Hidroxiapatita en conejos

International Journal of Biological Macromolecules 181 (2021) 369–377
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: http://www.elsevier.com/locate/ijbiomac
Preparation of nano-hydroxyapatite/chitosan/tilapia skin peptides
hydrogels and its burn wound treatment
Ouyang Qianqian a,b,c, Kong Songzhi d,⁎, Huang Yongmei a,b,c, Ju Xianghong d, Li Sidong d,
Li Puwang e, Luo Hui a,b,c,⁎⁎
a
Marine Biomedical Research Institute, the Key Lab of Zhanjiang for R&D Marine Microbial Resources in the Beibu Gulf Rim, Guangdong Medical University, Zhanjiang 524023, China
Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Zhanjiang 524023, China
The Marine Biomedical Research Institute of Guangdong Zhanjiang, Zhanjiang 524023, China
d
School of Chemistry and Environmental Science, Guangdong Ocean University, Zhanjiang 524088, China
e
Agricultural Product Processing Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang 524001, China
b
c
a r t i c l e
i n f o
Article history:
Received 29 December 2020
Received in revised form 28 February 2021
Accepted 14 March 2021
Available online 15 March 2021
Keywords:
Hydroxyapatite
Chitosan
Tilapia peptides
Burn healing
VEGF
a b s t r a c t
There is an urgent need for wound dressings to treat partial-thickness burns. Hydrogels are a promising material
that can maintain hydration to promote necrotic tissue removal. Tilapia peptides (TP) and hydroxyapatite (HA)
were incorporated into chitosan system to prepare new types of hydrogels. The hydrogels were cross-linking by
tannin (TA), which were developed to promote rapid wound healing in a New Zealand rabbit partial-thickness
burn model. Nanohydroxyapatite (NHA) was synthesized by coprecipitation method, which made hydrogels
have a highly porous structure comprised of interconnected pores, excellent water absorption and low hemolysis. Besides, the hydrogels showed excellent antimicrobial activities against both Escherichia coli (E. coli) and
Staphylococcus aureus (S. aureus), as well as the cytocompatibility on endothelial cells. Moreover, the hydrogels
promoted epithelial and dermal regeneration, reduce the expression of TNF-α and IL-6 and promote the skin regeneration by enhancing expression of collagen, STAT3, and VEGF.
© 2021 Published by Elsevier B.V.
1. Introduction
Skin, as the most significant human organ, plays a vital role in keeping homeostasis and preventing invasion from microorganisms and
chemical substances as the first line of defense against injuries, infections
and dehydration [1]. Clinically, more and more burns suffered nonhealing, which has made a huge economic impact on the world health
system [2,3]. There are diverse strategies that currently employed to
manage clinical aspects of burns, including hyperbaric oxygen therapy,
negative pressure wound therapy, growth factors, cell therapy, tissue
engineered skins, autograft transplantation and recently, experimental
biological dressings [4]. Biological dressings are new type of wound
dressing material based on wet healing theory [5]. The use of natural
polymers with different mechanical, physical, and biological properties
as wound dressings has been widely studied due to the many advantages of these macromolecules, such as good biocompatibility and their
non-irritant and nontoxic properties [6,7]. Wound dressings are also
⁎ Corresponding author.
⁎⁎ Correspondence to: L. Hui, Marine Biomedical Research Institute, the Key Lab of
Zhanjiang for R&D Marine Microbial Resources in the Beibu Gulf Rim, Guangdong
Medical University, Zhanjiang 524023, China.
E-mail addresses: [email protected] (K. Songzhi), [email protected] (L. Hui).
https://doi.org/10.1016/j.ijbiomac.2021.03.085
0141-8130/© 2021 Published by Elsevier B.V.
thought to prevent loss of body fluid, limit exudate buildup, protect
against external contamination, and have sufficient bactericidal effects
to inhibit infection and prepare an optimum bed for autografting [8].
