Materials & Design 218 (2022) 110687

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Materials & Design

journal homepage: www.elsevier.com/locate/matdes

Gelatine methacrylamide-based multifunctional bilayer hydrogels for

accelerating diabetic wound repair
Yang Hu a, Mingxuan Liu b, Daquan Zhou c, Feng Chen c, Qiang Cai b, Xiongwei Yan b, Jingfeng Li a,⇑
a
Department of Spine and Bone Tumor Surgery, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China
b
Department of Orthopedics, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei 441021, China
c
Department of Neurosurgery, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei 441021, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

A functional bilayer structure

comprising a porous lower layer and a
dense surface layer was successfully
prepared.

The outer layer of Gel-PLL/MeTro
hydrogel exhibited antibacterial
properties and excellent elasticity
and extensibility.

The inner layer of Gel-QK promoted
HUVEC proliferation and migration.

The bilayer hydrogel accelerated
diabetic skin wound healing.

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

Article history: Diabetic wounds are a common disease that plague many doctors in the clinic. Diabetic wounds are
Received 27 November 2021 caused by increased blood sugar levels, which leads to microvascular disease and skin dystrophy.
Revised 9 April 2022 Additionally, their occurrence can be accompanied by inflammation and a variety of bacterial infections.
Accepted 20 April 2022
Diabetic wounds are complex; therefore, achieving good results after a single treatment modality is often
Available online 25 April 2022
difficult. In this study, we used materials science engineering methods to integrate several factors
required for diabetic wound treatment, which resulted in the development of a bilayer hydrogel. First,
Keywords:
the upper layer of the hydrogel inhibits bacterial growth and promotes wound closing, which solves
Diabetic wound
Wound healing
the problems of wound infection and protection. Additionally, the lower layer of the hydrogel has a por-
Antibacterial ous structure, which promotes the adhesion and growth of wound healing-related cells. Moreover, the
Multifunctional materials lower layer of the hydrogel can release cytokines that promote vascular endothelial cell and fibroblast
Angiogenesis migration and stimulate skin tissue regeneration. We evaluated the ability of this double-layer hydrogel
to heal diabetic wounds in vivo and in vitro and found that it offered protection while promoting healing.
We also determined that a combination of multiple factors can provide better reparative effects to treat
chronic diabetic wounds.
Ó 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Diabetic skin ulcers are the most widely known complication of

diabetes, and the repair and healing of diabetic wounds with
⇑ Corresponding author. chronic nonhealing characteristics is still a great challenge [1–3].
E-mail address: jingfengli@whu.edu.cn (J. Li).

