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 Biomed. Mater. 19 (2024) 015008 https://doi.org/10.1088/1748-605X/ad0d85

Biomedical Materials

PAPER

An ECM-mimicking assembled gelatin/hyaluronic acid hydrogel

RECEIVED
11 July 2023
REVISED
with antibacterial and radical scavenging functions for
29 October 2023
accelerating open wound healing
ACCEPTED FOR PUBLICATION
15 November 2023
Xuebin Ma1, Wenli Ning2, Yiming Geng3, Huarong Shao1, Yang Liu1, Fei Liu1, Daizhou Zhang1, Bo Chi4,
Yali Hou3,∗ and Xiao Fu2,3,∗
PUBLISHED
7 December 2023
1
Shandong Provincial Key Laboratory of Biomedical Polymers, Shandong Provincial Key Laboratory of Biopharmaceuticals, Shandong
Academy of Pharmaceutical Sciences, Jinan, Shandong 250100, People’s Republic of China
2
School of Stomatology, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong 250021,
People’s Republic of China
3
Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan,
Shandong 250012, People’s Republic of China
4
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing Tech University,
Nanjing 211800, People’s Republic of China
∗
Authors to whom any correspondence should be addressed.
E-mail: n_houyali@sina.cn and fuxiao@sdfmu.edu.cn

Keywords: ECM-mimicking hydrogel, gelatin, hyaluronic acid, open wound healing

Supplementary material for this article is available online

Abstract
A multifunctional hydrogel dressing with hemostatic, antibacterial, and reactive oxygen species
(ROS)-removing properties is highly desirable for the clinical treatment of open wounds. Although
many wound dressings have been prepared, the modification of polymers is often involved in the
preparation process, and the uncertainty of biological safety and stability of modified polymers
hinders the clinical application of products. In this study, inspired by the composition and
crosslinking pattern of extracellular matrix (ECM), a deeply ECM-mimicking multifunctional
hydrogel dressing is created. Tannic acid (TA) and poly-ε-lysine (EPL) are added into a
gelatin/hyaluronic acid (Gel/HA) matrix, and a stable hydrogel is formed due to the formation of
the triple helix bundles of gelatin and hydrogen bonds between polymers. The introduction of TA
and EPL endows the ECM-mimicking hydrogel with stable rheological properties, as well as
antibacterial and hemostatic functions. The as-produced hydrogels have suitable swelling ratio,
enzyme degradability, and good biocompatibility. In addition, it also shows a significant ability to
eliminate ROS, which is confirmed by the elimination of 2,2-diphenyl-1-picrylhydrazyl free
radical. Full-thickness skin wound repair experiment and histological analysis of the healing site in
mice demonstrate that the developed ECM-mimicking Gel/HA hydrogels have a prominent effect
on ECM formation and promotion of wound closure. Taken together, these findings suggest that
the multifunctional hydrogels deeply mimicking the ECM are promising candidates for the clinical
treatment of open wounds.

1. Introduction 2.8%, respectively, and surgical wound infection was

The skin serves as both the first line of defense
and the largest organ of the human body, mak-
ing it particularly susceptible to damage [1, 2]. In
2014, about 14.5% of medicare beneficiaries in the
United States were diagnosed with at least one wound
or wound-related infection. Among them, surgical
wounds and traumatic wounds accounted for 3% and
the largest category with a prevalence rate of 4.0%
[3]. Common skin wounds can be wrapped or pressed
with gauze, bandage, or woundplast [4]. However,
for open skin wounds, sutures as the main wound
treatment method will adversely affect the surround-
ing tissues [5]. These wounds often act together with
bacterial infection and excessive inflammation [6, 7].
Bacterial infection at the wound site can lead to