Recently, much attention has been paid to the therapeutic effects of
chitosan-based hydrogels and hydrocolloid dressings to promote
wound healing [9–11]. These preparations maintain an ideal hydration
environment for wound healing while preserving the gaseous permeability of traditional gauze dressings [12]. They can absorb wound exudate and provide transparency, which facilitates clinical observation
[12]. They can also serve as a matrix for the controlled release of antibiotics and drugs impregnated during manufacturing [12–14]. However,
the chitosan hydrogels exhibit low mechanical strength and low antibacterial activity, which limits its applications in burs healing. Previously,
we have studied that the tilapia peptides combined with chitosan had
an excellent activity on wound repair [15]. However, the good fluidity
of hydrogel affects its adhesion to wounds. Many studies have showed
that nanohydroxyapatite has been used to build layered scaffolds to
achieve improved results in wound repair [17]. Nanohydroxyapatite particles have optimal antibacterial effects due to their high solubility and
rapid ion release in addition to their capacity for active oxygen species
production [18]. Additionally, nanohydroxyapatite is non-toxic and
produces minimal immunological reactions, and in vitro attachment.
Also, nanohydroxyapatite with crack free scaffolds promotes rapid cell
O. Qianqian, K. Songzhi, H. Yongmei et al.
International Journal of Biological Macromolecules 181 (2021) 369–377
2.4. Preparation of NHA/CS/TP composite hydrogels
proliferation, spreading, rapid bioabsorption and bone regeneration
in short time [19]. Given that incorporating nanohydroxyapatite
(NHA) into the chitosan hydrogel is able to generate a new type
of hydrogel with cross-linking by tannic acid to improve strength,
it will be feasible to use the hydrogel for wound healing. With
nanohydroxyapatite as the scaffold to form a stable hydrogel skeleton structure, it would be conducive to the effective components
of the hydrogel and the effect of the burn tissue, accelerate the repair of the burn tissue.
In the present study, we firstly prepared NHA by the method of
coprecipitation. Then the chitosan (CS) hydrogel was prepared by incorporating NHA and tilapia peptides (TP) into the hydrogel system. In
order to improve the mechanical properties and antibacterial properties,
the hydrogels were cross-linking by tannic acid. The morphological,
water absorption, biocompatibility and antibacterial activity of hydrogels
were also evaluated. Moreover, New Zealand rabbit back burn model
was established to assess the potential of NHA/CS/TP hydrogels as
burns dressing.
NHA/CS/TP composite hydrogels were prepared as follows. Briefly,
CS (2.0 g) and TP (3.0 g) were dissolved in 95 g of acetic acid (w/v,
1%) under continuous stirring to obtain a mixture solution with a final
concentration of 5% (CS/TP). Varied amounts of HA (0.5% (I) and 1.0%
(II) (w/w)) were added to the CS/TP solution to produce two kinds of
mixtures, respectively. Then, the mixtures were added to the tannin solution (5 w/v%) for crosslinking. Finally, the mixture was stirred for 1 h,
and poured into a mold coated with PTFE, which was then first frozen
for 24 h at −80 °C. To prepare the porous hydrogels, the frozen gels
were lyophilized using a freeze drier for 48 h at −54 °C. The composite
hydrogels were then unloaded from the mold, named NHA/CS/TP
(I) and NHA/CS/TP(II), respectively.
2.5. Hydrogel characterization
2.5.1. Morphological properties
The sample was frozen and brittle-broken in liquid nitrogen. The ultrastructure of NHA/CS/TP(I) and NHA/CS/TP(II) were analyzed by scanning electron microscope (SEM, S-4800, Japan).
2. Materials and methods
2.1. Materials
2.5.2. Water absorption ability
The water absorption ability of all the samples was tested. The water
absorption capacity test was conducted according to the previous
method [20]. NHA/CS/TP(I) and NHA/CS/TP(II) in dryness were accurately weighed and recorded as M0. The samples were immersed in
37 °C water for 30 min, and the wet samples were put on the filter
paper to absorb excess water. Then the wet samples were weighed
and recorded as M1, and the dry samples were weighed and recorded
as M0. Where X is stand for water absorption. The water absorption
ability of the samples was calculated as:
CS (average molecular weight = 100 kDa, degree of deacetylation
≥ 90%), was purchased from Qingdao Bozhi Huili Biotechnology Co.,
Ltd. (Qingdao, China). TP were prepared by our laboratory as described below, and its molecular was 500–1000 Da. Tannic acid, Ca
(NO 3 )2 ·4H2 O, (NH4 ) 2HPO4 , NH3 ·H2O were of analytical grade.
