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Bioactive Materials 39 (2024) 562–581
2452-199X/© 2024 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A cuttlesh ink nanoparticle-reinforced biopolymer hydrogel with robust
adhesive and immunomodulatory features for treating oral ulcers
in diabetes
Yajing Xiang
a
,
1
, Zhuge Pan
b
,
1
, Xiaoliang Qi
c
,
***
, XinXin Ge
a
, Junbo Xiang
d
, Hangbin Xu
a
,
Erya Cai
a
, Yulong Lan
d
, Xiaojing Chen
d
, Ying Li
d
, Yizuo Shi
a
, Jianliang Shen
c
,
d
,
**
,
Jinsong Liu
a
,
*
a
School & Hospital of Stomatology, Wenzhou Medical University, Wenzhou, Zhejiang, 325027, China
b
Department of Otolaryngology, Afliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, Zhejiang, 321000, China
c
National Engineering Research Center of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, 325027, China
d
Zhejiang Engineering Research Center for Tissue Repair Materials, Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang, 325001, China
ARTICLE INFO
Keywords:
Cuttlesh ink nanoparticles
Hydrogel patches
Tissue adhesives
Wound healing
Oral ulcers
ABSTRACT
Oral ulcers can be managed using a variety of biomaterials that deliver drugs or cytokines. However, many
patients experience minimal benets from certain medical treatments because of poor compliance, short
retention times in the oral cavity, and inadequate drug efcacy. Herein, we present a novel hydrogel patch
(SCE2) composed of a biopolymer matrix (featuring ultraviolet-triggered adhesion properties) loaded with
cuttlesh ink nanoparticles (possessing pro-healing functions). Applying a straightforward local method initiates
the formation of a hydrogel barrier that adheres to mucosal injuries under the inuence of ultraviolet light. SCE2
then demonstrates exceptional capabilities for near-infrared photothermal sterilization and neutralization of
reactive oxygen species. These properties contribute to the elimination of bacteria and the management of the
oxidation process, thus accelerating the healing phase’s progression from inammation to proliferation. In
studies involving diabetic rats with oral ulcers, the SCE2 adhesive patch signicantly quickens recovery by
altering the inamed state of the injured area, facilitating rapid re-epithelialization, and fostering angiogenesis.
In conclusion, this light-sensitive hydrogel patch offers a promising path to expedited wound healing, potentially
transforming treatment strategies for clinical oral ulcers.
1. Introduction
Oral ulcers, also known as injuries to the oral mucosa, represent
common and recurrent conditions in oral health, marked by the
persistent destruction or impairment of the oral epithelial tissue [1].
Globally, over 25 % of individuals have suffered or are currently
suffering from these ulcers [2,3]. Oral ulcers that are not treated or
poorly managed can cause the loss of epithelial tissue, creating cavities
or, in extreme situations, leading to tissue death. Such conditions make
fundamental activities such as eating, swallowing, talking, and digesting
difcult for individuals [4]. Furthermore, the absence of the natural
protective barrier of the oral mucosa renders these wounds prone to
bacterial infections, thereby diminishing the speed of ulcer healing [5,
6]. Although various treatments like oral ulcer patches (comprising
chitosan and avonoids), powders (containing vitamins), and ointments
(such as recombinant human epidermal growth factor gel) are available
to expedite the healing process, their effectiveness is limited [7–9]. The
constraint arises from their short-lived adhesion to the mucosal surface
Peer review under responsibility of KeAi Communications Co., Ltd.
* Corresponding author
** Corresponding authorNational Engineering Research Center of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang,
325027, China.
*** Corresponding author.
E-mail addresses: xiaoliangqi90@gmail.com (X. Qi), shenjl@wiucas.ac.cn (J. Shen), jinsong0719@wmu.edu.cn (J. Liu).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Bioactive Materials
journal homepage: www.keaipublishing.com/en/journals/bioactive-materials
https://doi.org/10.1016/j.bioactmat.2024.04.022
Received 20 January 2024; Received in revised form 17 April 2024; Accepted 21 April 2024
Bioactive Materials 39 (2024) 562–581
563
within the moist and dynamic environment of the oral cavity, typically
enduring for less than 2 h. Therefore, the development of biomaterials
with improved adhesive qualities is essential for accelerating the healing
process of oral ulcers.
Creating hydrogel patches that possess adhesive properties is
considered an effective strategy for addressing oral ulcers [10]. The
methods of adhesion for these hydrogels are generally categorized into
two types: chemical and physical, with the majority depending on a
transition from a liquid state to a solid one to achieve adherence [11,12].