© 2023 IOP Publishing Ltd

Biomed. Mater. 19 (2024) 015008
other serious complications [8]. Excessive inflam-
mation will trigger the release of matrix metallo-
proteinases and increase the risk of fibrotic scarring
[9]. In addition, a variety of reactive oxygen species
(ROS), such as hydroxyl radicals, hydrogen perox-
ide, and superoxide radicals, are normally produced
around the injured tissue, leading to protein and
DNA damage and hampering tissue regeneration [10,
11]. Therefore, multifunctional wound dressings with
antibacterial and antioxidant characteristics ought to
significantly speed up the healing process. Designing
novel functional dressing materials is the urgent task
for open wound treatment [12].
In order to accelerate wound healing, various bio-
materials have been used, including films, porous
foams, and functional hydrogels [13–15]. Because
of their excellent hydrophilicity, biocompatibility,
and three-dimensional network structure resembling
extracellular matrix (ECM), hydrogels have garnered
the most interest of these materials. It can be used as
a microbial barrier to keep a relatively moist envir-
onment at the skin defect site and maintain good
oxygen and water permeability, thus alleviating the
pain of patients to some extent [16–18]. However,
many functional hydrogel wound dressings (such as
gelatin hydrogels crosslinked by UV light, hyalur-
onic acid-based hydrogels crosslinked via Michael
addition reaction, Schiff base reaction, etc) involve
modification of polymers in the preparation pro-
cess, and the uncertainty of biological safety and
stability of modified polymers hinders the clinical
application of products [19–21]. In this work, we
intend to produce a unique type of multifunctional
hydrogel wound dressing through a physical blend-
ing strategy based on the prior hydrogel wound dress-
ing research of our group, which is the safest and
most close to ECM crosslinking method for hydro-
gel formation [22–24]. Inspired by the components
of ECM, gelatin and HA, as commonly used nat-
ural polymers, are selected as the primary ingredi-
ents of hydrogel wound dressings. We speculate that
the hydrogel designed to mimic ECM in terms of
composition and cross-linking strategy can provide
a favorable microenvironment for cell growth, and
this deep biomimetic design will be conducive to
cell proliferation, migration, and collagen deposition,
thereby accelerating wound healing. In order to avoid
wound infection, various antibacterial agents, includ-
ing antibiotics, silver nanoparticles, fullerenes, and
others, are embedded in hydrogels [25–27]. However,
the long-lasting antibacterial activity of hydrogels is
severely hampered by antibacterial substance leak-
age, which may even lead to metal ion toxicity
and allergic responses [28]. Poly-ε-lysine (EPL), a
naturally occurring biological metabolite contain-
ing about 30 l-lysine subunits, has broad-spectrum
antibacterial effects against both gram-positive and
gram-negative microorganisms [29, 30]. Especially,
2
X Ma et al
EPL with a molecular weight between 3600 and 4300
has strong antibacterial properties and is a strong can-
didate as an ingredient in antibacterial dressings [31].
Tannic acid (TA) is a natural polyphenol that has been
authorized by the Food and Drug Administration
(FDA) for use in medicine and food [32]. The
free radical scavenging activity of TA can protect
biomacromolecules from ROS attack and acceler-
ate the wound repair process [33–35]. Moreover, TA
has anti-inflammatory, antibacterial and hemostatic
properties [36–38]. These characteristics make TA a
good candidate for wound healing dressings.
In this study, the ECM-mimicking multifunc-
tional gelatin/hyaluronic acid (Gel/HA) hydro-
gels were prepared by a physical blending strategy
(figure 1). The introduction of TA and EPL makes
the Gel/HA hydrogels have excellent ROS scavenging
effect, antibacterial, and hemostatic ability. Notably,
the ECM-mimicking Gel/HA hydrogels exhibit good
wound healing performance confirmed by full-
thickness skin wound repair experiments. Overall,
this work presents a convenient and cost-effective
hydrogel dressing with promising clinical transla-
tional potential for the treatment of open wounds.
2. Experimental section
2.1. Materials
Gelatin (from porcine skin, Type A) and Calcein-
AM/Propidium Iodide Live/Dead kit were pur-
chased from Beyotime Biotech Co., Ltd (Shanghai,
China). HA in the form of sodium hyaluronate
(from microbial fermentation, MW ⩾ 1.8 MDa)
was obtained from Bloomage Biotechnology
Co. Ltd (Jinan, China). TA, trypsin and 2,2-
diphenyl-1-picrylhydrazyl (DPPH) were bought
from Shanghai Macklin Biochemical Co., Ltd