Burn cream (abbreviated MEBO) was purchased from MEBO Pharmaceuticals Co., Ltd. (Shantou, Guangdong, China). Tumor necrosis
factor-α (TNF-α) and interleukin-6 (IL-6) were measured by enzymelinked immunosorbent assay (ELISA) kits, obtained from eBioscience
(San Diego, CA, USA). STAT3 (#ab68153), VEGF (#ab27278) and immunoglobulin G- fluorescein isothiocyanate (IgG-FITC) were purchased
from abcam (Cambridge, UK).
X¼
M1−M0
100%
M0
All experiments were done at least in triplicate.
2.2. Preparation of hydroxyapatite (HA)
2.5.3. Hemolytic tests
Hemolytic tests were performed using rabbit hemocyte suspension
according to the reference [18]. Each sample (NHA/CS/TP(I) and NHA/
CS/TP(II)) was put into double distilled water for 24 h to obtain the material extract. 5 mL of material extract was mixed with 5 mL of 2% rabbit
hemocyte suspension (diluted with 0.9% NaCl solution (NS)), followed
by incubating at 37 °C for 3 h. Then the hemocyte suspensions were centrifuged at 1600 rpm for 10 min and detected on a UV/Vis spectrophotometer at 545 nm to determine the hemolytic ratio. Double distilled
water was employed as a positive control while normal saline (NS)
was employed as a negative control. The hemolytic ratio was calculated
according to the following equation:
Hydroxyapatite (HA) was synthesized by co-precipitation at
room temperature according to a modified method described by
Zhou et al. [17]. 200 mg of Ca (NO 3) 2 ·4H2O was dissolved in
350 mL of anhydrous ethanol to obtain a homogeneous solution
with a concentration of 0.2 M, and 5.52 g of (NH4)2 HPO 4 was dissolved in 210 mL of ultrapure water under simultaneous stirring to
obtain a homogeneous solution with a concentration of 0.2 M. The
pH of the two solutions was adjust with 25% of NH3·H2O to about
11. Then, (NH4)2HPO4 solution was added dropwise into Ca (NO3)
2 ·4H 2 O solution under simultaneous stirring. During the dripping
process, 25% of NH3·H2O solution was used to adjust the pH value
of the mixture, so that the pH value was always maintained at
about 10.5–11. After dripping, the mixture was stirred at a constant
speed for 24 h to disperse evenly, and then allowed to stand at
room temperature for 24 h to passivate the mixture. The mixture
was purified using a 0.22 μm filter membrane and washing with ultrapure water until pH was neutral. The filtered white hydroxyapatite was placed in a drying oven at a constant temperature of 60 °C
overnight, and the dried samples were ground into a fine powder
with a mortar for later use, called nanohydroxyapatite (NHA).
A¼
T−N
100%
P−N
where A is hemolytic ratio, T is the absorbance for the test sample, N is
the absorbance for the negative control, P is the absorbance for the positive control.
2.5.4. Antibacterial assay
The in vitro antibacterial assessment of hydrogels was examined
against using a representative Gram-negative Escherichia coli (E. coli,
ATCC25922) and a representative Gram-positive Staphylococcus aureus
(S. aureus, ATCC25723) as model microorganisms. Briefly, bacterial
cells were cultivated in Mueller-Hinton broth (MHB) overnight at
37 °C with 200 rpm in a shaker. Subsequently, 1 mL of bacterial suspension with concentrations of 1 × 107 CFU/mL were introduced to the surface of 24-well sized hydrogel for incubation of 24 h at 37 °C. The
2.3. Structure characterization of HA
The morphology of HA was viewed using high resolution transmission
electron microscopy (HRTEM, JEOL, Japan). Before the visualization, HA
was prepared by ultrasonic suspension in ethanol, then supported on a
Cu grid. X-ray diffraction (XRD) patterns of HA particles and gel power
samples were recorded on an X-ray diffractometer (Bruker D8 Advance).
