International Journal of Biological Macromolecules 138 (2019) 483–491

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International Journal of Biological Macromolecules

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

Physicochemical, antioxidant properties of giant croaker (Nibea japonica)

swim bladders collagen and wound healing evaluation
Yingyue Chen a, Huoxi Jin a, Fei Yang b, Shujie Jin a, Chenjuan Liu a, Liukai Zhang a, Ju Huang a, Shiguang Wang c,
Zhongyong Yan c, Xuwei Cai a, Rui Zhao a, Fangmiao Yu a, Zuisu Yang a, Guofang Ding a, Yunping Tang a,⁎
a
Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products, School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China
b
Hangzhou Obstetrics & Gynecology Hospital, Hangzhou 310008, China
c
Laboratory of Aquatic Products Processing and Quality Safety, Zhejiang Marine Fisheries Research Institution, Zhoushan 316021, China

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

Article history: Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were obtained from Nibea japonica swim
Received 30 December 2018 bladders. The denaturation temperature (Td) of ASC and PSC was approximately 33.8 °C. Sodium dodecyl sulfate-
Received in revised form 10 July 2019 polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR) analyses in-
Accepted 19 July 2019
dicated that ASC and PSC contained triple-helical type I collagen when compared to rat tail collagen type I. More-
Available online 19 July 2019
over, the microstructure of collagen sponges was uniform and porous. In addition, ASC and PSC exhibited
Keywords:
antioxidant properties and in vitro scratch assays showed that PSC at various concentrations (0, 12.5, 25, and
Nibea japonica 50 μg/mL) had significant effects on the scratch closure rate. Furthermore, collagen sponge from Nibea japonica
Swim bladders swim bladders exhibited an increased efficacy of wound healing when compared to the control mice. The levels
Collagen of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in the collagen sponge treated mice were signifi-
Antioxidant activity cantly decreased when compared to the control group. Thus, our results suggested that collagen sponge from
Wound healing Nibea japonica swim bladders has potential wound healing applications.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction Collagen, a vital structural protein, is found in connective tissue and

Wound healing is a multidimensional event that involves various
physiological regulatory processes. Once damaged, the skin goes
through a series of overlapping phases, including hemostasis, inflam-
mation, and proliferation [1,2]. During the inflammatory phase, the en-
zyme NADPH oxidase, which is highly active in inflammatory cells, can
produce large amounts of reactive oxygen species (ROS) [3,4]. Normally,
ROS are scavenged by antioxidants and there is a balance between ROS
production and neutralization [5]. However, this balance is disturbed in
a wound, where excessive ROS are produced. Excessive ROS production
can lead to oxidative stress, and the presence of free radicals may hinder
the wound healing process. Moreover, extracellular matrix compo-
nents, such as hyaluronic acid, collagen and proteoglycans can be dam-
aged by excessive ROS [5]. In addition, several growths factors and
cytokines play essential roles in the wound healing during inflamma-
tory and proliferative phases, such as interleukin (IL)-1β, IL-6 [6,7].
Therefore, for the wound healing process to proceed successfully, it is
necessary to maintain a balance between ROS production and antioxi-
dant activity, and to ensure adequate expression levels of growths fac-
tors and cytokines.
⁎ Corresponding author.
E-mail address: tangyunping1985@zjou.edu.cn (Y. Tang).
accounts for approximately 30% of the total protein composition in an
animal's body [8,9]. Collagen has been the focus of intensive research
given its wide spectrum of applications in various industrial fields, in-
cluding foods, pharmaceuticals, cosmetics and tissue engineering
[10–12]. Marine collagen has been gaining increasing attention due to
its unique advantages when compared with collagen extracted from
land animals, such as the lack of religious restrictions for its use, reduced
risk of disease transmission and low immunogenicity [13]. Massive
amounts of by-products are discarded by the booming marine products
processing industry or are simply used as animal feed or fertilizer.
Therefore, extracting collagen from these waste products would be a
better way of use, thereby preventing environmental pollution and cre-
ating enormous economic benefits [14,15]. Moreover, collagen is one of
the most effective wound healing biomaterials due to its outstanding bi-
ological properties, including biodegradability, lack of cytotoxicity, and
pro-proliferative and antioxidant effects [9,16]. For example, collagen
extracted from Miiuy Croaker swim bladders was shown to have 2,2-
diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical, superoxide
anion radical, and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid
(ABTS) radical scavenging activities [16]. Collagen membranes derived
from Chondrosia reniformis exhibited DPPH radical scavenging activity
and biocompatibility with both fibroblasts and keratinocytes [17]. In ad-
dition, the antioxidant, antihypertensive, antimicrobial, and