(Shanghai, China). EPL (MW: 3000–5000 Da) was
purchased from Aladdin Industrial Corporation
(Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), Dulbecco’s
modification of Eagle’s medium Dulbecco (DMEM)
and papain (500 U mg−1 ) were purchased
from Solarbio Life Sciences (Beijing, China).
Hyaluronidase (300–500 U mg−1 ) was obtained
from Shanghai Yuanye Biotechnology Co., Ltd
(Shanghai, China). Fetal bovine serum was bought
from Zhejiang Tianhang Biotechnology Co. Ltd
(Zhejiang, China).
2.2. Preparation and characterization of hydrogels
The hydrogels were prepared as follow: firstly, 500 µl
of a gelatin aqueous solution (80 mg ml−1 ) was mixed
with 100 µl of a HA aqueous solution (2 mg ml−1 ).
Then 50 µl of a TA aqueous solution (15 mg ml−1 )
was mixed with 50 µl of a EPL aqueous solu-
tion (15 mg ml−1 ). Afterwards, these two mix-
tures were mixed rapidly by an oscillator, followed
Biomed. Mater. 19 (2024) 015008
Figure 1. Schematic diagram of the ECM-mimicking multifunctional Gel/HA hydrogels and its application in open wound
healing.
by sonication to remove air bubbles in mixture.
The Gel/HA/TA/EPL (GHTE for short) hydrogel was
obtained after standing at 25 ◦ C for 24 h. For Gel/HA
hydrogel, TA and EPL were not introduced, and for
Gel/HA/TA (GHT for short) hydrogel, only TA was
added into Gel/HA hydrogel.
The lyophilized hydrogels’ microporous morpho-
logies were studied using scanning electron micro-
scopy (SEM). First, cylindrical hydrogel samples with
a radius of 0.5 cm and a height of 1 cm were pre-
pared, and then vacuum-lyophilized at −50 ◦ C for
48 h after being frozen at −80 ◦ C. The lyophilized
hydrogel samples were broken off and coated with
gold on their cross sections to improve their conduct-
ivity. Finally, the cross-sectional morphologies were
visualized under a voltage of 3 kV by a SEM (Carl
Zeiss, G300, Germany).
To investigate the swelling behavior of hydro-
gels, the hydrogels were first lyophilized and
weighed (W d ). The freeze-dried hydrogels were then
immersed in deionized water at 25 ◦ C. At differ-
ent time intervals, the swollen hydrogels were taken
out and weighed immediately after wiping the water
from the surface with filter paper. Swelling ratio was
denoted as (W s − W d )/W d .
For the enzymatic degradation of hydrogels,
the hydrogels were first lyophilized and weighed
(W d ). The freeze-dried hydrogels were then incub-
ated in phosphate buffer solution (PBS) containing
0.1 mg ml−1 hyaluronidase and 0.25 mg ml−1 papain
at 25 ◦ C. At predetermined time points (2, 5, 10, 18,
28, 38, 48, 72 h), the hydrogels were removed for lyo-
philization and then weighed (W s ).
Weight remaining(%) = (Ws /Wd ) × 100%
The porosity of hydrogels was studied using
the solvent displacement method [39]. The known
3
X Ma et al
weight (W d ) freeze-dried hydrogels were sub-
merged in 100% ethanol and maintained there until
saturation. Drowned hydrogels were taken out and
weighed (W s ) immediately. The percentage of poros-
ity was denoted as
(Ws − Wd )/(Vs × ρethanol )
where, V s is the volume of hydrogels after freeze-
drying and ρ is the density of ethanol.
Rheological characterization of hydrogels was
carried out using a rheometer (Anton Paar, MCR302,
Austria) with a 25 mm plate-plate sensor at 25 ◦ C.
First, strain sweep tests in the strain range from 0.1%
to 1000% were performed to determine the linear vis-
coelastic ranges of hydrogels at 6.28 rad s−1 . Next, the
oscillatory frequency sweeps were performed at a con-
stant strain of 1%. The storage modulus (G′ ) and the
loss modulus (G′′ ) were monitored in the frequency
range between 0.5 and 100 rad s−1 .
2.3. Antioxidant activity assay
The antioxidant capacity of GHTE hydrogels was
investigated via DPPH radical scavenging assay [40].
The 200 mg of hydrogels with different TA dosage
were added into the DPPH ethanol solution (4.0 ml,
100 µM). The solution was incubated for 30 min at
room temperature in the dark. The DPPH ethanol
solution (4 ml) was mixed with 0.2 ml of deionized
water for use as a control. The absorbance of the
treated solution was then measured using a UV–vis
spectrophotometer at 517 nm. The clearance ratio of
the DPPH radical was calculated based on the follow-
ing equation:
Antioxidant activity(%) = (Ac − As )/Ac × 100%
where, Ac and As are the absorbances of the control
and test sample, respectively.
Biomed. Mater. 19 (2024) 015008
2.4. Antibacterial assay
The broad-spectrum antibacterial effect of the hydro-
gel was evaluated through contact antibacterial test
[41]. Staphylococcus aureus [CMCC(B) 26 003] and
Escherichia coli [CMCC(B) 44 102] were selected as
model bacteria. Then 10 µl of the bacterial suspen-
sion (1 × 106 CFU ml−1 ) were pipetted onto the sur-