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International Journal of Biological Macromolecules 181 (2021) 369–377
manufacturer's instructions (Jiancheng Inst. of Biotechnology, Nanjing,
China). The level of hydroxyproline in the skin, which positively correlates with dermal collagen, was measured using in skin samples
(~100 mg each) by ELISA kit according to the manufacturer's instructions (Jiancheng Inst. of Biotechnology, Nanjing, China).
aliquots of culture medium and bacterial-attached hydrogel were transferred into sterilized tube containing 5 mL PBS and shook for 10 min
followed by a series of dilution, and then mixed solution was spread
on Luria-Bertani agar plates. The number of colonies was counted
Using a colony counter (Czone 7D, Hangzhou Xunshu Technology Co.,
Ltd., China) after incubation for 24 h in an anaerobic chamber.
2.10. The assessment of TNF-α and IL-6
2.6. Cytocompatibility evaluation
To prepare skin extracts for ELISAs, 0.3 g of each sample was homogenized (10,000 rpm, 20 s) with 9 times the volumes of PBS (4 °C) to produce 10% homogenate, which was centrifuged at 3000 rpm for 20 min at
4 °C. The supernatant was employed to estimate the TNF-α and IL-6
levels with ELISA kits (eBioscience) in accordance with the specifications.
The proliferation of HUVECs was assessed using CCK-8 assay. Briefly,
HUVECs were counted under a microscope using a hematocytometer,
and a concentration of 5 × 103 cells/mL was prepared in complete
media. Then, HUVECs were seeded in the 96-well plate. After 24 h of
cell adherence, the culture medium was replaced with different concentrations of samples. The samples were grouped as follows: (1) blank
control (control), (2) NHA/CS/TP(I), (3) NHA/CS/TP(II). After 24 h,
48 h and 72 h, cell viability was measured by CCK-8 assay. Then
100 μL of supernatants was extracted and read at 450 nm using a
plate reader (n = 3).
2.11. Immunohistochemical examination
The wounds and surrounding skin were harvested on day 21. Immunohistochemical staining as described by Huang et al. [21] was performed with rabbit antibodies against STAT3 (#ab68153, abcam),
VEGF (#ab27278, abcam) and IgG-FITC (abcam). The fluorescent images were collected with a CLSM as described above. The fluorescence
intensities of stained sections were quantified using Image-pro plus
6.0 software.
2.7. Burns repair
All animal procedures were performed in accordance with the
guidelines and ethics approval of Experimental Animal Care and Use
Committee of Guangdong Medical University. New Zealand rabbits
were commercially supplied by Guangdong Medical Laboratory Animal
Center (Foshan, China). The burns healing efficacies of composite films
were evaluated using New Zealand rabbit back burn model. New
Zealand rabbits were kept under monitored conditions at constant temperatures (25 ± 1 °C) with food and water ad libitum under a 12-h
dark/light cycle. The dorsal hair of New Zealand rabbits was shaved
and cleaned with 75% ethanol after anesthesia with 1.5% pentobarbital.
The scald wounds (3 cm2) were created in the dorsal skin of each animal
using a scalding temperature control instrument (YLS-5Q, Beijing
Zhongshidichuang Science & Technology Development Co. Ltd., Beijing,
China). Afterwards, MEBO, NHA/CS/TP(I) and NHA/CS/TP(II) were
applied to the burns. Burns treated with no materials under the same
conditions were served as the control. After 3, 7, 14 and 21 days, photographs of burn areas were taken, and the burn size at each time point
was determined by using Image J software (n = 5). Relative burn area
was calculated using the following formula:
S¼
2.12. Statistical analysis
The data were processed with IBM SPSS Statistics 22 software (IBM
Corp., Armonk, NY, USA) and analyzed using independent-samples ttests. Numerical data are expressed as mean ± SD, and differences
among groups were analyzed using one-way analysis of variance.
Differences were considered significant at p < 0.05.