https://doi.org/10.1016/j.ijbiomac.2019.07.111
0141-8130/© 2019 Elsevier B.V. All rights reserved.
484 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491
hypoglycemic activities of bioactive collagen peptides from marine bio-
logical sources have been extensively studied in the past [12,18].
Recently, the effects of collagen, collagen peptides or collagen com-
bined with other functional ingredients on wound healing have been
the focus of many studies due to their biocompatibility and outstanding
antimicrobial and antioxidant properties [19–21]. For example, both
in vitro and in vivo experiments showed that a novel fibrous collagen-
based cream manufactured with bovine collagen and succinic acid,
xanthan gum, glycerol, and lanolin could clinically be used to promote
wound healing [19]. Xiao et al. [20] synthesized the N-succinyl
chitosan-collagen peptide copolymer (NSC-COP) with microbial
transglutaminase and showed that the physicochemical and antioxi-
dant characteristics of the biomaterial were improved upon modifica-
tion, and associated with outstanding cell viability and wound healing
properties. Liu et al. [21] manufactured chitosan-collagen peptide (CS-
COP) with microbial transglutaminase and showed that this hydrogel
exhibited improved biocompatibility with NIH-3T3 cells, and potential
applicability as a wound healing promoting agent.
The giant croaker (Nibea japonica) is widely distributed along the
East China and South China Seas, has a strong ability to adapt to adverse
conditions, and is highly resistant to disease [9,13]. Nibea japonica swim
bladders are one of the by-products of the food processing industry
[9,13]. However, the properties and characteristics of collagen derived
from Nibea japonica swim bladders have not been studied. Here, we ob-
tained acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC)
from Nibea japonica swim bladders and investigated their physicochem-
ical, antioxidant, and in vitro and in vivo wound healing properties. Our
results indicated that collagen from Nibea japonica swim bladders had
the potential to promote wound healing.
2. Materials and methods
2.1. Raw materials
Nibea japonica swim bladders and NIH-3T3 fibroblasts were stored
in our laboratory [9]. Bovine collagen type I (cat. no. C8060) and rat
tail tendon collagen type I (cat. no. C8062) was purchased from Solarbio
(Beijing, China). Hematoxylin-eosin (H&E) staining kit was purchased
from Jiancheng Bioengineering Institute (Nanjing, China). The
enzyme-linked immunosorbent assay (ELISA) kits for detecting IL-1β,
IL-6 and tumor necrosis factor (TNF)-α were purchased from Boster Bi-
ological Technology Co., Ltd. (Wuhan, China). 30% Acr-Bis (29:1) was
obtained from Sangon Biotech (Shanghai, China). Other chemicals
were all of analytical grade.
2.2. Preparation of ASC and PSC
The swim bladders were mixed with 0.1 M NaOH (1:10, w/v) and
gently stirred for 24 h to remove non-collagenous proteins. Solutions
were replaced with fresh NaOH solution (0.1 M) every 8 h. Swim blad-
ders were washed with pre-cooled deionized water until the pH was
neutral. Then, 10% (v/v) n-butanol was added (1:10, w/v), and samples
were gently stirred for 24 h to remove fat from the swim bladders. ASC
was obtained by extracting with 0.5 M acetic acid (1:50, w/v) for 24 h,
while PSC was obtained by extracting with 0.5 M acetic acid (1:50, w/
v) combined with pepsin (1200 U/g) for 8 h. Collagen were then filtered,
introduced into dialysis bags (molecular weight cutoff: 10 kDa), and di-
alyzed with deionized water until the pH was neutral. The dialyzed col-
lagen suspension was freeze-dried and stored at −20 °C until use.
2.3. Amino acid analysis
The amino acid composition of collagen was analyzed according to
Tang et al. [9]. Collagen was hydrolyzed with HCl (6 N) at 110 °C for
24 h under vacuum. The hydrolyzed products were dissolved in citric
acid buffer and analyzed by Hitachi L-8800 amino acid analyzer
(Hitachi, Tokyo, Japan). The hydroxyproline content in the hydrolyzed
products was determined as described by Sun et al. [22].
2.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) analysis
SDS-PAGE was performed according to Tang et al. [9], using an 8.0%
resolving gel and a 5% stacking gel. Collagen (5 mg/mL) was dissolved in
0.1 M acetic acid, and total protein (30 μg) was then separated by SDS-
PAGE. The molecular weights of collagen were estimated using protein
weight makers, and the positive control was rat tail tendon collagen
type I.