Physical adhesion often utilizes hydrogen bonds, yet strong adhesion is
challenging to achieve and tends to fail in a moisture-rich oral envi-
ronment [13,14]. Consequently, approaches focused on creating
chemical connections with the tissue interface provide strong
attachment and support cellular movement at the location of adhesion
[15,16]. Research has shown that a hydrogel comprising N-(2-amino-
ethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy)butanamide
(NB)-modied hyaluronic acid (HA-NB) and methacrylated gelatin
(GelMA) displays several benets [17,18]. These include potent adhe-
sion to wet tissue, quick gelation, minimal swelling, and high biocom-
patibility, all crucial attributes for use in the moisture-laden
environment of the oral cavity [19]. HA-NB and GelMA, the funda-
mental components of the hydrogel, reect the extracellular matrix’s
(ECM) structure found in the oral mucosa, predominantly consisting of
proteins and polysaccharides [18,20]. As a result, such an
ECM-simulating adhesive hydrogel might serve as an ideal substance for
accelerating the repair of injuries in the oral mucosa.
Scheme 1. Fabrication and usage of SCE2 hydrogel for accelerated healing in oral ulcers among bacteria-infected diabetic rats. (A) Synthesis process of the SCE2
hydrogel. (B) Mechanisms of SCE2 hydrogel in enhancing wound recovery.
Y. Xiang et al.
Bioactive Materials 39 (2024) 562–581
564
After achieving adhesion properties, the precise coordination of
biological activities within hydrogel dressings is essential for the effec-
tive healing of wounds caused by oral ulcers. Like skin wound healing,
the recovery process of oral ulcers typically involves three consecutive
yet interrelated stages: inammation, cellular proliferation, and tissue
restructuring [21,22]. For the repair of oral mucosal injuries, oral
mucoadhesive hydrogels must independently adjust the wound’s
microenvironment. This adjustment involves tackling inammation,
bacterial invasions, and the activity of host cells, all while protecting the
wound’s surface within a saliva-rich environment over a prolonged
period (ideally exceeding 12 h), which is considered optimal for
addressing issues related to oral mucosal regeneration [23]. Lately,
various bioactive substances have been integrated into hydrogels for
skin wound healing, which speed up tissue repair through sterilization,
reduce inammation, and stimulate growth factor release, thus
bypassing the need for drugs, cell-based treatments, or cytokines [24,
25]. However, these bioactive agents frequently lack enduring adhesion
and stability in damp environments [2,26]. Moreover, the complexity of
their structural congurations, functional modications, and the
requirement for external interventions complicate their use in clinical
settings. Thus, there is an urgent demand for the creation of oral ulcer
dressings that exhibit strong adhesion (encompassing both chemical and
physical aspects) and potent healing (biological) properties [24].
In the current research, focused on enhancing the treatment of oral
ulcers, we aim to create an ECM-like hydrogel dressing, designated
SCE2, that combines a biopolymer matrix [consisting of methacrylate
silk broin (SFMA) and nitrobenzyl-modied chondroitin sulfate (CHS-
NB) with light-responsive gelling and adhesion properties] with cuttle-
sh ink nanoparticles (known for their healing benets) [27–29]. As
illustrated in Scheme 1, exposure to 365 nm light triggers the formation
of a safeguarding hydrogel coating on the wound via the radical poly-
merization of SFMA’s double bonds. Concurrently, the hydrogel dem-
onstrates robust adherence to moist tissues as the o-nitrobenzene group
in its precursor CHS-NB transforms into an aldehyde group under the
same light conditions. This transformation enables quick, strong
attachment to the tissue via imine bond interactions with the tissue’s
surface amino groups. Additionally, cuttlesh ink nanoparticles within
the SCE2 hydrogel exhibit exceptional abilities for near-infrared pho-
tothermal sterilization and neutralization of reactive oxygen species.
Such characteristics aid in the elimination of bacteria and the control of
oxidative conditions, thus accelerating the wound’s shift from an in-
ammatory stage to a proliferative phase. SCE2’s unique
three-dimensional network, composed of elements derived from natural
sources, potentially acts as a supportive reservoir, offering both nutri-
tional and mechanical assistance to cells. This environment fosters cell
attachment, movement, growth, and eventual angiogenesis in the pro-
cess of tissue reconstruction. The SCE2 hydrogel’s capacity to enhance
wound recovery was validated through experiments with a
streptozotocin-induced diabetic rat model. In summary, the SCE2
hydrogel exhibits considerable promise for its clinical use in swiftly
healing oral ulcers.
