Radiation-crosslinked scaffolds from gelatin/CM-chitin and gelatin/CM-chitosan hydrogels for adipose-derived stem cell culture

 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]: 
P (%) = (V1 − V3) × 100/(V2 − V3) (2) 
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 37C 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]. 
Biodegradability (%) = (Wo − W1) ×100/Wo
 (3) 
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 
RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN 
16 
with a ratio of 3 cm
2
/ml at 37C for 24 h, then 
centrifuged to obtain extraction media. The 
hADSCs with a density of 10
5
 cells/ml were 
incubated into the 96-well culture plate at 37C, 
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. 
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]. 
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 
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). 
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 
DANG VAN PHU et al. 
17 
G/CM-chitosan and G/CM-chitin scaffolds 
was believed to be appropriate for the scaffold 
requirements in the soft tissue engineering (10 
– 75 kPa in compressive modulus) [6, 11]. 
B. Porosity and pore size of scaffolds: 
Table I. The porosity and pore size of the scaffolds. 
Scaffolds Porosity, % Pore size, m 
Gelatin 50.0
b
 3.7 300 – 450 
G/CM-chitosan 73.3
a
 4.2 100 – 250 
G/CM-chitin 70.6
a
 5.8 120 – 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 
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 
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 
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]. 
Time, h 
S
w
el
li
n
g
 i
n
 P
B
S
, 
g
/g
0 
2 
4 
6 
8 
10 
0 2 4 6 8 10 12 
Gelatin 
G/CM-chitin 
G/CM-chitosan 
Scaffolds 
C
o
m
p
re
ss
iv
e 
m
o
d
u
lu
s,
 k
P
a
0 
40 
80 
120 
160 
200 
1 2 3 
1. Gelatin 
3. G/CM-chitin 
2. G/CM-chitosan 
Fig. 1. Swelling degree of scaffolds in phosphate 
buffer solution as a function time 
Fig. 2. Compressive modulus of 
the different scaffolds 
RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN 
18 
Fig. 3. SEM images and photographs of the scaffolds 
C. Biodegradation and cytotoxicity of the scaffolds 
Fig. 4. Enzymatic degradation of the scaffolds in collagenase solution 
 To be considered and applied in tissue 
engineering as a scaffold, the 
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 
almost completed degradation by collagenase 
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]. 
Gelatin G/CM-chitosan G/CM-chitin 
0 
20 
40 
60 
80 
100 
0 3 6 9 12 15 18 21 24 
Incubated time, h 
D
eg
ra
d
a
ti
o
n
 i
n
 c
o
ll
a
g
en
a
se
, 
%
G/CM-chitosan 
G/CM-chitin 
DANG VAN PHU et al. 
19 
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 
Fig. 6. Relative growth rate of hADSCs in extraction media 
Cytotoxicities of G/CM-chitosan and 
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). 
 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]. 
0 
20 
40 
60 
80 
100 
120 
140 
1 2 3 
Samples (scaffolds) 
R
G
R
, 
%
2. G/CM-chitosan 1. Latex 
3. G/CM-chitin 
1 2 3 4 
RADIATION-CROSSLINKED SCAFFOLDS FROM GELATIN/CM-CHITIN 
20 
IV. CONCLUSIONS 
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 
and gelatin/CM-chitin possessed the 
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. 
ACKNOWLEDGEMENTS 
 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. 
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