2.5. Determination of denaturation temperature (Td) values
The Td of collagen was determined with a Ubbelohde viscometer ac-
cording to Sun et al. [22], with some modifications. Briefly, collagen so-
lutions (0.5 mg/mL) were diluted with 0.1 M acetic acid for further
analysis. Samples were then heated from 20 to 40 °C with 2.5 °C heating
intervals. The viscosity at specific temperatures was obtained using the
formula described by Sun et al. [22].
2.6. Fourier transform infrared (FTIR) spectra
Under dry conditions, 1 mg of lyophilized samples was combined
with dry KBr (100 mg, spectrum pure), and pressed into thin sections
for later recording. Infrared spectra were determined with an FTIR Ten-
sor 27 spectrometer (Bruker, Rheinstetten, Germany). A wavenumber
range of 400–4000 cm−1 was recorded in a single scan at a resolution
of 1 cm−1.
2.7. Fabrication of collagen sponge and scanning electron microscopy
(SEM) analysis
Collagen solutions (10 mg/mL) without acetic acid were poured into
9 cm × 9 cm plates and then lyophilized. The ASC and PSC sponge mor-
phologies were analyzed with a high-resolution SEM (JSM-7800F, JEOL,
Japan). The samples were sprayed with gold and observed at a magnifi-
cation of 200×, 400× and 1000×.
2.8. Effects of pH and NaCl concentration on ASC and PSC solubility
The solubility of collagen was analyzed according to Yu et al. [13].
The lyophilized collagen was dissolved in acetic acid (0.5 M) to a final
concentration of 3 mg/mL. To ensure that the collagen was fully solubi-
lized, solutions were stirred at 25 °C for 2 h, and centrifuged
(12,000 rpm, 10 min) at 4 °C to remove any undissolved material.
The effect of pH on collagen solubility was determined as follows:
the pH of the supernatant was adjusted to 1.0–11.0 with HCl (6 N) or
NaOH (6 N), and deionized water was added to a final volume of
10 mL. The solutions were centrifuged (12,000 rpm, 10 min) at 4 °C
and supernatant was collected for the determination of protein
concentration.
The effect of NaCl concentration on collagen solubility was deter-
mined as follows: the supernatant was mixed with NaCl to a final con-
centration of 0–6% in 10 mL. The solutions were then centrifuged
(12,000 rpm, 10 min) at 4 °C, and the supernatant was collected for
the determination of protein concentration. The protein concentration
was calculated by the Bradford method, as described by Tang et al. [23].
2.9. Antioxidant activity
The DPPH, hydroxyl, superoxide anion and ATBS radical scavenging
activities of collagen from Nibea japonica swim bladders were deter-
mined according to Zhao et al. [16]. Ascorbic acid was used as positive
control.
 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491
2.9.1. DPPH radical scavenging activity
2 mL of sample with increasing concentrations (0.5–10 mg/mL) was
added into a series of centrifuge tubes (5 mL), and then 0.5 mL of DPPH
(0.1 mM) in ethanol and 1.0 mL of ethanol were added. The mixture was
placed in the dark for 30 min, and the absorbance was determined at
517 nm (As). The sample that substituted by deionized water was set
as the control group (Ac), and the DPPH that substituted by ethanol
was set as the blank group (Ab). The DPPH radical scavenging activity
of samples was obtained as follows:
DPPH radical scavenging activity ð%Þ ¼ ½1−ðAs −Ab Þ=Ac  100%
2.9.2. Hydroxyl radical scavenging activity
1.5 mL of 1, 10-phenanthroline solution (1.5 mM) was added into a
series of centrifuge tubes (5 mL), and then 1 mL of sample with increas-
ing concentrations (0.5–10 mg/mL) and 1.5 mL of FeSO4 solution
(1.5 mM) were added. Finally, 1.0 mL of H2O2 solution (0.03% v/v)
was added to initiate the reaction. The mixture was incubated at 37 °C
for 90 min, and the absorbance was determined at 536 nm (As). The
sample that substituted by deionized water was set as the control
group (Ac), and the H2O2 solution that substituted by deionized water
was set as the blank group (Ab). The hydroxyl radical scavenging activ-
ity of samples was obtained as follows:
Hydroxyl radical scavenging activity ð%Þ ¼ ½1−ðAs −Ab Þ=Ac  100%
2.9.3. Superoxide anion radical scavenging activity
1 mL of sample with increasing concentrations (0.5–10 mg/mL) was
added into a series of centrifuge tubes (5 mL), and then 1 mL of
nitrotetrazolium blue chloride (NBT) solution (2.52 mM) and 1 mL of
NADH (624 mM) were added. Finally, 1.0 mL of phenazine methosulfate
(PMS) solution (0.12 mM) was added to initiate the reaction. The mix-
ture was incubated at 25 °C for 5 min, and the absorbance was deter-