2. Materials and methods
2.1. Materials
Silkworm cocoons were obtained from the Sericulture Technology
Extension Station (Chongqing, China). N-(2-aminoethyl)-4-[4-(hydrox-
ymethyl)-2-methoxy-5-nitrophenoxy]butanamide (NB–NH
2
) was ob-
tained from Chaocheng Technology (Haining, Zhejiang). Aladdin
(Shanghai, China) provided N-hydroxysuccinimide (NHS), chondroitin
sulfate (sourced from sharks with purity >99 %, and M
w
=340 kDa), 4-
(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium chloride,
glycidyl methacrylate (GMA), sodium carbonate (Na
2
CO
3
), dimethyl
sulfoxide, lithium bromide (LiBr), N-(3-dimethylaminopropyl)-N
′
-eth-
ylcarbodiimide hydrochloride (EDC⋅HCl), glycidyl methacylate, and
lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP). 2-Morpholi-
noethanesulfonic acid was provided by Macklin (Shanghai, China).
Hydrogen peroxide (H
2
O
2
) was provided by Sigma (Shanghai, China).
Innochem (Beijing, China) provided 1,1-diphenyl-2-picrylhydrazyl
(DPPH) and calcein acetoxymethyl ester. Yeasen (Shanghai, China)
was the provider of propidium iodide (PI) and streptozotocin (STZ).
Beyotime (Nanjing, China) supplied 2,2ʹ-azino-bis (3-ethyl-
benzothiazoline-6-sulfonic acid), Cell Counting Kit (CCK)-8, and 2
′
,7
′
-
dichlorouorescein diacetate. SYTO9 was provided by Thermo Fisher
(Waltham, USA). Gibco (NY, USA) was responsible for supplying fetal
bovine serum, penicillin-streptomycin, Dulbecco’s modied Eagle’s
medium (DMEM), and phosphate buffer saline (PBS). Hopebiol (Qing-
dao, China) was responsible for supplying Tryptic Soy Broth, agar
powder, and Luria-Bertani Broth. All additional reagents and solvents,
unless noted otherwise, were used in their original state and were of
analytical reagent grade.
2.2. Synthesis of cuttlesh ink nanoparticles
Nanoparticles derived from cuttlesh ink (CFI) were extracted using
differential centrifugation from a fresh cuttlesh’s ink sac. To begin, the
mixture with CFI nanoparticles underwent centrifugation at 2100 rpm
for a duration of 5 min, aiming to eliminate sizable precipitates.
Following this, it was centrifuged once more at 12,000 rpm for 10 min to
harvest the CFI nanoparticles. Finally, the CFI nanoparticles were
thoroughly rinsed with double distilled water (DDW) and subsequently
suspended in DDW for later use.
2.3. Synthesis of methacrylate silk broin (SFMA) and nitrobenzyl-
modied chondroitin sulfate
Synthesis of SFMA: Silk broin was fabricated by initially cutting
silkworm cocoons into broken strips, which were then boiled in 0.02 M
Na
2
CO
3
for 30 min to eliminate the sericin layer. Post-degumming, the
acquired bers underwent rinsing with DDW, followed by air-drying at
37 ◦C. These were then dissolved in a 9.3 M LiBr solution at 65 ◦C for a
period of 4 h. Subsequently, the obtained silk broin solution was
moved into a dialysis bag (MWCO, 3.5 kDa) for dialysis. This process,
conducted against DDW for 48 h, included six changes of DDW, facili-
tating the removal of lithium bromide. After dialysis, the silk broin
solution underwent centrifugation at 9000 rpm for 15 min at 4 ◦C,
repeated twice. The nal step involved acquiring silk broin through
freeze-drying.
To synthesize SFMA, 10 g of freeze-dried silk broin was combined
with 40 mL of PBS (pH =8.5) and stirred for 3 h. Meanwhile, in another
vessel, 2.93 g of GMA was merged into 100 mL of PBS solution (pH =
8.5) under constant agitation. Following this, the GMA mixture was
slowly added to the silk broin blend, creating the reaction mixture.
After allowing the reaction between GMA and silk broin at room
temperature overnight, the mixture was subjected to dialysis using bags
(molecular weight =3.5 kDa) in DDW for a week. The dialyzed SFMA
was then freeze-dried for subsequent use.