face of the hydrogels (1.0 cm × 1.0 cm) in a dish and
10 µl of bacterial suspension without materials was
used as a control. After incubating the plate at 25 ◦ C
for 6 h, 1 ml of sterile PBS was added into each well to
re-suspend the surviving bacteria. Finally, the treated
bacterial suspension (50 µl) formed colonies,
Bacterial viability(%)
= (Survivor CFUs on patch)/(CFUs of control)
× 100%.
2.5. Cytocompatibility of hydrogels
The GHTE hydrogel extracts were used to evaluate
the cytocompatibility of hydrogels by MTT assay and
live/dead assay. L929 mouse fibroblasts (L929 cells)
were selected for test due to their vital effect during
the wound healing process. The detailed experimental
procedures are described in supporting information.
2.6. In vitro cell scratch assay
L929 cells were first seeded into a 24 well plate for
24 h, and the formed monolayer cell was scratched by
a 200 µl pipet tip. The dropped cells were then washed
away with PBS solutions. Next, the L929 cells were co-
incubated with GHTE hydrogels in DMEM medium
containing 2% FBS. At predetermined time points
(0, 12, 24 h), the L929 cells were photographed by
an inversed fluorescent microscope (SOPTOP, ICX41,
China).
2.7. Hemocompatibility of hydrogels
The dried hydrogels were gently mixed with eryth-
rocyte suspension in a tube. After an hour of co-
incubation, the absorbance of the supernatant was
recorded at 540 nm by a UV–vis spectrophotometer.
Water and PBS were served as the positive control and
negative control groups, respectively. The detailed
experimental procedures are described in supporting
information.
2.8. Hemostatic effect evaluation
The hemostatic effect of hydrogels was evaluated by a
mouse-tail amputation model. The tail-cut mice were
treated with GHTE hydrogels, and the group without
any treatment was used as a control. The detailed
experimental procedures are described in supporting
information.
2.9. In vivo wound healing assessment
The effect of GHTE hydrogels on open wound
repair was evaluated by a full-thickness skin wound
model in the back of mice. In this experiment, 15
4
X Ma et al
female Kunming mice (8 weeks old) were randomly
divided into three groups (5 animals per group),
including a wound without treatment group, a
TegadermTM -treated group, and a GHTE hydrogel-
treated group. The animals were paralyzed with
10 wt% chloral hydrate and then fixed to a surgical
corkboard. After shaving the hair of animals, a full-
thickness round wound with a diameter of 1 cm was
created in the dorsal region of mice. After surgery
and covering the wounds of different groups with
TegadermTM and GHTE hydrogel, the animals were
fed separately to prevent touching the wound tissue.
Wounds were monitored on days 0, 3, 7, and 14 after
surgery and digital photographs were taken to meas-
ure the wound surface area using ImageJ software.
To evaluate the healing process and inflammation
reaction of wound, the regenerated tissues were fixed
with paraformaldehyde (4%) on day 14, and then
entrenched in paraffin for pathological slice. After
Hematoxylin and Eosin (H&E) and Masson’s tri-
chrome staining, the histological analysis was imple-
mented under an inverted microscope. The wound
healing rate was measured based on the following
equation:
The wound closure(%) = (S0 − St )/S0 × 100%
where S0 represents the area of the initial wound and
St is the wound area on days 3, 7, and 14.
3. Statistical analysis
Experimental data were expressed as the
mean ± standard deviation. Statistical analyses
were performed by one-way ANOVA followed using
Tukey’s multiple comparison tests, and statistical sig-
nificance was accepted at P < 0.05.
4. Results and discussion
4.1. Preparation and characterization of hydrogels
Glycosaminoglycans and proteins are the main com-
ponents of ECM, among which gelatin and HA
receive more attention. Gelatin is a hydrolysate of
collagen, and the presence of signal peptides such
as Arg-Gly-Asp (RGD) in the backbone of gelatin
can increase re-epithelialization and collagen depos-
ition, which is essential for skin regeneration [42,
43]. HA is a major constituent of the ECM in the
human body, it plays an important role in supporting
cells, recognition by specific surface receptors, colla-
gen deposition, and angiogenesis during the wound
healing process [44–46]. Therefore, gelatin and HA
were selected to prepare bionic ECM hydrogel in
this work. Furthermore, TA with natural antioxidant
function and EPL with spectral antibacterial function
were introduced to achieve the preparation of ECM-
mimicking multifunctional Gel/HA hydrogels. Here,
the multifunctional Gel/HA hydrogels were prepared
Biomed. Mater. 19 (2024) 015008 X Ma et al