mined at 560 nm (As). The sample that substituted by deionized water
was set as the control group (Ac). The superoxide anion radical scaveng-
ing activity of samples was obtained as follows:
Superoxide anion radical scavenging activity ð%Þ
¼ ðAs −Ac Þ=Ac  100%
2.9.4. ABTS radical scavenging activity
1 mL of diluted ABTS radical solution and 1 mL of sample with in-
creasing concentrations (0.5–10 mg/mL) was added into a series of cen-
trifuge tubes (5 mL). The mixture was incubated at 25 °C for 10 min in
the dark, and the absorbance was determined at 734 nm (As). The sam-
ple that substituted by deionized water was set as the control group
(Ac). The ABTS radical scavenging activity of samples was obtained as
follows:
ABTS radical scavenging activity ð%Þ ¼ ðAc −As Þ=Ac  100%
2.10. In vitro scratch closure assay
NIH-3T3 fibroblasts were seeded into 6-well plates (2 × 105 cells per
well), and incubated at 37 °C in 5% CO2 until the cell confluence reached
approximately 80–90%. A scratch wound was then created with a pi-
pette tip (200 μL), and the wound debris was washed away with phos-
phate salt buffer (PBS). Cells were then treated with PSC (0, 12.5, 25, or
50 μg/mL), and cultured for 12 or 24 h. Cells without PSC treatment
were set as the blank control, and cells with bovine collagen type I (50
μg/mL) treatment were set as positive control. The phase-contrast mi-
croscope (CKX41-A32PH, Olympus, Tokyo, Japan) was used to observe
the scratch closure, and the scratch area was calculated by Image J
485
software. The scratch closure rate (%) was obtained as follows:
Scratch closure rate ð%Þ ¼ ðA0 −At Þ=A0  100%
where A0 is the scratch area at 0 h and At is the scratch area at the spec-
ified time point.
2.11. Animals and wound creation
ICR mice were purchased from Zhejiang Academy of Medical Sci-
ences (Hangzhou, China). The animal experiments were performed ac-
cording to the guidelines of Zhejiang Ocean University ethics
committee. Twenty male ICR mice (6–8 weeks old, 22–24 g) were ran-
domly divided into two groups: the wound control group and PSC
sponge treated group. The mice were anesthetized with chloral hydrate
(4%) during surgery. Then, the fur of mice was quickly removed and an
8 mm diameter wound area was created with sterile surgical scissors. In
the PSC sponge treated group, the wound area was covered with PSC
sponge which was sterilized by ultraviolet irradiation. In the wound
control group, the wound area was just treated with physiological sa-
line. A digital camera was used for taking photographs to observe the
wound healing pattern on days 0, 7 and 12, and the wound area was cal-
culated by Image J software. The wound closure rate (%) was obtained as
follows:
Wound closure rate ð%Þ ¼ ðA0 −At Þ=A0  100%
where A0 is the wound area at 0 day and At is the wound area at a par-
ticular day.
2.12. Histopathological analysis
For histopathological analysis, fixed skin tissues were embedded in
paraffin solution and cut into 5-μm thick pieces. The paraffin sections
were then stained with H&E for histopathological analysis. Light micros-
copy (Olympus CX31, Japan) was used for observing the stained
sections.
2.13. Measurement of inflammatory factors
Mice from each group were euthanized, and wound tissues were
dissected for further experiment. The wound tissues were homogenized
with physiological saline (w/v, 1:9), and the supernatant was obtained
after centrifugation. The levels of IL-1β, IL-6 and TNF-α in the superna-
tant were measured according to the instructions of the ELISA kit
(Boster, Wuhan, China).
2.14. Statistical analysis
All tests were performed in triplicate and the results are expressed
as the mean ± standard deviation (SD). Data were analyzed by SPSS
19.0 software (IBM SPSS Statistics, Ehningen, Germany).
3. Results and discussion
3.1. Amino acid composition
The yield of ASC and PSC extracted from Nibea japonica swim blad-
ders was approximately 11.33% and 15.35% (by dry weight), respec-
tively. The amino acid content of collagen from Nibea japonica swim
bladders is presented in Table 1. It is apparent from the chart that gly-
cine represents nearly one-third of the total amino acid content in
both ASC and PSC. Glycine, which in most cases is found every three res-
idues in collagen, has been found to be cytoprotective against ROS and
regulates the inflammatory response [24–26]. Moreover, the total
imino acid content of ASC was 181.1 residues/1000 residues and for
486 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491

