
~100.000 Da, and pI 8-9), carboxymethyl-
chitosan (CAS 83512-85-0, a deacetylation
degree of ~95%, a molecular weight ~60.000
Da, and a substitute degree of 0.87), and
carboxymethyl-chitin (CAS 52108-64-2, a
deacetylation degree of ~95%, a molecular
weight ~110.000 Da, and a substitute degree of
0.62) were purchased from Sigma-Aldrich,
Merck, Germany. PBS buffer (pH 7.4) was
supplied by Elabscience, USA. The tetrazolium
dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide was bought from
Sigma Aldrich, USA. Dulbecco’s Modified
Eagle’s Medium (DMEM)/F12 cell culture
medium was purchased from Gibco Co., USA.
The collagenase enzyme, type I (activity 2
U/ml) was a product of Sigma Aldrich, USA.
Human adipose-derived stem cells (hADSCs)
in the 4th generation were provided by the
Faculty of Biology and Biotechnology,
University of Science – Vietnam National
University Ho Chi Minh City.
B. Methods
Preparation and sterilization of
scaffolds: The solutions with a total polymer
concentration 15% (w/v) of gelatin and both
composites of gelatin/carboxymethyl-chitosan
(G/CM-chitosan) and gelatin/carboxymethyl-
14
DANG VAN PHU et al.
chitin (G/CM-chitin) with the weight ratio of
magnification of 1000 for post sputter-coated
samples. The pore sizes and morphological
shapes of porous scaffolds were also
determined using SEM images. The porosity of
the scaffolds was measured by the liquid
supplanted method [2, 3]. Briefly, the porosity
is conventionally equal to the volume of
trapped ethanol in the porous scaffolds. The
scaffold samples were put into a measuring
cylinder containing an exact volume of
absolute ethanol (V1). After 24 h of soaking,
read the mixture volume of ethanol with
immersed scaffolds (V2). Afterward, the
scaffold samples were taken out and the
residual ethanol volume was determined (V3).
The porosity (P, %) was calculated by the Eq.
2 as follows [2]:
8.5/1.5 were prepared by completely dissolving
in distilled water at 50C for 2 h. The solutions
were poured into Petri dishes (diameter 10 cm,
thickness 1 cm) and cooled to solidify in
pseudo-gel at room temperature. The pseudo-
gels were irradiated at a dose of ~35 kGy to
obtain hydrogels with crosslinked bonds [10,
11]. The hydrogels were cut into cubic
specimens 101010 mm3 in size and
lyophilized (freeze-drying) to create porous
scaffolds [5]. Afterward, the porous scaffolds
were subjected to -irradiation with ~25 kGy
for sterilization [1].
Swelling in PBS and mechanical
property of scaffolds: Swelling behavior of
porous scaffolds in PBS (0.1 M, pH 7.4) at
37C as a function of immersion-time was
P (%) = (V1 − V3) × 100/(V2 − V3)
(2)
evaluated by
calculated using Eq. (1) as follows [3, 13]:
a
weighting method and
Biodegradation and cytotoxicity tests:
Enzymatic degradation of the scaffolds was
evaluated through the weight loss after
immerging in PBS solution containing
colagenase. Briefly, scaffold sample of about
40 – 60 mg (Wo) was incubated in 5 ml
enzyme solution, which was prepared by
diluting stock collagenase in PBS (0.1 M, pH
7.4) to an activity concentration of 2 U/ml. The
biodegradation of scaffolds was carried out in
vitro at 37C for a period of 21 h. At destined
time interludes, the remainder of the samples
was removed to dry by freeze-drying and
weighed (W1). The biodegradability was
figured out using Eq. 3 [3, 11].
Swelling degree (g/g) = Ws/Wd
(1)
Where Ws and Wd are respectively the
weights of swollen scaffolds and dried
scaffolds.
The compressive modulus, a parameter
representing the mechanical property of
scaffolds, was measured using a texture tester
(INSTRON 5582, USA) equipped with a 1000
N load cell. To determine the compress
modulus, scaffolds were swollen in PBS for 24
h and cut into cylinders (diameter 10 mm,
height 10 mm) and applied a constant stain rate
of 60 mm/min [5, 11].