https://doi.org/10.1016/j.matdes.2022.110687
0264-1275/Ó 2022 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Hu, M. Liu, D. Zhou et al.
Diabetic skin ulcers are one of the main causes of disability and
death in diabetic patients and a burden to both patients and soci-
ety [4]. Therefore, new methods should be constantly explored to
solve the problems of diabetic wound healing. The mechanism of
poor wound healing in patients with diabetes is still unclear, but
many studies have shown that it is mainly related to hypoxia,
impaired angiogenesis, metabolic changes, and high levels of reac-
tive oxygen species (ROS) [5]. Conventional treatments in the clinic
are the control of blood sugar levels, the use of antibiotics, the
debridement of the ulcer and autologous skin grafts [6,7]. How-
ever, the curative effects of these methods are unsatisfactory. Sta-
tistically, 31% of patients experience recurrence after using the
abovementioned traditional treatment methods. Thus, the treat-
ment of diabetic ulcer wounds is an urgent and difficult problem.
Recently, dressings for the treatment of nonhealing wounds have
attracted increased attention and have shown great efficacy. There-
fore, the development of highly active biomaterial-based wound
dressings is essential to promote wound healing in diabetic
patients.Scheme 1..
Polymer hydrogels have shown great potential as dressings for
diabetic wound healing. Recently, natural or synthetic hydrogels
have been combined with other bioactive factors to better address
the problem of diabetic wound healing. Natural polymers (such as
chitosan, hyaluronic acid, alginate, collagen or elastin) are particu-
larly conducive to the preparation of wound dressings because of
their good biocompatibility and degradability. Ng et al. developed
an antimicrobial hydrogel for promoting wound healing because
the healing process is delayed due to a variety of mechanisms, such
as the continuous production of inflammatory mediators when
microbial infection occurs in a wound [8]. Zhang et al prepared a
Scheme 1. Schematic illustration of GelMA-based bilayer hydrogels with antibacterial and angiogenic properties designed to accelerate diabetic wound repair.
2
Materials & Design 218 (2022) 110687
series of hyaluronic acid-based composite hydrogels, which pro-
moted wound coagulation and prevented the formation of fibrotic
scar tissue [9]. However, such hydrogel dressings often have lim-
ited application in dynamic mechanical environments due to their
insufficient mechanical properties. Relatively, synthetic hydrogels,
such as poly (ethylene glycol) (PEG) and poly (vinyl alcohol) (PVA),
are increasingly used because of their excellent mechanical proper-
ties. Yang et al. reported highly stretchable, adhesive
polydopamine/polyacrylamide-based hydrogel dressings for
wound healing. The hydrogels exhibited high tensile strength, high
tensile strain and ideal compressive property, which helped enable
the dressing to adapt to the dynamic mechanical changes of the
skin at the wound [10]. Such hydrogels are often combined with
other factors due to their lack of bioactive factors.
Although important progress has been made in some aspects of
hydrogels, in the face of the complexity of diabetic wounds, new
hydrogel dressings with versatile repair functions are needed.
The ideal wound dressing should have the following properties:
(1) the capability to absorb exudate and toxic components from
the wound surface; (2) the ability to allow gas exchange; (3) the
capacity to protect the wound from bacteria; (4) good biosafety;
(5) easy removal without causing damage to the wound; and (6)
biological functions such as vascularization and anti-infective
properties. Compared with a single dressing lacking multifunction-
ality, double-layer composite membranes composed of different
functional layers can better promote the healing process and pro-
duce various biological effects. Usually, the upper layer is com-
posed of a dense film, while the lower layer consists of a porous
hydrogel [11 12]. Additionally, the outer layer may act to control
water vapour penetration, while the inner layer is designed to
Y. Hu, M. Liu, D. Zhou et al.
attach to wound tissue and drain wound exudate [13,14]. Based on
these ideas, several bilayer synthetic dressings, such as sponge
composite membranes [15–17], have been developed in recent
years.