PSC the figure was 187.2 residues/1000 residues. The total imino acid
content of ASC and PSC from Nibea japonica swim bladders was similar
to the content found in ASC and PSC from Miiuy Croaker (approximately
196.7 and 199.5 residues/1000 residues, respectively) [16], but was
lower than the content found in calf skin collagen (approximately 215
residues/1000 residues), porcine skin collagen (approximately 220 res-
idues/1000 residues), and human collagen type I (approximately 223
residues/1000 residues) [13]. The imino acid content of collagen has
been linked to its thermal stability, which is a critical characteristic for
medical applications. The difference in total imino acid and hydroxypro-
line content may be species-specific and related to the habitat temper-
ature of the species.

3.2. SDS-PAGE analysis

The SDS-PAGE analysis of collagen is shown in Fig. 1. The ASC and

PSC band patterns were very similar, with both samples containing α-
chains (α1 and α2), β-chains (dimer) as well as a small quantity of γ-
chains (trimer). As the major constituent, both contained two α-
chains (α1 and α2), and the molecular weight of the α1-chain (near
130 kDa) was higher when compared to that of the α2-chain (between
95 kDa and 130 kDa). The protein patterns of collagen obtained from
Nibea japonica swim bladders were similar to those of type I collagen
from calf and rat (Fig. 1), and suggested that collagen from Nibea japon-
ica swim bladders belong to collagen type I. The protein pattern of col-
lagen extracted from Nibea japonica swim bladders was similar to that
of collagen obtained from Miiuy Croaker [16], rohu [27], and grass
carp [28] swim bladders.
3.3. Determination of the Td
Collagen has important biomedical applications, and is involved in
bone repair [29], articular cartilage regeneration [30], wound healing
[31], and hemostasis [32]. In order to meet biomedical and tissue engi-
neering requirements, the thermal stability of collagen must be taken
into account. The physical characteristics of collagen, such as its solubil-
ity and viscosity change when it is subjected to high temperatures.
Under these conditions, the solubility of collagen may decrease and col-
lagen may precipitate. Therefore, the viscosity of collagen is often used
to determine its denaturation temperature (Td) [9,24].
In this study, the Td value of collagen extracted from Nibea japonica
swim bladders was calculated and the thermal transition curves are
Table 1
Amino acid composition of ASC and PSC from Nibea japonica swim bladders (results are
expressed as residues/1000 residues).
Fig. 1. SDS-PAGE analysis of ASC and PSC from Nibea japonica swim bladders. M: molecular
weight markers; 1: rat tail tendon collagen type I; 2: ASC extracted from Nibea japonica
swim bladders; 3: PSC extracted from Nibea japonica swim bladders.
illustrated in Fig. 2. The thermal transition curves of ASC and PSC were
very similar, and the Td value in both cases was approximately 33.8
°C, which is very similar to the value obtained for collagen extracted
from yellowfin tuna swim bladders (32.97 °C for ASC and 33.92 °C for
PSC) [33]. Recently, the Td values of collagen obtained from the swim
bladders of other fish species were reported. For example, ASC and
PSC were isolated from Miiuy Croaker swim bladders and their Td
values were 24.7 °C and 26.7 °C, respectively [16]. Kaewdang et al.
[33] extracted collagen from yellowfin tuna swim bladders, and the Td
values of ASC and PSC were 32.97 °C and 33.92 °C, respectively. More-
over, Pal et al. [34] extracted collagen from carp swim bladder and re-
ported a Td of 38.88 °C for ASC and of 39.38 °C for PSC. On the other
hand, collagen isolated from rohu swim bladders exhibited good ther-
mal stability and the Td of PSC was 42.16 °C [27].
As indicated above, the Td of collagen obtained from Nibea japonica
swim bladders was higher compared to that of collagen obtained from
marine fish swim bladders but lower than that of collagen extracted
from freshwater fish swim bladders. The difference in Td values is re-
lated to the ambient temperature in which these species live. The pro-
line and hydroxyproline content of collagen plays an essential role in

Amino acid ASCa PSCb Calf skin Porcine skin Human type I
collagen [13] collagen [13] collagen [13]