Synthesis of nitrobenzyl-modied chondroitin sulfate (CHS-NB):
Dissolving 2 g of chondroitin sulfate in 100 mL of 2-morpholinoethane-
sulfonic acid solution (0.01 M, pH 5.17) initiated the process. After that,
60 mg (0.18 mmol) of NB-NH
2
, previously dissolved in dimethyl sulf-
oxide, was incorporated into the mixture. In a separate step, 1.2 g (1.36
mmol) of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium
chloride, after being dissolved in DDW, was divided and introduced in
three equal segments, each separated by a period of 0.5 h. The acquired
mixture was then stirred in a dark environment at 35 ◦C for 3 h. Next,
dialysis of the mixture against a 0.1 M sodium chloride solution took
place over three days, leading to its subsequent freezing and
lyophilization.
Y. Xiang et al.
Bioactive Materials 39 (2024) 562–581
565
2.4. Preparation of SCE2 hydrogel
In this research, we developed hybrid hydrogels integrating SFMA,
CHS-NB, and CFI nanoparticles (abbreviated as SCE). The process began
with mixing 1 mL of a 10 % (w/v) SFMA solution containing 8 mg of LAP
photoinitiator with 1 mL of a 2 % (w/v) CHS-NB solution, which
included varying amounts of CFI nanoparticles. This mixture was then
exposed to ultraviolet light for 1 min, triggering a free radical poly-
merization reaction due to the residual double bonds, resulting in the
formation of a hydrogel. Four different SCE hydrogels were produced,
each with distinct CFI nanoparticle concentrations of 0, 0.5, 1, and 2
mg/mL, labeled SCE0, SCE1, SCE2, and SCE3, respectively.
2.5. Characterization of physicochemical traits
2.5.1. Spectra of proton nuclear magnetic resonance (
1
H NMR)
Deuterium oxide served as the solvent for preparing samples inten-
ded for
1
H NMR analysis, which were examined using a Quantum-I 400
MHz instrument from China. The
1
H NMR spectra obtained were sub-
sequently processed and analyzed with Mestrenova software.
2.5.2. Thermogravimetric analysis (TGA)
TGA was conducted using a TGA8000 PerkinElmer thermo-analyzer
(USA). In an air environment, the samples underwent examination. The
TGA curves were traced from 50 to 600 ◦C, applying a consistent heating
rate of 20 ◦C per minute.
2.5.3. Fourier transform infrared (FTIR) spectra
The functional groups of the CFI nanoparticles and SCE hydrogels
were assessed employing the Bruker Tensor II FTIR spectrometer from
Germany. The scan range for these samples was adjusted to span from
4000 to 400 cm
−1
.
2.5.4. Zeta potentials
The zeta potentials of the samples were determined using a Multi-
sizer 4e Coulter Counter (USA).
2.5.5. Ultraviolet–visible–near-infrared (UV–VIS–NIR) test
UV–VIS–NIR spectrum was carried out using a Cary5000 spectro-
photometer (Agilent, USA) at ambient temperature.
2.5.6. Dynamic light scattering (DLS) measurements
DLS was performed using a Zetasizer nano series instrument (Mal-
vern, UK), which features a He−Ne laser (633 nm, 4.0 mW). For
calculating the average diameters of particles, analyses of the autocor-
relation functions were performed using the cumulants approach. A
minimum of three assessments were conducted for each specimen at
25 ◦C, following a 3-min stabilization period before starting the
measurements.
2.5.7. Scanning electron microscope (SEM)
The surface of the CFI nanoparticles and freeze-dried hydrogels was
sprayed with platinum using an EM ACE600 from Leica (Germany).
Subsequently, the sample surface morphology was observed using a eld
emission SEM from Hitachi (SU8010, Japan), which operated at an ac-
celeration voltage of 5 kV. The pore diameter of the SCE hydrogels was
quantied using ImageJ software.
2.5.8. Transmission electron microscope (TEM)
For TEM analysis, samples were examined with a Jeol JEM-1230
TEM from Japan, which was set to function at 100 kV.
2.5.9. Swelling
Initially, SCE hydrogel samples, each with a predetermined weight,
were placed in PBS at 25 ◦C to reach a state of equilibrium. Throughout
the swelling process, the SCE hydrogels were periodically removed at set
intervals. Moisture on the hydrogel surfaces was removed by gently
pressing with damp lter papers. Subsequently, the wet weight of each
SCE hydrogel was precisely determined using a BSA224S-CW electronic
scale (Sartorius, Germany). To calculate the swelling ratio, the formula
(M
1
– M
2
)/M
2
was used, where M
1
is the weight of the hydrogel in its
swollen state, and M
2
is its weight when dry [30].
2.5.10. Water retention
The freeze-dried SCE hydrogels were immersed in deionized water
until a state of equilibrium was attained. The weights of these fully
saturated SCE hydrogels were documented (W
f
). The SCE hydrogels
were then positioned in an oven set at 37 ◦C. At specied intervals, the
weights of the SCE specimens were registered (W
t
), and the water
retention percentage was computed employing the following equation:
Water Retention (%) =W
t
/W
f
×100 % [31].