The purpose of this research was to develop a multifunctional
bilayer wound dressing for diabetic skin ulcers in an attempt to
achieve better skin repair effects. To solve this clinical problem
by combining a variety of factors, we used materials science engi-
neering methods. In this study, we integrated therapeutic ele-
ments, such as antibacterial properties, mechanical contraction,
and vascular regeneration promotion, into a bilayer hydrogel. Gela-
tine methacrylamide (GelMA) was used as the base material
because GelMA-based hydrogels are transparent, biodegradable,
and biocompatible materials suitable for the adhesion and prolifer-
ation of keratinocytes that can support epidermal delamination
[18]. Biomacromolecules, growth factors and other small-
molecule drugs can be delivered through polymer scaffolds to pro-
mote wound healing and prevent infection. In particular, studies
have shown that antibacterial activity can be achieved by integrat-
ing different types of bactericides, including metal nanoparticles
[19], cationic polymers [20] and antibacterial peptides. e-Poly-L-
lysine (PLL) is a water-soluble, biodegradable, biocompatible and
low-cost cationic polypeptide that has been approved by the FDA
for clinical use or use as a food-grade cationic antibacterial agent
[21]. The upper layer of the hydrogel prepared herein consists of
a PLL layer with strong antibacterial properties that is used to pro-
tect the wound and reduce infection [22]. The upper layer of the
hydrogel also contains elastin, which can promote wound contrac-
tion and increase the rate of wound healing [23]. The lower layer of
the bilayer hydrogel has a porous structure, which is conducive to
the adhesion and migration of wound healing-related cells, vascu-
lar endothelial cells, fibroblasts, etc. Moreover, the lower layer con-
tains VEGF-mimetic peptides, which can accelerate angiogenesis
and collagen fibre regeneration [24]. We examined the mechanical
and structural properties of the bilayer hydrogel and determined
its effects on cells in vitro. Finally, we applied the bilayer hydrogel
in a rat diabetic wound model to observe its effects on wound
healing.
2. Materials and methods
2.1. Materials
GelMA (82% degree of substitution) and PLL were purchased
from Aladdin Industrial Corporation (Shanghai, China). The QK
peptide (a 15-mer VEGF mimetic peptide; Ac-
KLTWQELYQLKYKGI-NH2) was custom prepared by a commercial
manufacturer (GLS, Shanghai, China) and supplied in 95% purity.
Human tropoelastin and lithium acylphosphinate salt (LAP) were
purchased from Sigma–Aldrich (USA). Dulbecco’s modified Eagle’s
medium (DMEM) was purchased from GIBCO (Life Technologies).
The CD31 antibody was purchased from Abcam (UK).
2.2. Synthesis of the Gel-PLL and MeTro polymers
PLL-modified GelMA polymers (Gel-PLL) were synthesized by
the Michael addition approach. Briefly, 250 mg of GelMA and
35 mg of PLL were dissolved in water under a nitrogen atmosphere
at 50 °C and allowed to react for 24 h. Methacryloyl-substituted
recombinant tropoelastin (MeTro) was synthesized as described
elsewhere [25]. Briefly, tropoelastin was methacrylated by the
addition of methacrylate anhydride in PBS (10%, wt/vol) and
allowed to react for 12 h at 4 °C.
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Materials & Design 218 (2022) 110687
2.3. Synthesis and characterization of the Gel-QK polymer
The Gel-QK polymer containing the VEGF mimetic peptide was
generated via free radical polymerization of the double bonds
under blue light with the photoinitiator LAP. In brief, 1% (wt/vol,
10 mg) GelMA dissolved in PBS was added to 80 lg of the QK pep-
tide (our preliminary studies indicated that there were too few
double bonds in GelMA to crosslink if the content of QK peptide
exceeded 80 lg) and 5 mg of LAP. The polymer solutions were
induced to react by exposure to 405 nm blue light at 10 mW/cm2
for 3 min. The 1H NMR spectrum of Gel-QK was obtained with a
Bruker Avance II 600 spectrometer (Bruker Corporation, Switzer-
land). Fourier transform infrared (FTIR) spectra were obtained with
an FTIR Excalibur Series instrument (FTIR, Nicolet 6700) at
wavenumbers in the range of 4000–400 cm1.
2.4. Preparation of the Gel-QK and Gel-PLL/MeTro hydrogels
The Gel-QK hydrogel was prepared by radical polymerization of
the double bonds. To prepare the Gel-QK hydrogel, 10% (wt/vol,