Aspartic acid 43.9 43.3 45 44 43

Threonine 16.7 16.8 18 16 17
Serine 25.4 25.3 33 33 33
Glutamic acid 46.6 45.0 75 72 71
Glycine 322.4 327.5 330 341 335
Alanine 98.2 100.1 119 115 111
Cysteine 0 0 0 0 0
Valine 13.5 13.4 21 22 26
Methionine 9.0 8.4 6 6 6
Isoleucine 8.4 8.0 11 10 9
Leucine 16.2 15.8 23 22 23
Tyrosine 2.0 1.3 3 1 2
Phenylalanine 9.1 9.2 3 12 12
Histidine 9.0 8.9 5 5 6
Lysine 23.4 24.3 26 27 23
Arginine 46.9 47.1 50 48 50
Proline 107.7 112.4 121 123 120
Hydroxyproline 73.4 74.8 94 97 103
Imino acid 181.1 187.2 215 220 223
a
ASC from Nibea japonica swim bladders.
b
PSC from Nibea japonica swim bladders. Fig. 2. Thermal denaturation curves of ASC and PSC from Nibea japonica swim bladders.
 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491
Fig. 3. FTIR analysis of ASC (A) and PSC (B) from Nibea japonica swim bladders.
determining its thermal stability, with higher proline hydroxylation im-
proving the thermal stability. Our findings are in agreement with the
composition of imino acid mentioned above.
3.4. FTIR spectra
The FTIR spectra of collagen from Nibea japonica swim bladders are
shown in Fig. 3. ASC and PSC showed almost identical spectral positions
despite their different absorbance intensities. The characteristic amide
A, B, I, II and III bands are also presented in Fig. 3. The free N\\H
stretching vibration can be seen within the 3400–3440 cm−1 range,
with a shift near 3300 cm−1 due to the existence of hydrogen bonds
[9,35]. The amide A band, which is usually related to the N\\H
stretching frequency, appeared at 3418.47 and 3443.35 cm−1 in ASC
and PSC, respectively. The amide B band, associated with CH2 asymmet-
rical stretching, appeared at 2926.25 and 2926.57 cm−1 in ASC and PSC,
respectively. The amide I band, associated with the polypeptide back-
bone C\\O stretching vibration, showed the characteristic wavenumber
between 1600 and 1700 cm−1, with the peak absorption in ASC and PSC
occurring at 1655.86 and 1654.23 cm−1, respectively. The absorption
peaks at 1554.94 cm−1 in ASC and 1556.01 cm−1 in PSC coincided
with the position of amide II bands (1550–1600 cm−1). The amide III
Fig. 4. SEM images of ASC and PSC from Nibea japonica swim bladders (magnification: 200×, 400×, and 1000×).
487
peaks are associated with intermolecular collagen interactions and ap-
peared at 1240.07 cm−1 in ASC and 1240.38 cm−1 in PSC, suggesting
that they adopted a helical structure [9,36].
3.5. SEM analysis
Collagen is being widely applied in biomedical engineering as a
sponge for wound dressings, as a hemostatic agent or as a matrix for
cell proliferation [19,32]. Therefore, it is important to understand the
microstructure of collagen. The collagen solution extracted from Nibea
japonica swim bladders was lyophilized and the microstructure of colla-
gen sponges was analyzed by SEM. The white, uniform, and pyknotic
sponges are shown in Fig. 4. We found that the superficial morphology
of ASC was identical to that of PSC. The SEM images of ASC and PSC
showed a sophisticated, multi-layered, clustered fibrous external mesh-
work. A loose, fibrous, and porous structure was observed at a magnifi-
cation of 200×, 400×, and 1000×. These results suggested that collagen
extracted from Nibea japonica swim bladders may be utilized as a bio-
polymer in hemostatic agents, for wound healing, or drug delivery.
3.6. Effects of pH and NaCl on collagen solubility
The effects of pH and NaCl concentration on the solubility of collagen
were investigated and the results are shown in Fig. 5. Both ASC and PSC
were highly soluble at low pH (1–4), with maximal solubility observed
at pH 2 (Fig. 5A). When the pH value is higher than 5, the solubility of
collagen decreases rapidly. Precipitation is closely related to hydropho-
bic interaction between collagen molecules. When the pH reaches the
isoelectric point, the total net charge of proteins became zero, allowing
proteins to aggregate [37,38]. On the contrary, a repulsive effect be-
tween protein chains is found when the pH differs from their character-
istic isoelectric point, which may explain the mild increase in solubility
at pH values between 9 and 11. Due to slight differences in the proper-
ties and conformation of ASC and PSC molecules, PSC showed better sol-
ubility than ASC.
The solubility of collagen tended to slightly decrease (with some
fluctuations) at NaCl concentrations between 0 and 2%, but significantly
decreased when the NaCl concentration reached approximately 3%
(Fig. 5B). The increased NaCl concentration enhanced the interactions
between protein chains, leading to greater hydrophobic interactions
and precipitation. The solubility of PSC was relatively higher compared
488 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491

Fig. 5. Relative solubility of ASC and PSC from Nibea japonica swim bladders under different (A) pH and (B) NaCl concentration conditions.