Biodegradability (%) = (Wo − W1) ×100/Wo
(3)
Porosity and pore size determination:
The porous scaffolds in the dry state were cut
into cubic pieces with the sizes of 101010
mm3 for measuring porosity. Microscopic
structural morphology of the scaffold was
observed by the scanning electron microscopy
(S-4800, HITACHI, Japan) with an
Cytotoxicity of the scaffolds was
appraised by an indirect contact method
through extracts and MTT assay according to
ISO 10993-5:2009 [11]. The scaffold
extraction was prepared by incubating the
scaffolds in the DMEM/F12 medium
containing 10% FBS and 1% specific antibody
accelerating voltage of 10 kV and
a
15
RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN…
with a ratio of 3 cm2/ml at 37C for 24 h, then
centrifuged to obtain extraction media. The
hADSCs with a density of 105 cells/ml were
incubated into the 96-well culture plate at 37C,
5% CO2 for 24 h. After that, the culture
medium in the cell-incubated wells was
replaced with the extraction media and then
incubated for 24 h. The cells cultured with the
DMEM/F12 medium set out as the negative
control group and latex rubber extraction that
was a cytotoxic compound was as a positive
control group [9]. The relative growth rate
(RGR, %) of the cells in treatments was
determined using the MTT assay at 24 h and
calculated according to Eq. 4 [4]. The
absorbance of MTT-dyed cell suspensions was
measured at the wavelength of 570 nm using a
spectroscopy equipped microplate reader.
penetration, the infiltration of culturing cells,
and the excretion of secretions [3]. Fig. 1
describes the swelling of the porous scaffolds
in the PBS solution. The swelling degree of
the gelatin scaffold was the lowest (about 4.8
g/g) in comparison to the G/CM-chitin (about
7.0 g/g) and the G/CM-chitosan (around 9.1
g/g). These differences may be caused by
inconformity in crosslinking density and the
number of hydrophilic groups in the scaffold,
particularly the lower the crosslinking density
and the higher the hydrophilic groups, the
higher the swelling degree. Yang et al. [11]
substantiated that CM-chitosan in a mixture of
gelatin/CM-chitosan was able to improve the
swelling degree of irradiation-crosslinked
gelatin hydrogels based on reducing the
crosslinking density and increasing the
hydrophilic groups. In addition, Zhao et al.
[13] supported that the irradiation-crosslinked
extent of CM-chitin was higher than that of
CM-chitosan, which could also be the basis
for the explanation of the difference in
swelling of the G/CM-chitosan and G/CM-
chitin (Fig. 1).
RGR (%) = As ×100/Anc
(4)
Where As and Anc are the optical
density of the test sample and the negative
control, respectively. The samples with an
RGR value higher than 70% are considered
non-cytotoxicity or quality [4].
In the more notable worth, the CM-
chitosan and CM-chitin not only ameliorated
to the swelling proportion but also improved
the mechanical property of the resulted
scaffolds from G/CM-chitosan and G/CM-
chitin. The compressive modulus or stiffness
of scaffolds plays a significant role in the
regulation of stem cell fate as well as
adhesion, migration, and differentiation. The
crosslinking density of the hydrogels is the
main element to affect scaffold stiffness [6].
Fig. 2 shows the compressive modulus of the
prepared scaffolds. The attained results
indicated that the compressive modulus of the
gelatin scaffold (about 167.5 kPa) was
effectively diminished to 45.6 kPa and 66.4
kPa for G/CM-chitosan and G/CM-chitin
scaffolds, respectively. The stiffness of the
III. RESULTS AND DISCUSSION
In these experiments, the polymeric
formulation and the radiation-crosslinking dose
were applied based on the findings as reported
in the previous paper [10]. And the porous
scaffolds from gelatin, gelatin/CM-chitin and
gelatin/CM-chitosan hydrogels prepared by
radiation-crosslinking in combination with
freeze-drying
and
gamma
irradiation
sterilization were characterized.
A. Swelling in PBS and mechanical property
of scaffolds:
The swelling behavior and compressive
modulus of scaffolds are the principal
parameters. The swelling ability of scaffolds
is closely relative to the essential nutrition
16
DANG VAN PHU et al.
G/CM-chitosan and G/CM-chitin scaffolds
requirements in the soft tissue engineering (10
was believed to be appropriate for the scaffold
– 75 kPa in compressive modulus) [6, 11].