Figure 2. Physical and chemical properties of multifunctional Gel/HA hydrogels. (a) Swelling ratio of Gel/HA, GHT, and GHTE
hydrogels in deionized water. (b) Degradation curves of GHTE hydrogels incubated in PBS and enzyme solutions containing
hyaluronidase (0.1 mg ml−1 ) and papain (0.25 mg ml−1 ). (c) Porosity percentage of Gel/HA, GHT, and GHTE hydrogels. (d)
SEM image of freeze-dried GHTE hydrogel. The red circles and arrows display the morphology of the porous interconnections.
(e) Strain sweep and (f) frequency sweep of Gel/HA, GHT, and GHTE hydrogels. (g) Storage moduli of Gel/HA, GHT, and GHTE
hydrogels at 10 rad s−1 . (h) Color change of the DPPH solution containing GHTE hydrogels with different TA dosage and (i)
their corresponding percentage of scavenging effect.

by a physical blending strategy (figure 1). Firstly,
HA solution was added into the hot gelatin solu-
tion to prepare the mixed solution of gelatin and
HA. Then the TA and EPL mixed solution was added
into the aforementioned mixed hot solution. Finally,
the mixture was vortexed fully and left overnight
at 25 ◦ C. After that, the ECM-mimicking Gel/HA
hydrogels formed due to the formation of the triple
helix bundles of gelatin and hydrogen bonds between
polymers [39, 47–49].
The physical properties of hydrogels play an
important role in wound healing, such as the ability
to absorb water, which facilitates the wound healing
process by absorbing wound efflux [28, 36]. In this
study, the swelling ratios of hydrogels were approx-
imately 15 times their initial weight (figure 2(a)),
which shows a high water absorption capacity. After
incubation for about 23 h, hydrogels reached swell-
ing equilibrium. Compared with Gel/HA hydrogel,
GHT hydrogel shows a lower swelling ratios, which
may be due to the increased crosslink degree of the
Gel/HA hydrogel induced by the introduction of TA.
GHTE hydrogel shows the lowest equilibrium swell-
ing ratios, indicating that the introduction of EPL
to the GHT hydrogel further increased the cross-
link degree [50]. The biodegradability of hydrogels is
essential for tissue regeneration, which can provide
space for cell migration, infiltration, and proliferation
[51]. As shown in figure 2(b), the residual weight
of GHTE hydrogel was less than 20% after 3 d
of enzymatic digestion in the solution containing
hyaluronidase and papain, while the residual weight
was still high at 80% in PBS, showing a good bio-
degradation performance. High porosity has a great
impact on cell migration, nutrient and oxygen sup-
ply, and waste removal [52]. In this work, the poros-
ity was measured via ethanol displacement method.
Ethanol can enter into the freeze-dried hydrogels
through capillary force, but the volume of the freeze-
dried hydrogels will change after soaking in ethanol,
so the porosity measured by the ethanol displacement
method is only a reference value, not the actual value
of the hydrogel [39, 53]. The porosity percentage
was greater than 50% for all samples, and the poros-
ity of GHT hydrogel and GHTE hydrogel showed a
decreasing trend (figure 2(c)), which may be attrib-
uted to the hydrogen bonds between TA and poly-
mer and the electrostatic attraction between EPL and