100 mg) GelMA dissolved in PBS was added to 800 lg of the QK
peptide and 5 mg of LAP. The polymer solutions were crosslinked,
and the hydrogel was generated by exposure to 405 nm blue light
at 10 mW/cm2 for 3 min. Then, the Gel-PLL/MeTro hydrogels were
prepared by radical polymerization of double bonds in the Gel-PLL
and MeTro polymers. MeTro is a highly elastic photocrosslinkable
bioelastomer protein. The Gel-PLL polymer was endowed with
antimicrobial activity from the cationic polypeptide polymer PLL.
Due to their biocompatibility and highly tuneable mechanical
properties, the crosslinkable MeTro and Gel-PLL biopolymers were
explored here as the outer layer of the hydrogels to provide good
mechanical strength and antimicrobial activity. MeTro and Gel-
PLL were diluted in PBS to concentrations of 1% and 20%, respec-
tively. Polymer solution (10% Gel-QK or 20% Gel-PLL/MeTro, wt/
vol) was sprayed onto pig skin surface wounds or used to fabricate
rectangular samples that were placed in polydimethylsiloxane
(PDMS) (25  5  2.5 mm) moulds for tensile tests.
2.5. Characterization of the mechanical properties
The polymer solutions in PDMS moulds were photocrosslinked
via exposure to blue light (405 nm; Long Wave Ultraviolet Lamp
(Upland, CA)) for 180 s. To calculate the tensile elastic modulus
and extensibility, each sample was placed between two pieces of
double-sided tape within the tension grips of the instrument and
extended at a rate of 1 mm/min until failure.
2.6. Scanning electron microscopy (SEM) analysis
SEM imaging was conducted to evaluate the porosity of the dif-
ferent hydrogels. SEM images were obtained from lyophilized
hydrogel samples (Gel-QK, Gel-PLL/MeTro and bilayer) using a
Hitachi S-4800 scanning electron microscope.
2.7. Bioactivity evaluations
HUVECs or L929 cells were seeded into 96-well tissue culture
plates or on different hydrogel surfaces. Culture medium without
hydrogels served as a negative control. At regular time intervals
(1, 3, 5 days), 30 lL of MTT (5 mg/mL) solution was added to each
well and then incubated for another 4 h. Thereafter, all medium
was removed, and 150 lL of dimethyl sulfoxide (DMSO) was added
with stirring to dissolve the crystals. The optical density at 490 nm
was measured using a microplate reader.
Y. Hu, M. Liu, D. Zhou et al.
2.8. Transwell chamber assay
Transwell chambers (Falcon, Corning, USA) were used to
observe HUVEC and L929 cell migration. The upper chamber con-
tained 200 lL of serum-free medium and cells, and the lower
chamber contained 500 lL of complete medium and different
hydrogels. After 24 h of incubation, the cells remaining in the
upper chamber were removed, and the migrated cells were fixed
with 4% paraformaldehyde (PFA) for 10 min, followed by staining
with 0.1% crystal violet solution for 30 min. An inverted fluores-
cence microscope was used for cell observation and data acquisi-
tion, and the cell number per field was counted.
2.9. In vitro evaluation of antimicrobial activity
The antibacterial activity of the hydrogels against Staphylococ-
cus aureus (ATCC 29213) and Escherichia coli (ATCC 8739) was
determined on the surface of the hydrogels. In brief, 150 lL of each
hydrogel precursor was poured into a 48-well microplate and
exposed to Blue light (405nm) for 10 min. After complete gelation
and sterilization, 10 lL of bacterial suspension in sterilized PBS
(104 colony-forming units (CFU)/mL) was added to the surface of
each hydrogel in the 48-well microplate. The hydrogels were incu-
bated with bacteria in a shaking incubator for 2 h (37 °C, 180 rpm).
Then, 1 mL of sterilized PBS was added to each well of the 48-well
microplate containing the hydrogels to resuspend the surviving
bacteria on the hydrogel surface. Ten microlitres of bacterial sus-
pension in sterilized PBS (104 CFU/mL) resuspended in 1 mL of
sterilized PBS was used as a negative control. The CFUs on the agar
plate were calculated after incubation for 18 to 24 h at 37 °C. All
tests were repeated three times for each group.
2.10. Wound healing assay
All animal procedures in this study were approved by the Ani-
mal Care and Experimental Committee of Zhongnan Hospital affil-
iated with Wuhan University School of Medicine. Forty Sprague–
Dawley rats (aged 8 weeks) were injected with streptozotocin
(65 mg/kg b.w., i.p.), and their blood sugar levels were determined
7 days later to confirm diabetic model achievement. Then, all rats