10
8
200
1. Gelatin
160
2. G/CM-chitosan
3. G/CM-chitin
120
6
4
80
40
0
G/CM-chitosan
G/CM-chitin
Gelatin
2
0
2
6
1
0
4
8
10 12
2
3
Time, h
Scaffolds
Fig. 1. Swelling degree of scaffolds in phosphate
Fig. 2. Compressive modulus of
buffer solution as a function time
the different scaffolds
B. Porosity and pore size of scaffolds:
Table I. The porosity and pore size of the scaffolds.
Scaffolds
Porosity, %
50.0b 3.7
73.3a 4.2
70.6a 5.8
Pore size, m
300 – 450
Gelatin
G/CM-chitosan
G/CM-chitin
100 – 250
120 – 300
In a column, the means followed by the same letter are not different significantly (P <0.05).
The scaffolds with high porosity and
adequate pore size are required to provide a
microenvironment for cell-cell and cell-
surrounding interactions. The interactions
influence an attachment, migration, and
penetration of cellular in-growth as well as
pore size in the range of 100 – 300 m
with thin pore walls and abundance of
interconnects. Furthermore, these scaffolds
had a significantly higher porosity (around
70 – 73%) than that of the gelatin one
(about 50%). From these results, it was
perspicuous that CM-chitin and CM-
chitosan in blend with gelatin had a
vascularization
in
them
[8].
The
microstructural properties, ascertained
from SEM images such as the pore sizes
and porosity of the produced scaffolds,
were audibly manifested in Table 1 and Fig.
3. The SEM images revealed that the
gelatin scaffold possessed a large pore size
(300 – 450 m), thick walls, and almost no
interconnecting channels among the pores.
Meanwhile, both the scaffolds of the
G/CM-chitosan and G/CM-chitin owned a
satisfactory
effect
on
the
porous
microstructural properties of the gelatin
scaffold, which could also be mainly due to
the change in a crosslinking behavior.
More specifically, the porous properties of
G/CM-chitosan and G/CM-chitin scaffolds
were adequately assumed to culture the
hADSCs
and
generate
soft
tissue
engineering [3, 5, 7].
17
RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN…
Gelatin
G/CM-chitosan
G/CM-chitin
Fig. 3. SEM images and photographs of the scaffolds
C. Biodegradation and cytotoxicity of the scaffolds
100
80
60
40
20
0
G/CM-chitosan
G/CM-chitin
0
3
6
9
12 15 18 21 24
Incubated time, h
Fig. 4. Enzymatic degradation of the scaffolds in collagenase solution
To be considered and applied in tissue
engineering as scaffold, the
almost completed degradation by collagenase
a
enzyme activity. After 21 h incubation, the
biodegradability of both the scaffolds was
around 81 – 83% evenly. However, the
biodegradability of the G/CM-chitosan was
slightly faster than that of the G/CM-chitin in
the first 15 h. The reason could be explained
that the swelling rate and the porosity of the
G/CM-chitosan scaffold were moderately
higher than that of the G/CM-chitin one,
leading to a more favorable invasion of the
enzyme [7, 11].
biodegradability and non-cytotoxicity are
fundamental requirements that need to be
satisfied. According to Yang et al [11], the
collagenase enzyme is commonly used for
evaluation of scaffold biodegradability in
vitro due to it is able to degrade hydrogels
containing proteins. Fig. 4 illustrates the
relationship of the biodegradability of the
scaffolds with inoculation time. The gained
results showed that the scaffolds were to be
18
DANG VAN PHU et al.
1
2
3
4
Fig. 5. The confocal micrographs of hADSCs cultured in extraction media and MTT tested: 1) Fresh culture
medium (control), 2) Latex extract (control +), 3) G/CM-chitosan scaffold extract, and 4) G/CM-chitin
scaffold extract
140
1. Latex 2. G/CM-chitosan
120
100
80
60
40
20
0
3. G/CM-chitin
1
2
3
Samples (scaffolds)
Fig. 6. Relative growth rate of hADSCs in extraction media
Cytotoxicities of G/CM-chitosan and
From these results, it may be suggested
that the G/CM-chitosan and G/CM-chitin
scaffold exhibited non-cytotoxicity for the
hADSCs. Concerning cell ingrowths, the
RGR% values of the hADSCs after an
incubation period of 24 h with the different
extracts are described in Fig. 6. The RGR
values were of 96.2% and 97.1% respectively
for G/CM-chitosan and G/CM-chitin samples,
appropriated 7.3-fold higher than that of the
positive control group. These values could be
assorted to scoring 1 (non-cytotoxicity) and
qualified [4, 9]. The superb non-cytotoxicity or
cytocompatibility of these scaffolds was
assigned to the biocompatibility of the gelatin,
CM-chitosan, and CM-chitin [11], as well as a
suitable 3 D microenvironment provided by the
scaffold materials for the hADSCs [3].