5
Biomed. Mater. 19 (2024) 015008
Figure 3. Antibacterial activity of the multifunctional Gel/HA hydrogels. (a) Representative images of agar plates formed by S.
aureus and E. coli after treatment with PBS, Gel/HA, GHT, and GHTE hydrogels and (b), (c) their corresponding percentage of
antibacterial effect.
polymer, increasing the degree of crosslinking of the
hydrogel and thus reducing the porosity of hydro-
gel. In addition, SEM image of GHTE hydrogel after
freeze-drying showed highly porous structures and
high interconnection (figure 2(d)), which would be
conducive to nutrient transport and waste discharge.
In addition, the GHTE hydrogel has almost no wet
adhesion ability (figure S1), which can avoid second-
ary injury to the wound when the dressing is changed.
The rheological properties of hydrogels were ana-
lyzed through the strain sweep and frequency sweep.
Figure 2(e) illustrates that the G′ (storage modulus)
values of hydrogels in strain sweep showed a linear
viscoelastic range from 0.1% to 30% strain, while G′
and G′′ (loss modulus) curve cut at a point of 200%,
meaning that if the strain exceeded 200%, the hydro-
gel structure was totally destroyed [4]. The frequency
sweep reveals that G′ was frequency independent and
greater than G′′ across the entire frequency range,
demonstrating the elastic solid nature of hydrogels
(figure 2(f)) [54]. The average G′ of Gel/HA hydrogel
(at 10 Hz) was lower than that of the GHT and GHTE
6
X Ma et al
hydrogels, demonstrating that the addition of TA and
EPL increased the strength of hydrogels (figure 2(g)).
The results of the swelling and porosity are consistent
with this outcome.
4.2. Radical scavenging activity of hydrogels
Dressings with antioxidant properties may enhance
wound healing by scavenging excess ROS [55]. In this
study, the DPPH free radical scavenging capability of
hydrogels was used to assess their antioxidant prop-
erties. The gallol and catechol groups impart TA with
a high radical scavenging ability [56]. As shown in
figure 2(h), the dark purple of DPPH solution became
colorless when GHTE hydrogel was added into DPPH
solution because of the quenching of DPPH. The
antioxidant efficiency increased with increasing TA
dosage in the hydrogel. When the TA dosage was
increased to 50 µg, 200 mg of GHTE hydrogel elim-
inated about 90% of the free radicals from 4.0 ml
of DPPH ethanol solution (100 M) (figure 2(i)), and
200 mg of TA aqueous solution eliminated 98.3% of
free radicals (figure S2). These results demonstrated
Biomed. Mater. 19 (2024) 015008
Figure 4. Cytocompatibility of GHTE hydrogel. (a)–(c) Viability of L929 cells cultured with different concentrations of GHTE
hydrogel extract for 24, 48, and 72 h. (d) Fluorescence images of live (green)/dead (red) L929 cells cultured with GHTE hydrogel
extracts (5 mg ml−1 ) for 1, 2 and 3 d, respectively. Cultures in medium without GHTE hydrogel extracts were used as a control.
that the other components of GHTE hydrogels had
negligible effects on the antioxidant activity of TA,
and a strong antioxidant activity of GHTE hydrogels.
4.3. Antibacterial property of hydrogels
One of the main challenges of wound healing con-
tinues to be infection. Dressings with antimicrobial
activity will help avoid the infection of wounds with
dangerous microorganisms and accelerate wound
healing. Here, the antibacterial properties of hydro-
gels were tested using S. aureus and E. coli by the
spread plate approach [57]. Figures 3(a)–(c) shows
the results and the matched colony counts. Compared
to the PBS group, Gel/HA hydrogel had no bac-
teriostatic effect. Antibacterial activity got better after
adding TA into Gel/HA hydrogel, GHT hydrogel
showed obvious bacteriostatic effect on S. aureus but
no obvious inhibitory effect on E. coli. When EPL was
further introduced, the GHTE hydrogel killed 100%
of bacteria and no bacterial clones were found on
the agar plates, indicating the excellent antibacterial
potential of GHTE hydrogels against E. coli and S.
aureus.
4.4. Cytocompatibility and migration evaluation
Wound dressings used for wound repair should have
sufficient biocompatibility. The cell biocompatibility
of GHTE hydrogels was first evaluated by an MTT
7
X Ma et al
assay. As can be seen in figures 4(a)–(c), the
proliferation and viability of L929 cells were eval-
uated at 24, 48 and 72 h with various concentra-
tions of GHTE hydrogel extract. The results showed
that the cell viability treated with GHTE hydrogel
extract was no less than 90% of that of the PBS
control group, which proved that GHTE hydrogel
had no cytotoxicity. In addition, the cytocompatib-
ility was also evaluated visually by a Calcein-AM/PI
live/dead staining assay. As shown in figure 4(d), the
L929 cells co-incubated with both PBS and GHTE
hydrogel extracts exhibited a high-density, spindle-
like morphology, and only a few cells dead, indicating
that GHTE hydrogel had no cytotoxicity. Therefore,
the cell viability and Calcein-AM/PI staining res-
ults of L929 cells indicated GHET hydrogels have
high cytocompatibility and application potential in
biomedicine.
The cell scratch assay was performed to invest-
igate the effects of hydrogels on the migration and
motility of L929 cells (figure 5(a)). After 12 h of co-
culture, there were clear differences in cell mobility
between the hydrogel group and the control group.
Compared to the control group, L929 cells incub-
ated with GHTE hydrogel showed higher cell mobil-
ity after 24 h (figure 5(b)). This may be due to
the inherent elastic properties of GHTE hydrogels
and the deeply biomimetic ECM components, which
Biomed. Mater. 19 (2024) 015008 X Ma et al