3. Results and discussions
3.1. Characterization of hydroxyapatite
The hydroxyapatite particles were characterized by XRD and TEM,
and the results were shown in Fig. 1. TEM images show that dispersed
HA crystals have different morphology, usually in the form of stick. At
the same time, some HA crystals form fusiform or irregular aggregates.
The size of HA crystals and their aggregates was tens of nanometers.
The XRD patterns of HA crystals show strong diffraction peaks at
26.32°, 32.9°, and 34.1°, and these peaks correspond exactly to the overlapping diffraction of HA and the characteristic peaks of the lattice plane
[17]. Nanohydroxyapatite is non-toxic with high mechanical strength
and produces minimal immunological reactions, and in vitro attachment [22,23]. Also, nanohydroxyapatite with crack free scaffolds promotes rapid cell proliferation, spreading, rapid bioabsorption and bone
regeneration in short time [19].
S0−S1
100%
S0
Here, S0 represents the original burn area, S1 represents the unhealed burn area, and S represents relative wound area, respectively.
2.8. Histological and histometric analysis
3.2. Characterization of hydrogels
After the New Zealand rabbits were euthanized at each time point,
the surrounding skin and muscle around the wound area were extracted and fixed in 4% paraformaldehyde. Then the tissue samples
were stained with hematoxylin and eosin (H&E) and observed under
a fluorescence microscope (IX73, Olympus, Tokyo, Japan). The tissue
samples on day 21 were stained with Masson's trichrome to analyze
total collagen content (calculated as a percentage of the aniline blue
staining in dermis). Stained slides were observed and photographed
with a digital slice scanning system (Pannoramic MIDI, 3D HISTECH,
Budapest, Hungary), and then quantified by Image-pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
The SEM image was used to microscopically observe the morphology of NHA/CS/TP-I and NHA/CS/TP-II hydrogels and evaluate their microstructure (Fig. 2A–B). The SEM image revealed that the prepared
hydrogel had a highly porous structure comprised of interconnected
pores. The pores of NHA/CS/TP-II are smaller and the voids are more
uniform. The pore size was in the range of 42–86 μm, which was conducive to locking moisture and keeping a wet environment for wound
healing and favorable for the cell attachment and migration.
The results of water absorption are shown in Fig. 2C. The interactions
between chitosan and water molecules could cause the polymer to be
swelled while tannin protects the polymer from dissolution. While absorbing water, the swelling of hydrogels can provide a suitable environment for supporting cell survival, proliferation, and migration. The
results showed that hydrogels had excellent water absorption, which
2.9. Total protein and hydroxyproline content estimation
The level of total protein in the skin was measured using supernatant for skin tissue homogenization by ELISA kit according to the
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Fig. 1. (A) The XRD pattern of HA; (B) the TEM image of HA.
indicating that the dressing has good blood compatibility and high
safety [25–27]. The compatibility of prepared wound dresser with
blood cells, is regarded as a prerequisite for wound healing [28]. Hemolysis tests were conducted using 2% rabbit hemocyte suspension. The results were showed in Fig. 2D, obvious hemolysis was appeared in
distilled water, but no evident hemolysis was detected in normal saline
(NS). However, between NHA/CS/TP-I and NHA/CS/TP-II, low hemolysis
was appeared, NHA/CS/TP-II showed better compatibility than NHA/CS/
TP-I with a hemolysis rate of 4.37%.
is important to obtain a sustained drug delivery in the wound healing
site [24]. Additionally, combined with SEM results, it can be seen that
the NHA/CS/TP-II hydrogel had higher cross-linking degree and more
compact spatial structure.
The hemolysis rate acts as one way to evaluate the material biocompatibility. Wound healing dressings are usually indirect contact with
wounds and blood, so the dressings must meet safety standards to prevent hemolysis after contact with blood. The lower the hemolysis rate of
the dressing is, the less the ability to destroy red blood cells will be,
Fig. 2. (A) The SEM image of NHA/CS/TP-I; (B) the SEM image of NHA/CS/TP-II; (C) the water absorption capacity of hydrogels; (D) the results of hemolysis tests of hydrogels; All values are
expressed as the means ± SD for each group (n = 3).