to that of ASC, which could be attributed to the enzymatic activity of
pepsin that changes the collagen structure.
Similar outcomes were reported for collagen extracted from unicorn
leatherjacket skin [38], Spanish mackerel skin and bone [37], and Nibea
japonica skin [13]. However, our results showing how the solubility of
collagen changes with increasing pH and NaCl concentration are likely
to find practical applicability in the design of the extraction process
and for functional correlations.
3.7. Antioxidant activity
Marine collagen or collagen active peptides are often used in the skin
care industry and to promote wound healing due to their antioxidant
properties and other desirable characteristics [18,20,39]. During
wound healing, it is desirable to neutralize ROS, which are implicated
in tissue injury [2,20]. Therefore, it is important to study the radical
scavenging properties of wound healing biomaterials. To evaluate the
antioxidant properties of collagen extracted from Nibea japonica swim
bladders, we used four traditional methods, including DPPH and ABTS
assays and hydroxyl and superoxide anion radical scavenging assays.
As illustrated in Fig. 6, both ASC and PSC (0.25–10 mg/mL) obtained
from Nibea japonica swim bladders could scavenge DPPH, hydroxyl, su-
peroxide anion, and ABTS radicals. The radical scavenging activity of PSC
was slightly better compared to that of ASC. On the other hand, ASC and
PSC appeared to better scavenge DPPH and ABTS radicals than hydroxyl
and superoxide anion radicals.
Previous studies have described marine-derived collagen prepara-
tions with antioxidant activity [16,40]. For example, ASC and PSC

Fig. 6. (A) DPPH, (B) hydroxyl (C) superoxide anion, and (D) ATBS radical scavenging activities of ASC and PSC from Nibea japonica swim bladders.
 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491
Fig. 7. Effects of PSC from Nibea japonica swim bladders on the scratch closure rate. (A) PSC
promoted cell migration was evaluated using a scratch wound healing assay. (B) Wound
closure rate. Positive control: 50 μg/mL of bovine collagen type I. *p b 0.05 and **p b
0.001 vs. control.
obtained from Miiuy Croaker swim bladders were found to scavenge
DPPH, hydroxyl, superoxide anion, and ABTS radicals at concentrations
between 0.25 and 3 mg/mL [16]. In addition, ASC and PSC extracted
from whale shark cartilage were shown to exhibit antioxidant activity
against DPPH radicals. The activity of PSC from whale shark cartilage
was also higher compared to that of ASC [40]. Our findings were in
agreement with the results from those studies. However, as shown in
Fig. 6, the antioxidant activities of collagen were relative low (approxi-
mately 0–50% at concentrations between 0.25 and 10 mg/mL), and
should be improved before incorporating them in wound healing appli-
cations by adding other functional ingredients, such as chitosan or
chemically modified chitosan [20,41]. For example, Xiao et al. [20] syn-
thesized NSC-COP with microbial transglutaminase and found that the
Fig. 8. Effects of PSC sponge on wound healing. (A) Representative photographs on days 0, 7 and 12. (B) Wound contraction (%) on days 7 and 12. Data are expressed as the mean ± SD (n
= 5). *p b 0.05 and **p b 0.001 vs. control.
489
antioxidant activity improved, therefore, it could be used as a wound
healing biomaterial. Also, Fan et al. [41] manufactured collagen-
chitosan with microbial transglutaminase and reported strong DPPH
and H2O2 scavenging activities at collagen-chitosan concentrations of
0.15–2.50 mg/mL.
3.8. In vitro effects of PSC on scratch closure
During wound healing, fibroblast proliferation, and migration accel-
erate the re-epithelialization process and promote wound closure
[1,42]. Scratch tests are commonly used to estimate the wound healing
rate in vitro [1,42]. Therefore, the effects of collagen obtained from Nibea
japonica swim bladders on the wound healing rate were evaluated by
the scratch test using NIH-3T3 fibroblast cells. According to the high an-
tioxidant activity of PSC when compared to ASC, and considering the al-
lergenic possibility of ASC, we chose PSC for cell culture experiment.
The scratch closure rate was calculated, and the results are illus-
trated in Fig. 7A. Compared to the control, the wound scratch area was
significantly reduced in the presence of PSC, which was dose-
dependent. The scratch closure rate at PSC concentrations of 12.5, 25,
and 50 μg/mL was significantly higher when compared to the control
(Fig. 7B), and the scratch closure rate at PSC concentrations of 50
μg/mL was similar when compared to the 50 μg/mL of bovine collagen
type I (Fig. 7B). In particular, the scratch was almost completely closed
after 24 h of treatment. The in vitro wound closure assay results sup-
ported the conclusion that PSC from Nibea japonica swim bladders in-
duced NIH-3T3 cell migration.
3.9. Effect of PSC sponge in wound closure in animals
Wound healing pattern was observed visually by using a digital cam-
era for taking photographs on days 0, 7 and 12. Compared with the con-
trol group, the PSC sponge from Nibea japonica swim bladders had a
significant wound healing effect (Fig. 8A). A wound area reduction of
approximately 88.7% and 98.2% was observed in the PSC sponge treated
group on days 7 and 12, respectively (Fig. 8B). However, it was approx-
imately 76.8% and 96.39% in the wound control group on days 7 and 12,
respectively (Fig. 8B). Our results indicated that PSC sponge from Nibea
japonica swim bladders has the potential for promoting wound healing.
3.10. Histopathological examination
In order to investigate the wound healing ability of collagen sponge,
we collected the wound tissue for histological analysis by H&E staining
on days 7 and 12 [43,44]. As shown in Fig. 9A, after 7 days in the control
group, there were only 1–3 layers of epidermal cells, and a large number
of inflammatory cells infiltrated in dermis. Few amounts of new blood
vessels, fibroblasts and collagen fibers were observed in the control
group on day 7. There were also few amounts of sebaceous gland cells
and hair follicle tissues, but the formation of granulation tissues were
490 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491