Figure 5. Assessment of cell migration, hemolysis and hemostasis. (a) Schematic diagram of the co-culture model of L929 cells
with GHTE hydrogels for cell migration. (b) Migration images of L929 cells treated with GHTE hydrogels. The control group was
treated with FBS supplemented culturing medium without hydrogels. (c), (d) Digital images of hemolysis test and hemolysis ratio
after treated with water, PBS, Gel/HA, GHT, and GHTE hydrogels. (e), (f) Hemostatic images of control group and GHTE
hydrogel group after rat tail amputation, and their corresponding collected blood mass.

can promote cell migration [40, 58]. These results
suggested that GHTE hydrogel could significantly
facilitate cell migration and further accelerate wound
healing.
4.5. Hemocompatibility of hydrogels
Hematotoxicity is one of the factors limiting the use of
biomaterials in vivo as it can raise the amount of free
hemoglobin in the blood. Therefore, blood compat-
ibility must be checked when developing a new bio-
material. Here, the blood compatibility of hydrogels
was evaluated by in vitro hemolysis tests to determ-
ine whether they meet the requirements for in vivo
applications. As shown in figure 5(c), the red blood
cells dispersed in deionized water were quickly lysed
under hypertonic conditions and the supernatant was
red, while both the hydrogel group and the negat-
ive control group were transparent. The hemolysis
rates of Gel/HA, GHT, and GHTE hydrogels were
0.6%, 0.5%, and 0.7%, respectively (figure 5(d)),
which was below than the international penetration
threshold of 5% for biomaterials [59], demonstrat-
ing that the addition of TA and EPL did not increase
8
the hemolysis of Gel/HA hydrogel, and GHTE hydro-
gels could be used to further study the hemostatic
properties.
4.6. In vivo wound healing study
4.6.1. Animal hemostasis experiments
The first stage of wound healing is hemostasis,
so potential wound dressings should have effective
hemostasis [60]. Here, the rat tail excision model
was used to verify the hemostatic efficacy of GHTE
hydrogel (figure 5(e)). As shown in figure 5(f),
the blood loss of GHTE hydrogel group was only
26.2 ± 3.4 mg, while the no-treatment control group
was 95.1 ± 5.1 mg. There was a substantial differ-
ence in the amount of blood loss between the GHTE
hydrogel group and the control group (P < 0.01),
indicating that GHTE hydrogel has a good hemo-
static ability. This may be due to following factors: the
high-water absorption of the GHTE hydrogel, which
helps to form dense clots at the bleeding site. The
presence of signal peptides in the gelatin structure,
such as RGD, facilitates better adhesion of cells to the
hydrogel, and the cationic groups in the gelatin help
Biomed. Mater. 19 (2024) 015008 X Ma et al

Figure 6. In vivo accelerated wound healing of GHTE hydrogels. (a) Photographs of skin wounds on the 0th, 3th, 7th, and 14th
days treated with TegadermTM , GHTE hydrogels, and control group. (b) Wound closure and (c) traces of wound area on day 0, 3,
7, and 14. (d) H&E and Masson’s Trichrome staining results of healed wound tissues on day 14. Black, yellow and blue arrows
represent epithelial tissue, hair follicles, and collagen fibers, respectively.