2.5.11. Hydrogel adhesion performance
The adhesive strength of SCE hydrogels to biological tissues was
assessed using fresh pig skin in accordance with the ASTM standards
(F2255–05 and F2258-05). Before the tests, slices of pork skin were
prepared and treated overnight with an EDC/NHS solution. A 200
μ
L
quantity of the hydrogel precursor was applied to a 1 cm ×1 cm section
of two pig skin pieces and then cross-linked under ultraviolet light for 1
min. The resulting SCE hydrogels were subjected to tensile tests to the
point of failure. These tests were conducted on an Instron 5944 machine,
which applied a stretching speed of 8 mm/min.
2.5.12. Rheological properties
The rheological characteristics of the synthesized SCE hydrogels
were investigated at 25 ◦C employing a DHR-2 rheometer (TA, USA) in a
stress-controlled mode. In the experiments, to prepare a sample
compatible with the instrument’s shear disc, the SCE hydrogel precursor
was dispensed into a plastic mold with dimensions of 25 mm in diameter
and 1 mm in height. Strain sweep tests ranged from 0.1 % to 2000 %
oscillatory strains at a constant frequency (1 Hz) to explore the linear
viscoelastic regions. Based on the strain sweep outcomes, the oscillation
strain was set at 1 %. Sweep analyses were conducted to examine the
relationship between modulus and frequency (0.01–100 Hz). The SCE
hydrogels’ capacity for self-repair was evaluated through dynamic step-
strain analyses, alternating the oscillatory strain between 1000 % and 1
% for every cycle. Investigations into the shear-thinning properties of
the SCE samples were carried out employing steady-rate sweep analyses,
where shear rates ranged between 0.1 and 100 1/s.
2.5.13. Investigating photothermal characteristics
Photothermal properties of CFI nanoparticles and SCE hydrogels
underwent assessment through an experiment utilizing NIR laser light.
To investigate the photothermal impact under 808 nm laser light,
samples at different concentrations (2, 1.0, and 0.5 mg/mL) of CFI
nanoparticles and SCE hydrogels were organized into 1.5 mL tubes.
Monitoring and recording of temperature alterations in these materials
were executed continuously using an E4 FLIR infrared camera (USA).
Subsequent evaluations were made on how varying NIR laser powers
(2.0, 1.0, and 0.5 W/cm
2
) affected the photothermal efciency of the
materials. Additionally, the resilience of CFI nanoparticles and hydro-
gels under photothermal conditions was examined across four laser on-
and-off cycles. The photothermal conversion efciency (
η
) of the created
samples was determined by subjecting them to 808 nm NIR light at 1 W/
cm
2
for 5 min and then turning off the laser. Temperature variations in
the materials were closely tracked with a FLIR thermal imaging camera.
2.5.14. Reactive oxygen species (ROS) scavenging capability
CFI nanoparticles and SCE2 hydrogel’s antioxidant abilities were
assessed by conducting a DPPH radical scavenging assay, which
measured their capacity to neutralize free radicals. A new solution of
DPPH in ethanol (0.1 mM) was mixed for the test. Subsequently,
Y. Xiang et al.
Bioactive Materials 39 (2024) 562–581
566
specimens were immersed in the DPPH solution and kept in the dark at
37 ◦C for a set duration. Then, the reactive mixture’s absorbance was
gauged at a wavelength of 517 nm. The DPPH scavenging ability per-
centage was determined with the equation: Inhibition (%) =(1 – A
a
/A
b
)
×100 %. In this formula, A
b
represents the initial absorbance of the
DPPH solution, while A
a
denotes the DPPH’s absorbance after being
mixed with the samples under test for a set duration.
Besides, the overall antioxidant capacity of the CFI nanoparticles and
SCE2 hydrogel underwent evaluation through the 2,2ʹ-azino-bis (3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. In a summarized
procedure, hydrogels weighing 100 mg, containing varying concentra-
tions of nanoparticles, were submerged in 1.0 mL of culture medium for
30 min. Adhering to the instructions supplied by the kit’s producer, the
extracts from these hydrogels were subjected to a total antioxidant ca-
pacity assay. The subsequent step involved measuring and comparing
the solution absorbance at 734 nm.