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International Journal of Biological Macromolecules 181 (2021) 369–377
Fig. 3. (A) Survival rate of E. coli after contact with different hydrogels for 24 h. (B) Survival rate of S. aureus after contact with different hydrogels for 24 h. All values are expressed as the
means ± SD for each group (n = 3), * indicates significant difference from the control group, **p < 0.01.
viability of NHA/CS/TP-II is significantly higher than NHA/CS/TP-I and
the control group. However, in 72 h, the cell viability of NHA/CS/TP-II
is significantly higher than the control group, while the cell viability of
NHA/CS/TP-I is significantly lower than the control group. Therefore,
during 72 h, NHA/CS/TP-II hydrogel has been suitable for cell proliferation and better compatibility due to its tremendous positive effect on
cell growth.
3.3. Antibacterial property of hydrogels
Since many failure cases in terms of biomaterials-based therapy are
caused by bacterial infections, biomaterials with microbe inhibition currently are getting more attentions in clinic applications, particularly
wound dressing. Thus, the antibacterial performance of as-prepared hydrogel in this study against two representative microbes (Gram-negative E. coli and Gram-positive S. aureus) was investigated in vitro. The
results showed that, both NHA/CS/TP-I and NHA/CS/TP-II have excellent
antibacterial properties (Fig. 3), however, the antibacterial performance
of NHA/CS/TP-I and NHA/CS/TP-II does not have significant difference.
Such excellent inherent antibacterial capacity in hydrogels can be ascribed to the combined antibacterial ability from chitosan, TA and HA.
Therefore, the hydrogels are attractive for wound healing applications.
3.5. In vivo wound healing study of hydrogels
After burn injury on the dorsal skin of New Zealand rabbits, we observed tissue softening, necrosis, edema, and interstitial fluid exudation
with gradually expanding wound edges and red and swollen surrounding tissues (Fig. 5A). On day 3, the wounds remained soft and pale, with
no scabbing but obvious suppuration. On day 7, no scabbing, but suppuration and obvious exudation of interstitial fluid were observed in the
control group. In the other groups, the peripheral scabs began to come
off, and the wound area was significantly smaller than in the control
group. On day 14, the wounds were scabbed all groups, the peripheral
scabs began to come off, and the area continued to reduce. The decreases in wound area were greater in the NHA/CS/TP-I and NHA/CS/
TP-II groups than in the control and MEBO groups. On day 21, the
scabs had completely come off in the NHA/CS/TP-I and NHA/CS/TP-II
groups but not in the control group. The best outcomes were observed
in the NHA/CS/TP-II and MEBO groups, where hair began to grow in
the wound area.
The changes in healing rates of burn wounds in New Zealand rabbits
over time are shown in Fig. 5B. On day 3, the healing rate of each group
was negative as the wound area increased. On day 7, the wound area
was smaller than on day 3, with the slowest healing rate was observed
in the control group. The healing rate of the NHA/CS/TP-II group was significantly faster than in the control group, but not significantly different
from the MEBO group. On day 14, the healing rates of the NHA/CS/TP-I
and NHA/CS/TP-II groups were significantly faster than the control
group. The healing rate of the NHA/CS/TP-II group was also significantly
better than that of the MEBO group. The changes observed on day 21
were similar to that on day 14, and the NHA/CS/TP-II group healing rate
was 5.40–24.47% higher than the other groups, which was significant.
Wound healing is a complex process involving hemostasis, inflammation, cell proliferation and migration, angiogenesis, and remodeling
[29]. To evaluate the growth rate and quality of regenerated tissues, histopathological analyses were performed on H&E-stained wound tissue.
The results are shown in Fig. 5C. On day 14, there was still a moderate
inflammatory reaction, the scabs had not come off, and epithelial regeneration was not obvious in the control and MEBO groups. By contrast,
mild inflammation and extensive angiogenesis were observed in the
NHA/CS/TP-I and NHA/CS/TP-II groups. Moreover, complete epidermal
3.4. Cytocompatibility of hydrogels
For acting as a wound dressing, hydrogels that are biocompatible are
a prerequisite in biomedical application. The cytocompatibility of
hydrogels was examined using CCK-8 assay (Fig. 4). HUVECs were selected in this study because they play an essential role in wound healing.