trap negatively charged red blood cells in the blood
clot [61]. In addition, TA in the GHTE hydrogel has
a vasoconstrictive effect and can interact with blood
proteins [62], which has a synergistic effect on the
hemostatic ability of GHTE hydrogel.
4.6.2. Wound regenerating assessment
In order to further assess the capacity of GHTE
hydrogel to promote closure and healing of open
wounds, a full-layer skin wound model was estab-
lished in Kunming mice. Each mouse’s wound healing
phase was documented using pictures, and the wound
closure traces were carefully examined. Figure 6(a)
displays the wound closure of the TegadermTM (a
commercial thin film dressing) treatment group, the
GHTE hydrogel treatment group, and the control
group on days 0, 3, 7, and 14. The wound areas of all
three groups became smaller with increasing postsur-
gery time. Compared to the control and TegadermTM
dressing group, the GHTE hydrogel group showed the
fast wound contraction speed during the whole repair
process. On the 14th day, the GHTE hydrogel treat-
ment group showed completely closed, even some of
the mice presented hair coverage. Quantitative ana-
lysis of the wound area on the 7th day showed that
the wounds covered with GHTE hydrogel had a heal-
ing ratio of 84%, which was significantly higher than
the control (60%) and TegadermTM dressing (65%)
group (figures 6(b) and (c)). The GHTE hydrogel
group’s wound healing ratios were around 100% after

9
Biomed. Mater. 19 (2024) 015008
14 d of therapy, which is similar to the reported data
on HA or gelatin-based hydrogels to promote healing
[39, 63, 64].
H&E and Masson’s trichrome stain were used to
assess the effect of the wound healing process fur-
ther. The TegadermTM dressing group and the con-
trol group both had an abundance of inflammat-
ory cells surrounding the wound healing site accord-
ing to the results of H&E staining, and no obvious
inflammatory reaction was observed in the GHTE
hydrogel treatment group (figure 6(d)), indicating
that GHTE hydrogel did not cause foreign body reac-
tion. Moreover, the wound treated with GHTE hydro-
gel was covered with epithelial tissue closer to nor-
mal skin, and the hair follicles and even hair could
be clearly observed. Newly formed collagen deposits
in regenerated skin tissue were detected by Masson’s
trichrome staining. As shown in figure 6(d), after
14 d of treatment, collagen fiber density was higher
in the GHTE hydrogel treated group compared to the
other groups. These results showed that GHTE hydro-
gels enhanced the formation of ECM during wound
regeneration and promoted wound healing, but rapid
wound closure can lead to disordered deposition of
collagen at the site of injury, which is not condu-
cive to attenuating scar formation, and this aspect of
the risk needs to be further investigated in our future
work. Overall, based on the ability to promote healing
of full-thickness skin injuries, the prepared hydrogels
will have a large potential for skin defect trauma and
surgical wound treatment.
5. Conclusion
In this study, an ECM-mimicking multifunctional
hydrogel was prepared using gelatin, hyaluronic acid,
TA and EPL via a physical blend strategy, which
is one of the safest methods for hydrogel forma-
tion and has the advantages of environmental pro-
tection, economy and industrial feasibility. The res-
ulting hydrogel showed appropriate swelling ratio,
enzyme degradability, and stable rheological proper-
ties. Moreover, the GHTE hydrogel exhibited good
cytocompatibility, hemocompatibility, and the abil-
ity to promote cell migration as assessed by live/dead
cell staining, MTT assay, hemocompatibility, and
cell scratch assay. Additionally, the GHTE hydrogel
demonstrated outstanding antibacterial efficacy and
significantly ROS scavenging capacity, which could
effectively inhibit the growth of S. aureus and E. coli,
and neutralize DPPH free radicals. More signific-
antly, in vivo open wound healing results presented
accelerated skin tissue regeneration and more col-
lagen deposition at the end of 14 d. Therefore, the
developed ECM-mimicking multifunctional hydro-
gel dressing has great application potential in the clin-
ical treatment of open wounds.
10
X Ma et al
Data availability statement
All data that support the findings of this study are
included within the article (and any supplementary
files).
Acknowledgments
This work is financially supported by the
Shandong Provincial Natural Science Foundation
(ZR2021QH255, ZR2021MH219), National key R&D
Plan Key Research Projects (2021YFC2103100),
Shandong Province Key R&D Program
(Major Technological Innovation Project)
(2021CXGC010501), Shenzhen Basic Research
Project (JCYJ20190809160209449).
Ethics information
All procedures performed in studies involving animal
experimentation were approved by the Experimental
Animal Ethics Committee of Shandong First Medical
University Affiliated Provincial Hospital (No. 2021-
099). This article does not contain any studies involve
human participants.
ORCID iD
Xiao Fu  https://orcid.org/0000-0001-9005-6881
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