Fig. 9. Histopathological examination of wounded skin tissue of the control and PSC sponge treated groups with hematoxylin-eosin (H&E) staining on days 7 and 12 (magnification: 200×).

not obvious in the control group on day 7. As shown in Fig. 9C, epider-
mal cells in the PSC sponge treated group on day 7 could reach to 4–6
layers, including the basal layer, spinous layer cells and granular layer
cells. In dermis, a large number of inflammatory cells infiltrated, and vis-
ible new blood vessels, fibroblasts and collagen fibers could be observed
in the PSC sponge treated group on day 7. Furthermore, there were
more sebaceous gland cells and hair follicle tissues in the PSC sponge
treated group, and the granulation tissues were obviously formed on
day 7.
As shown in Fig. 9B, after 12 days in the control group, there were
only 3–5 layers of epidermal cells, and a large number of inflammatory
cells infiltrated in dermis. In addition, more neovascularization, fibro-
blasts and collagen fibers appeared in dermis, and sebaceous gland
cells and hair follicle tissues began to form. As shown in Fig. 9D, the epi-
dermal cells could reach to 6–9 layers in the PSC sponge treated group
on day 12, including basal layer, spinous layer, granular layer, hyaline
layer and keratinocytes. There were a lot of new blood vessels, fibro-
blasts and collagen fibers in dermis, and few inflammatory cells infil-
trated in dermis. Furthermore, a large number of sebaceous gland cells
and hair follicle tissues could be observed in the PSC sponge treated
group on day 12. Our results indicated that PSC sponge from Nibea ja-
ponica swim bladders could speed up the wound healing process
when compared to the control group.
healing properties. The Td of ASC and PSC was approximately 33.8 °C.
SDS-PAGE and FTIR analyses indicated that ASC and PSC contained
triple-helical collagen type I. Both collagen extracts were highly soluble
in acidic solutions with zero or low NaCl concentrations and appeared to
have better thermal stability than collagens isolated from the bladders
of other marine fish. In addition, the fluffy structure, antioxidant,
in vitro and in vivo wound healing properties of PSC sponge from
Nibea japonica swim bladders suggested that this PSC sponge has poten-
tial wound healing applications. Our future in vitro and in vivo studies
will focus on the signaling mechanisms that promote wound healing.
Declaration of Competing Interest
The authors declare no conflict of interest in this work.
Acknowledgements
This work was financially supported by the National Natural Science
Foundation of China (grant no. 41806153), the National Undergraduate
Training Program for Innovation and Entrepreneurship (grant no.

3.11. Effect of PSC sponge on inflammatory factors in animals

Pro-inflammatory cytokines play an essential role in wound healing,

such as IL-1β, IL-6 and TNF-α [43,44]. These cytokines are involved in
stimulating fibroblast proliferation and chemotaxis. Previous studies
have shown that healing time in IL-6 knockout or IL-1 receptor antago-
nist mice was longer than the controls [45,46]. As shown in Fig. 10, com-
pared to the wound control group, the levels of the IL-1β, IL-6 and TNF-
α were reduced in the PSC sponge treated group. Our results were con-
sistent with the previous reports which showed that the PSC sponge
could reduce the levels of inflammatory factors, and exhibited the abil-
ity for wound healing application.

4. Conclusions

In this study, we extracted ASC and PSC from Nibea japonica swim Fig. 10. Effects of PSC sponge on levels of IL-1β, IL-6 and TNF-α. Data are expressed as the
bladders and analyzed their physicochemical, antioxidant, and wound mean ± SD (n = 5). *p b 0.05 vs. control.
 Y. Chen et al. / International Journal of Biological Macromolecules 138 (2019) 483–491
201810340015), and the Zhejiang Xin-miao Talents Program (grant no.
2018R411052).
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