were randomly divided into one of 4 groups: the control, Gel-QK
hydrogel, Gel-PLL/MeTro hydrogel and bilayer hydrogel groups
(each group consisted of 10 rats). After anaesthetization by i.p.
injection of 3% phenobarbital and shaving, the rats underwent
full-thickness skin excision. Two full-thickness skin defects
(Ø=2.0 cm) were made on each rat’s back, and then the rats were
treated according to the protocol for their particular group.
Wounds were protected by a skin patch. On days 0, 7 and 14 after
the procedure, the wounds were photographed using a digital cam-
era (Canon, Japan). The wound margins and areas were determined
using image analysis software (NIH Image). The degree of wound
closure was determined using the formula percentage wound
closure = [(A0-At)/A0]  100%, where A0 is the initial wound area,
and At is the wound area at time point t. At 7 and 14 days after the
procedure, 5 rats in each group (5 on days 7 and 5 on day 14) were
sacrificed via anaesthesia overdose, and the wound tissue was har-
vested and preserved in 4% paraformaldehyde for histological
analysis.
2.11. Histological evaluation
For histological analysis, samples were dehydrated, embedded
in paraffin and sliced into sections (6 mm thick). Neoepithelium
length and collagen deposition were observed via haematoxylin
and eosin (H&E) and Masson’s trichrome staining. Immunohisto-
chemistry was employed to observe angiogenesis in the wound
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Materials & Design 218 (2022) 110687
field. First, sections were rehydrated and underwent antigen retrie-
val. After incubation with a primary antibody against CD31 (1:200,
Abcam, Cambridge, UK) at 4 °C overnight, sections were incubated
with a biotinylated secondary antibody and ABC complex and
stained with 3,30 -diaminobenzidine (DAB) substrate. All sections
were counterstained with haematoxylin and observed under a
light microscope. Angiogenesis was determined in six sections
from different samples. For each section, six high-power fields
were randomly observed, and the number of new blood vessels
was counted.
2.12. Statistical analysis
All data are recorded as the mean ± standard deviation (SD).
One-way ANOVA and Student–Newman–Keuls post hoc tests were
applied to assess significance. P values < 0.05 were considered to
be significant. Statistical comparison of the data was performed
using GraphPad Prism 9 statistics software.
3. Results and discussion
3.1. Fabrication of the bilayer hydrogels
3.1.1. Synthesis and characterization of the Gel-QK polymer
The FTIR spectrum of Gel-QK is shown in Fig. 1A. The character-
istic peaks of GelMA are clearly present in both the GelMA and Gel-
QK spectra, and the appearance of a conjugated double bond
stretching vibration at 3080 cm1 in both the QK and Gel-QK sam-
ples could be attributed to the benzene ring moiety. Similarly,
methacrylated groups (CH2 = CH2) appeared in the 1H NMR spectra
at the characteristic shifts of d = 5.3 ppm and 5.6 ppm (Fig. 1B).
Additionally, the signals at chemical shifts from 7.0 to 7.5 ppm
were attributed to the aromatic protons of Trp and Tyr, which indi-
cated that the peptide QK successfully interacted with GelMA.
3.1.2. Fabrication of the bilayer hydrogels
In this study, we prepared a double-layer hydrogel to achieve
multifunctional dressings. The Gel-QK hydrogel of the bottom layer
has vascular and porous properties, and the outer Gel-PLL/Metro
hydrogel has elastic and antibacterial properties. We successfully
fabricated bilayer wound dressings consisting of an outer layer
with high elasticity and antimicrobial activity and a base layer
with a porous microstructure and angiogenic properties. As shown
in Fig. 1C, the two layers exhibited a transparent appearance and
could be sprayed from a bottle, which made them easy to store
and apply. After exposure to blue light (405 nm) for 180 s, the liq-
uid solidified at the wound to block it and did not increase in size
when the wound moved.
3.2. Evaluation of the in vitro swelling ratio
For diabetic wound healing hydrogel dressings, swelling prop-
erties are also important. It is hoped that the hydrogel can absorb
the exudate and maintain stability, which can prevent the wound
from cracking. The swelling ratios of the different hydrogels were
determined after 24 h of incubation in PBS at 37 °C. The results