As displayed in Fig. 4, the results of cell viability showed hydrogels' good
cytocompatibility. In 24 h, the cell viabilities of NHA/CS/TP-I and NHA/
CS/TP-II are both significantly higher than the control. In 48 h, the cell
Fig. 4. The results of cytotoxicity tests. All values are expressed as the mean ± SD for each
group (n = 3), * indicates significant difference from the control group, *p < 0.05.
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Fig. 5. (A) Digital photos of burns that were covered with control (physiological saline solution), MEBO, NHA/CS/TP-I and NHA/CS/TP-II within 21 days; (B) percentage of burns size over
time. Compared with control, *p < 0.05, **p < 0.01; compared with MEBO, #p < 0.05.
Hydroxyproline is a precursor amino acid necessary for procollagen
and collagen synthesis. As it indirectly reflects collagen content and is
related to tissue healing ability, it is an important indicator for evaluating wound healing [31]. Changes in the hydroxyproline contents of
granulation tissue after 21 days are shown in Fig. 6B. Compared with
the control group, there was a significant increase in the NHA/CS/TP-II
group (p < 0.05). Compared with the MEBO group, there were no differences in the NHA/CS/TP-I group, while a significant increase was observed in the NHA/CS/TP-II group (p < 0.05). These findings suggest
that NHA/CS/TP-II increases hydroxyproline content in granulation tissue and promotes wound healing.
TNF-α is an inflammatory factor produced by macrophages. IL-6 is a
lymphokine produced by activated T cells and fibroblasts that activates
acute response proteins and plays a pro-inflammatory role. Increased expression of TNF-α and IL-6 during wound healing can lead to an inflammatory imbalance in the body. TNF-α and IL-6 levels in the wound tissue
at 21 days are shown in Fig. 6C and D. Both were decreased in the NHA/
regeneration was observed in the NHA/CS/TP-II group, suggesting that
the formula promotes wound healing.
3.6. The expression of total protein, hydroxyproline, TNF-α and IL-6
The amount of protein synthesis directly affects the growth of granulation tissue and the final wound healing. Moreover, the total protein content in wound granulation tissue also reflects cell proliferation, with a
higher total protein content indicating better granulation tissue growth
[30]. Fig. 6A shows changes in total protein content in granulation tissue
in each group at 21 days. Compared with the control group, total protein
content in the granulation tissue was significantly increased in the NHA/
CS/TP-I and NHA/CS/TP-II groups (both p < 0.05). However, no significant
differences with the MEBO group were observed in the NHA/CS/TP-I
group, but there was a significant increase in the NHA/CS/TP-II group
(p < 0.05). The result indicates that NHA/CS/TP-II promotes protein synthesis in granulation tissue and accelerates wound healing.
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International Journal of Biological Macromolecules 181 (2021) 369–377
Fig. 6. (A) The content of total protein in wound tissues in each group; (B) the content of hydroxyproline in wound tissues in each group; (C) the expression of TNF-α in wound tissue;
(D) the expression of IL-6 in wound tissue. Compared with the control group, *p < 0.05; compared with MEBO group, #p < 0.05; Data represent means ± SD, n = 6.
CS/TP-II groups compared to the control group. Notably, STAT3 and
VEGF expression in the NHA/CS/TP-II group were more similar to levels
in the normal group.
CS/TP-I and NHA/CS/TP-II groups, which showed the lowest expression
with significant differences from the control and MEBO groups. This indicates that NHA/CS/TP-II reduces the local inflammatory response to promote wound healing.
4. Conclusions
3.7. The expression of collagen, STAT3 and VEGF
In summary, hydroxyapatite crystals with particle size of tens of
nanometers were synthesized by coprecipitation method. Then we
have successfully fabricated a nanohydroxyapatite-reinforced NHA/CS/
TP-I and NHA/CS/TP-II hydrogels for burns healing. NHA/CS/TP-I and
NHA/CS/TP-II hydrogels displayed porous structure that is similar to extracellular matrix (ECM). NHA/CS/TP-II hydrogel showed significantly
water absorption, antibacterial, cytocompatibility, those of which were
extremely beneficial for accelerating the process of burns healing.