CM-chitin based scaffolds were assessed
according to ISO 10993-5:2009 by incubating
hADSCs with the extracts of the scaffolds [7,
9]. As a well-known, the MTT assay is a
colorimetric test method based on the color
intensity of a purple formazan crystal, which
depends on the living cell's metabolic activity.
The morphology properties and density of
cells from the testing extracts after performing
MTT assay are presented in Fig. 5. The
morphology and cell density for treatments of
the G/CM-chitosan and G/CM-chitin were
seemingly unchanged from that of the
negative control group, while the cells with
shrinkage, flaking off, and almost no forming
formazan crystals were found in the positive
control (latex extract).
19
RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN…
gelatin/CM-chitosan/β-tricalcium phosphate
IV. CONCLUSIONS
composite scaffold for bone tissue
engineering”, Mater. Sci. Eng. C, 32(4),
994, 2012.
The mixture scaffolds of gelatin/CM-
chitosan and gelatin/CM-chitin were
successfully fabricated using radiation-
crosslinking combined with freeze-drying.
The performance in the same behaviors of
ameliorating the inherent properties of the
gelatin scaffold by CM-chitosan and CM-
chitin was evaluated and compared. Both
scaffold materials of gelatin/CM-chitosan
[3]. T. Agarwal, R. Narayan, S. Maji et al.,
“Gelatin/Carboxymethyl
chitosan
based
scaffolds for dermal tissue engineering
applicationˮ, Int. J. Biol. Macromol., 93(Pt B),
1499, 2016.
[4]. W. Xu, Z. Wang, T. Li et al., “Membranes
based on carboxymethyl chitin as potential
and gelatin/CM-chitin
possessed the
scaffolds
for
corneal
endothelial
swelling degree, porosity and pore size,
mechanical strength, biodegradability, and
non-cytotoxicity in suitability for human
adipose-derived stem cell culture for soft
tissue regeneration. The entire results of this
work propound a potential of the scaffolds
in the application to hADSC culture.
Nevertheless, other prime biological-
features, such as proliferation and
differentiation of hADSCs on the scaffolds
should be further explored.
transplantation”, Polym. J., 49, 789, 2017.
[5]. N. Sagar, V.P. Soni and J.R. Bellare,
“Influence of carboxymethyl chitin on
stability and biocompatibility of 3D
nanohydroxyapatite/gelatin/carboxymethyl
chitin
composite
for
bone
tissue
engineering”, J. Biomed. Mater. Res. Part B,
100B(3), 264, 2011.
[6]. Y-H. Tsou, J. Khoneisser, P-C. Huang and X.
Xu, “Hydrogel as a bioactive material to
regulate stem cell fate”, Bioact. Mater., 1(1),
39, 2016.
ACKNOWLEDGEMENTS
[7]. K-H. Wu, C. Mai, C-W. Lin, K-C. Yang
This work was financially supported
by the Ministry of Science and Technology
(MOST), Vietnam under Project No.
“DTCB.05/19/TTNCTK” in the period 2019 –
2020. The authors would like to thank
VINATOM, VINAGAMMA, and HCMUS for
providing the favorable conditions during
project implementation.
and J. Yu, “The influence of bubble size
on
chondrogenic
differentiation
of
adipose-derived stem cells in gelatin
microbubble scaffolds”, J. Mater. Chem. B,
6(1), 125, 2017.
[8]. H-T. Lu, T-W. Lu, C-H. Chen, K-Y. Lu and F-
L. Mi, “Development of nanocomposite
scaffolds based on biomineralization of N,O-
carboxymethyl chitosan/fucoidan conjugates
for bone tissue engineering”, Int. J. Biol.
Macromol., 120(Pt B), 2335, 2018.
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