showed a higher swelling ratio for the Gel-QK hydrogel than for
the Gel-PLL/MeTro hydrogel with the outer layer (Fig. 1D). The
greater swelling property is advantageous for clearing wound exu-
date and maintaining a moist environment at the wound site [26].
The low swelling of the outer Gel-PLL/MeTro hydrogel was suffi-
cient to maintain wound healing.
Y. Hu, M. Liu, D. Zhou et al.
Fig. 1. Physicochemical properties of the modified gels and layers. (A) FTIR spectra of GelMA, Gel-QK and the QK peptide. (B) 1H NMR spectra of GelMA, Gel-QK and the QK
peptide. (C) Observation of the two layers and illustration of the spraying properties. (D) Water absorption characteristics of the different layers.
3.3. Mechanical properties of the hydrogels
Due to the elastic characteristics of skin tissue and the dynamic
stress environment of the wound, wound dressings need suitable
mechanical properties. The mechanical properties of the MeTro/
GelMA composite hydrogels were characterized through tensile
testing. Tensile tests on the Gel-PLL/MeTro and Gel-QK hydrogels
revealed that the elastic modulus (Fig. 2A) and extensibility
(Fig. 2B) of the Gel-PLL/MeTro hydrogel exceeded those of the
Gel-QK hydrogel. Although the elastic modulus of the Gel-PLL/
MeTro hydrogel was lower than that of native skin (88 kPa 
300 kPa) [27], the extensibility of Gel-PLL/MeTro was greater than
80%, which is similar to that of native skin [28]. Therefore, it is
expected that due to its excellent mechanical properties, this
bilayer wound dressing would be able to adapt to the dynamic
stress of skin.
3.4. SEM analysis
Previous studies have investigated the influence of the
microstructural features of hydrogel scaffolds on the regenera-
tion and repair of target tissues and found that cells from the
surrounding tissues can infiltrate the porous gel and deposit
autologous extracellular matrix (ECM) components to form tis-
sue [29]. Therefore, SEM images were acquired from lyophilized
Gel-PLL/MeTro, Gel-QK and bilayer hydrogels (Fig. 2C). Our
results showed an interconnected macroporous structure in the
Gel-QK layer; in contrast, Gel-PLL/MeTro was dense with almost
no porous structure. The cross-sectional overview of the bilayer
scaffold confirmed that both layers were intact, as observed in
the SEM image. The bilayer hydrogel clearly showed a two-
layer interface.
5
Materials & Design 218 (2022) 110687
3.5. Gel-QK promotes cell proliferation and migration
Angiogenesis is the biological process of new blood vessel for-
mation, involving endothelial cell proliferation, migration, and
tube formation, and can determine the outcome of diabetic wound
healing because newly formed vessels can transport oxygen and
nutrients into wound sites [30]. To evaluate the effects of Gel-QK
on the proliferation and migration of angiogenesis-related cells,
HUVECs and L929 cells were plated on the surfaces of different
materials to examine the proliferative effects. Moreover, the num-
ber of migrating cells was determined by Transwell observation to
assess the promotion of cell migration. As shown in Fig. 3A, the
Gel-QK hydrogel promoted HUVEC and L929 proliferation com-
pared to the hydrogels in the control and Gel-PLL/MeTro groups.
Moreover, the Gel-QK hydrogels supported HUVEC and L929 cell
migration better than the control, GelMA and Gel-PLL/Metro
groups. However, there was lower migration of HUVECs and
L929 cells in the Gel-QK group than in the free QK peptide group
with the same QK content, which was because the QK released
from the Gel-QK hydrogel promoted cell migration through VEGF
and VEGF receptor binding (Fig. 3B, C). These observed phenomena
demonstrated that the VEGF-mimetic peptide released by the Gel-
QK layer can effectively promote the migration of vascular-related
cells, which is conducive to angiogenesis and wound healing. In the
early healing stage, without sufficient angiogenesis, high concen-
trations of glucose will accumulate in the wound site, leading to
ischaemia and tissue necrosis [31]. Therefore, reconstruction of
the diabetic wound vascular network during the early stage of
healing helps to prevent wound expansion and ulceration.
3.6. Gel-PLL/MeTro has antibacterial effects
Impaired white blood cell function in diabetic patients leads to
metabolic abnormalities, which increase vulnerability to wound
Y. Hu, M. Liu, D. Zhou et al. Materials & Design 218 (2022) 110687