More importantly, NHA/CS/TP-II hydrogel promotes total protein synthesis in granulation tissue, substantially increases hydroxyproline content, reduces the local wound inflammatory response, accelerates
angiogenesis and epithelialization, promotes collagen fiber production,
and significantly increases STAT3 and VEGF expression, thereby promoting granulation tissue formation and burns healing. Therefore,
NHA/CS/TP-II hydrogel has great potential as an ideal burns dressing
to promote wound healing.
Changes in collagen contents on day 21 were observed by Masson
staining (Fig. 7). Collagen fibers are stained blue, and muscle fibers,
cytoplasm, and red blood cells appear red. The collagen fibers were
arranged in an orderly fashion with uniform density in the normal
group (no burn) and the MEBO, NHA/CS/TP-I and NHA/CS/TP-II
groups. By contrast, they were disordered in the control group. The
collagen content (represented by average optical density) in the dermis layer of the burn wound was calculated, and the results were
shown in Fig. 7. The average optical densities of collagen in the
NHA/CS/TP-I and NHA/CS/TP-II groups were significantly higher
than in the control and MEBO groups and similar to that in the normal group.
The transcription factor STAT3 can bind to DNA and regulates multiple signal transduction pathways. VEGF is a key factor in angiogenesis
that can serve as a highly specific mitogen to promote endothelial cell
proliferation and induce angiogenesis. VEGF can also increase the release of angiogenetic factors by inducing macrophage chemotaxis,
playing an important role in granulation tissue formation in the early
stage of healing [32,33]. It has been reported that the promoter region
of the VEGF gene has a binding site for STAT3, and continuous activation
of STAT3 can induce VEGF expression [34]. As shown in Fig. 5, STAT3 and
VEGF were expressed in the wounds in all groups 21 days after treatment. The expression levels of STAT3 and VEGF (represented by average
optical density) in the wounds in each group were calculated with
Image-plus 6.0 software. The results showed that the expression of
both proteins was significantly higher in the NHA/CS/TP-I and NHA/
CRediT authorship contribution statement
Qianqian Ouyang: Investigation, Methodology, Validation.
Kong Songzhi: Conceptualization, Funding acquisition.
Huang Yongmei: Software, Data curation, Validation.
Ju Xianghong: Software, Validation.
Li Sidong: Writing - Review & editing.
Li Puwang: Supervision, Project administration, Resources.
Luo Hui: Conceptualization, Funding acquisition, Writing- Review &
editing, Project administration.
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O. Qianqian, K. Songzhi, H. Yongmei et al.
International Journal of Biological Macromolecules 181 (2021) 369–377
Fig. 7. (A) The expression of collagen, STAT3 and VEGF in normal skin (a) or wounds that were treated with physiological saline solution (b), MEBO (c), NHA/CS/TP-I (d) or NHA/CS/TP-II
(e); (B) the mean densities of collagen content (compared with the control group, *p < 0.05 and **p < 0.01; Data represent means ± SD, n = 6); (C) the mean densities of STAT3
expression in each group on day 21 (compared with the control group, *p < 0.05; Data represent means ± SD, n = 6); (D) the mean densities of STAT3 expression in each group on
day 21 (compared with the control group, *p < 0.05 and **p < 0.01; Data represent means ± SD, n = 6).
Declaration of competing interest
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The authors declare no conflict of interest.
Acknowledgments
This work was supported financially by Project of Science and Technology Plan of Guangdong Province (2017A010103023, 2018A030307015),
the National Natural Science Foundation of China (No. 81803684), the
Guangdong University Youth Innovation Talent Project(2020KQNCX023),
the public service platform of south China Sea for R&D marine biomedicine
resources (2017C8A), the Fund of Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (ZJW-2019-007), the Science and
Technology Program of Guangdong Province (2019B090905011).
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