Furthermore, the scavenging efciency of the CFI nanoparticles and
SCE2 hydrogel for superoxide radicals (O
2
•−
) was determined by the
inhibition rate of nitro blue tetrazolium (NBT). A reaction mixture in
PBS solution was prepared, containing NBT (336
μ
M), nicotinamide
adenine dinucleotide (936
μ
M), phenazine methosulfate (120
μ
M), and
the prepared materials. After removing the tested samples, the mixture
was maintained at 37 ◦C for 6 min. Next, the absorbance of the speci-
mens at 560 nm was documented. The scavenging capacity for O
2
•−
in
each sample was calculated with the equation: Inhibition (%) =(1 – A
t
/
A
c
) ×100 %. Here, A
c
refers to the absorbance in the blank control
group, while A
t
indicates the absorbance in samples containing CFI
nanoparticles or SCE2 hydrogel.
2.6. In vitro cell assays
2.6.1. CCK-8 experiments
The cytotoxicity of the SCE hydrogels was assessed via the CCK-8
assay. Initially, all samples were immersed in cell culture media for
24 h to extract the SCE leachate. Then, RAW 264.7 and RS1 cells were
planted in 96-well plates at a density of 5 ×10
3
cells per well, utilizing
DMEM enriched with 10 % fetal bovine serum. Following a 24-h period,
the medium for cell culture was discarded. Subsequently, cells under-
went exposure to leachate from the hydrogel, undergoing incubation
periods of 1, 3, and 5 days. At specic intervals, the medium was
replenished with fresh media containing 10 % CCK-8. After incubating
the solution in darkness for 2 h, its absorbance was measured at 450 nm
with a Thermo Fisher spectrophotometer (1530, USA). Cell viability was
calculated using the formula Cell Viability (%) =A
t
/A
i
×100 %, where
A
i
represents the absorbance of the control, and A
t
is the absorbance of
the treatment group. This process involved ve sets of parallel experi-
ments, each conducted in duplicate.
Besides, cell viability was evaluated through live/dead cell staining
to observe the survival state of the cells. Specically, RAW 264.7, RS1,
and HGF cells were initially plated at a density of 5 ×10
4
cells per well
in confocal dishes and incubated for 12 h. These dishes subsequently
received treatments with hydrogel leaching solutions. Post a culture
period of three days, staining of both live and dead cells was performed
utilizing a calcein acetoxymethyl ester (calcein-AM)/PI staining kit.
Observations of the cellular uorescence were made with a Leica
confocal microscope.
2.6.2. Antioxidant tests
The protective effects of CFI nanoparticles and SCE hydrogels against
intracellular oxidative stress were investigated by evaluating cell sur-
vival following exposure to H
2
O
2
and treatments with CFI nanoparticles
or hydrogels. RS1 cells and RAW 264.7 macrophages were incubated in
96-well plates at a concentration of 1 ×10
4
cells per well and were
permitted to proliferate overnight. Cells cultured in media supple-
mented with H
2
O
2
served as the positive control. Subsequently, cultures
were treated with variously processed H
2
O
2
, followed by a 6-h
incubation. Cell viability was then determined using the CCK-8 assay.
To further investigate the intracellular antioxidant capabilities of the
hydrogels, a green uorescent probe for ROS, 2
′
,7
′
-dichlorouorescein
diacetate (DCFH-DA), was employed to investigate intracellular ROS
levels. HGF, RAW 264.7, and RS1 cells were cultured in confocal dishes
and allowed to develop overnight. Following the removal of the cell
culture medium, adherent cells were treated with the same volume of
H
2
O
2
or hydrogel extract for 6 h. The uorescent probe DCFH-DA was
then introduced. Afterward, uorescence microscopy was used for cell
imaging. For quantitative assessments, cells in 6-well plates underwent
the same treatment previously outlined, aimed at detecting intracellular
ROS. Following a 30-min DCFH-DA exposure period, these cells under-
went trypsinization and were washed three times with PBS. The uo-
rescence intensity in the cells was later quantied through ow
cytometry.
2.6.3. Cell migration
RS1 cells were planted at a concentration of 3 ×10
5
cells per well.
When achieving 90 % conuence, an articial gap was generated among
the cells by creating a linear scratch with a 10
μ
L pipette tip. Subse-
quently, cells that had detached were eliminated by washing thrice with
pre-warmed PBS. Following this, RS1 cells were transferred to a fresh
medium and treated with the same volume of either H
2
O
2
or hydrogel
extract. Observations of the cells were made under a microscope after
incubation periods of 0, 12, and 24 h.