Fig. 2. Characterization of the mechanical properties and pores of the bilayer hydrogels. (A) Elastic modulus and (B) extensibility. (C) Representative SEM images of the
hydrogels.

Fig. 3. Proliferation and cell migration of HUVECs and L929 cells affected by different hydrogels. (A) Proliferation curves of HUVECs and L929 cells obtained from the MTT
assay. (B-C) Transwell images and quantitative analysis of cell migration from the Transwell chamber assay. * p < 0.05, ** p < 0.01 compared with the control group; # p < 0.05,
## p < 0.01 compared with the GelMA group; & p < 0.05, && p < 0.01 compared with the QK group.

infections. Generally, antibacterial properties should be considered
to prevent bacterial invasion. Ideally, wound healing hydrogels
should not only promote angiogenesis and enhance the wound
repair process but also prevent bacterial infection [32]. The antimi-
crobial properties of these hydrogels were evaluated by colony for-
mation assays. The results demonstrated that the Gel-PLL/MeTro
hydrogels effectively prevented both S. aureus and E. coli coloniza-
tion, as shown by the reduction in CFU compared to the Gel-QK
hydrogel group (Fig. 4A, B). The introduction of the outer layer to
6
form the bilayer wound dressing enhanced the antibacterial
activity.
3.7. The bilayer hydrogel accelerates diabetic skin wound healing
After determining the antibacterial and mechanical properties
of the double-layer hydrogel as well as its ability to promote cell
proliferation and migration through in vitro experiments, we
applied the double-layer hydrogel to a rat diabetic skin defect
Y. Hu, M. Liu, D. Zhou et al.
Fig. 4. Determination of the antibacterial activity against E. coli and S. aureus. (A)
Representative images of E. coli and S. aureus colonies from a colony survival assay.
(B) Quantitative analysis of the E. coli and S. aureus colonies from a colony survival
assay.
model. After confirming successful establishment of the rat dia-
betes model by blood glucose measurements, we placed the
double-layer hydrogel in skin defects on the backs of rats and
Fig. 5. In vivo evaluation of wound healing after treatment with different hydrogels. (A) Wound images from the four individual groups (blank control, Gel-PLL/MeTro, Gel-
QK and bilayer) at 0, 7 and 14 days. (B) Traces of wound closure. (C) Statistical results of the wound healing rates. **P < 0.01.
7
Materials & Design 218 (2022) 110687
observed the wounds for 14 days. During the observation period,
the rats showed no abnormal behaviours, suggesting that the
hydrogel has no systemic toxicity. There were no signs of inflam-
mation or infection around the wound, suggesting that the
double-layer hydrogel did not irritate the skin. We first observed
wounds with the naked eye and found that on the 7th day, the
Gel-PLL/MeTro group had a higher wound healing rate than the
control group, suggesting that the antibacterial effects or the
wound contraction effects of elastin were beneficial to wound
healing. The double-layer hydrogels showed the greatest amount
of wound shrinkage, suggesting that as the upper hydrogel con-
tracted, the cell growth-promoting properties of the lower hydro-
gel played a role in healing. On the 14th day, the double-layer
hydrogel group showed the best repair effects, and the Gel-QK
group also showed better reparative effects than the other two
groups (Fig. 5A-C). The above results suggest that in the early stage
of repair, antibacterial properties play a major role in accelerating
wound healing, while in the later stage of repair, cell proliferation
plays a major role; thus, the hydrogel that combined the two prop-
erties displayed the best reparative effects. The H&E staining
results further confirmed the naked eye observations (Fig. 6). Col-
lagen fibre deposition and angiogenesis are two important physio-
logical processes during wound healing (Fig. 6). Normal capillary
formation is very important for the transportation of oxygen and
nutrients during wound repair, and thus, revascularization is very
important for diabetic patients; however, high sugar contents hin-
der blood vessel regeneration, leading to ischaemia and tissue
necrosis. We used Masson staining and immunohistochemistry
(CD31 staining) (Fig. 7) to observe collagen fibre deposition and
angiogenesis at the wound site. The experimental results indicated
that the Gel-QK and the double-layer hydrogels exhibited greater
collagen fibre deposition and angiogenesis than the control and
Gel-PLL/MeTro groups, suggesting that the porous structure and
VEGF mimetic peptide in the Gel-QK layer of the hydrogel played
the expected role. Comparing the double-layer hydrogel group
with the Gel-QK group showed that the double-layer hydrogel
group displayed more collagen fibre deposition and angiogenesis,
suggesting that the antibacterial and physical closing effects acted
synergistically on collagen fibre deposition and angiogenesis.
Hence, improving angiogenesis and revascularization in diabetic
wounds is crucial to accelerate chronic wound healing [33–35].
Thus, by combining the physical closing and antibacterial proper-
ties of the Gel-PLL/MeTro layer with the cell-promoting attributes
Y. Hu, M. Liu, D. Zhou et al. Materials & Design 218 (2022) 110687