2.6.4. Angiogenesis
Angiogenesis tests were performed to assess the cell’s capacity to
promote the formation of new blood vessels in conditions of oxidative
stress. Usually, 50
μ
L of Matrigel was spread uniformly over a 96-well
plate and solidied after incubating for 1 h. After this, human umbili-
cal vein endothelial cells (HUVECs) were planted on the Matrigel with a
density of 2 ×10
4
cells per well. Once adherence was achieved, either
hydrogel extract or H
2
O
2
was introduced. Afterward, the cells under-
went an 8-h cultivation period. In the nal step, they were stained for 20
min using a calcein-AM staining kit and then observed through a
microscope.
2.6.5. Transcriptome sequencing
RAW 264.7 macrophages were processed to extract total RNA uti-
lizing Invitrogen Trizol reagent (California, USA). A Thermo Scientic
spectrophotometer (NanoDrop 2000, USA) was employed to ascertain
RNA’s purity and concentration. Additionally, the RNA’s integrity was
veried via the Santa Clara Bioanalyzer (Agilent 2100, USA). Subse-
quently, RNA-seq library construction was performed using the VAHTS
Universal V6 RNA-seq Library Prep Kit.
2.7. In vitro blood compatibility assay
The blood compatibility of SCE hydrogels was evaluated through a
hemolytic assay. First, fresh whole blood was washed thrice with PBS at
3000 rpm for 5 min each. Blood that had been washed and resuspended
to a volume of 0.6 mL received treatment with 60 mg of hydrogel placed
in a 1.5 mL centrifuge tube for 2 h. For control sets, 60 mg of DDW and
60 mg of PBS were added to separate 0.6 mL blood samples, serving as
negative and positive controls, respectively. Following treatment, each
sample was subjected to centrifugation (3000 rpm for 4 min). Subse-
quently, the absorbance of the resultant solutions was determined at
545 nm utilizing a spectrophotometer.
2.8. In vitro antibacterial assays
2.8.1. Bacterial preparation
The antibacterial activity of the SCE hydrogels was assessed
employing methicillin-resistant Staphylococcus aureus (MRSA, ATCC
43310) and multidrug-resistant Pseudomonas aeruginosa (MRPA, ATCC
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27853). The bacterial solution was rst revived and cultured at 37 ◦C for
24 h. Subsequently, 10 mL of growth medium was combined with 100
μ
L
of stock bacteria solution (either MRSA or MRPA, 5 ×10
9
CFU/mL). The
mixture was then incubated overnight at 37 ◦C in a shaking incubator at
200 rotations per minute. Following this, the bacteria were diluted at a
ratio of 1:1000 (v/v) for subsequent applications.
2.8.2. Studying photothermal antibacterial performance
100 mg of the samples (hydrogels or PBS) were mixed into 1 mL of
the bacterial solution. After incubation and different treatments, 20
μ
L
of this bacterial solution was uniformly spread onto agar plates. The
plates were then incubated overnight to allow for bacterial colony for-
mation. The colonies were subsequently counted and photographed for
analysis.
2.8.3. Bacterial morphology observation
Morphological changes in bacteria after antibacterial assays were
monitored using SEM and TEM imaging methods. In the SEM protocol,
bacterial cultures (MRSA and MRPA) were collected, washed three times
with PBS, and xed in 2.5 % glutaraldehyde at 4 ◦C overnight. A
dehydration sequence using increasing concentrations of ethanol was
conducted prior to SEM examination. For TEM analysis, bacteria were
rst xed in 2.5 % glutaraldehyde at 4 ◦C, followed by post-xation with
1 % osmium tetroxide. Subsequently, the specimens were washed three
times in PBS for 15 min each. Dehydration followed, using a series of
ethanol treatments for 15 min at each concentration step. The prepared
samples were then analyzed and imaged using TEM.
2.8.4. Bacterial live/dead staining
Additionally, the antibacterial properties of SCE hydrogels were
examined through a bacterial live/dead staining assay. Post various
treatments, the bacterial cells were co-stained with SYTO9 and PI for a
duration of 20 min in the dark, followed by three washes with PBS.
Following the manufacturer’s instructions, all bacteria stained with
SYTO9 presented green uorescence, while those stained by PI, indi-
cating dead bacteria, exhibited red uorescence. The concluding step
was to capture uorescence images using a confocal microscope (Nikon,
Japan).
2.9. Wound healing in vivo
2.9.1. Constructing the type 1 diabetic rat model
The animal study adhered to the policies and norms set by the
Wenzhou Medical University Animal Experiment Center (authorization
number Wydw7019-0134).
Male Sprague-Dawley rats, procured from Beijing Laboratory Animal
Technology Co., Ltd (Vital River, China), were used. For the develop-
ment of the type 1 diabetic wound model, male Sprague-Dawley rats
received a 1 % STZ intraperitoneal injection after fasting for 12 h,
thereby creating a model of diabetes in rats. One week after injection,
blood glucose levels were randomly monitored by sampling blood from
the tail vein. Rats exhibiting blood glucose levels exceeding 16.7 mM
were considered successfully modeled for type 1 diabetes.