Fig. 6. Histological analysis of collagen in wounds after treatment with the hydrogels. (A) H&E staining images of skin tissue sections. Vertical lines indicate wound edges. (B)
Statistical analysis of the total lengths of the wounds after 7 and 14 days of treatment. (C) Area positive for collagen fibre deposition.

Fig. 7. Immunohistochemical (IHC) staining analysis of CD31 after 7 and 14 days of treatment. (A-B) Representative images and statistical results of the number of
CD31 + cells at 7 and 14 days. * P < 0.05, ** P < 0.001; scale bar: 50 lm.

of the Gel-QK layer, the best diabetic wound repair effects can be
achieved. Through in vivo experiments, we clarified the role of
these double-layer hydrogels in promoting diabetic wound heal-
ing. Moreover, we found that integrating multiple repair factors
had a synergistic effect compared to each individual factor. Com-
bining a variety of therapeutic factors through engineering meth-
ods and the use of biological materials can lead to the best repair
of diabetic wounds.
4. Conclusion
The occurrence of chronic diabetic wounds is a complex process
involving multiple factors, including skin nutritional disorders,
bacterial infections, and inflammation. Traditional treatment
methods, such as the use of antibiotics or blood sugar control pro-
grammes, can solve only a single pathogenic factor; therefore,
these individual methods often fail to achieve a good therapeutic
effect. In this study, we used engineering methods to develop a
bilayer hydrogel that integrates several related factors that pro-
mote diabetic wound healing, including inhibiting bacterial growth
and promoting the activity of vascular endothelial cells and fibrob-
lasts. Through in vivo and in vitro experiments, we showed the
therapeutic effects of this bilayer hydrogel on chronic diabetic
wounds. Moreover, we found that inhibiting bacterial growth or
promoting cell activity alone cannot sufficiently promote wound
healing, and only the combination of these two factors can lead
to better reparative effects to treat chronic diabetic wounds.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
None
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