2.9.2. Constructing MRSA-infected back skin wounds
Prior to initiating the SCE hydrogel therapy, diabetic rodents un-
derwent a fasting period of 4 h. After administering anesthesia, we
shaved the rodents’ backs and created full-thickness incisions along both
sides of the spinal midline. Subsequently, full-thickness cutaneous le-
sions (8 mm in diameter) on the rodents received a 10
μ
L application of
MRSA solution (concentration: 1 ×10
7
CFU/mL) to develop an infection
in the wound model. We categorized the infected skin wounds into ve
distinct groups: one receiving only PBS (baseline group), another with 3
M Tegaderm lm (positive control), one with SCE0 hydrogel, another
with SCE2 hydrogel, and the last group received SCE2 hydrogel along
with laser exposure (1.0 W/cm
2
, duration: 3 min). We applied 100 mg of
the designated substances to each group. Measurements of the wound
sizes were recorded on days 0, 3, 7, and 14.
2.9.3. Constructing MRSA-infected oral ulcer wounds
In order to establish the oral mucositis model in rats, we employed a
modied version of the conventional technique. Once the diabetic rats
were under anesthesia, we dried the mandibular mucosa of the Sprague-
Dawley rats with sterile cotton. Subsequently, a square piece of lter
paper (3 mm by 3 mm) was soaked in 50 % glacial acetic acid for 5 s and
then quickly placed on the gingival mucosa. The lter paper remained
on the mucosa for 30 s before removal, and the area was then cleansed
employing a sterilized cotton ball dampened in PBS to eliminate any
remaining acetic acid. The lter paper was then soaked in MRSA solu-
tion (concentration: 1 ×10
7
CFU/mL) for another 5 s. The ulcers
generated were divided into ve categories: one baseline group treated
with PBS, a positive control group receiving dexamethasone membrane
therapy, one set administered SCE0 hydrogel, another provided with
SCE2 hydrogel, and the last group treated with SCE2 hydrogel before
undergoing laser therapy (1.0 W/cm
2
for 3 min). Daily photographs
were taken, and notes on the ulcers were documented.
2.9.4. Hemostatic assay
Initially, the hemostatic capabilities of the developed SCE2 hydrogel
were assessed using a rat tail hemostasis model. The procedure involved
cutting the rat’s tail at the lower third with surgical scissors. After a 15-s
interval, different materials (each weighing 200 mg) were applied to the
tail wounds. The treatment procedure was captured in photographs, and
the volume of blood loss was meticulously recorded.
To further explore the SCE2 hydrogel dressing’s hemostatic effec-
tiveness, a liver hemorrhage model was established. The rat’s abdominal
cavity was carefully opened through surgery, revealing the liver. We
used absorbent paper to meticulously soak up any uid seeping out.
Next, a scalpel was employed to create an 8 mm in length and 1.5 mm in
depth incision on the surface of the liver. Following this, we applied 200
mg of various substances to the damaged area on the rat’s liver. This
procedure was documented with photographs.
2.9.5. Photothermal performance
Diabetic rats were randomly assigned into three equal sets, each
comprising three rats: SCE2, SCE0, and PBS. For the groups involving
SCE0 and SCE2 hydrogels, the wound areas received NIR light treatment
(808 nm, 1 W/cm
2
, for 3 min). Thermal imagery of the areas was
captured using infrared camera technology.
2.9.6. Biosafety evaluation
Following the in vivo studies on the healing of wounds infected with
bacteria, blood specimens were collected from the rats to conduct
biochemical analyses and routine blood examinations. Concurrently,
key organs (heart, liver, spleen, kidney, and lung) of the type 1 diabetic
rats were extracted and subjected to hematoxylin-eosin (H&E) staining
for histological assessment.
2.9.7. Histological analysis
At the conclusion of the experiment, tissues from the dorsal skin and
oral mucosa were harvested for histological analysis. A Leica microtome
(Germany) facilitated the slicing of tissues into sections 40
μ
m thick.
These samples were then xed in 4 % paraformaldehyde for a full day
and embedded in parafn. Subsequently, the derived tissue sections
underwent staining with H&E, as well as Masson, myeloperoxidase
(MPO), tumor necrosis factor-alpha (TNF-
α
), CD86 (M1 macrophage
marker), vascular endothelial growth factor (VEGF), CD206 (M2
macrophage marker) and platelet endothelial cell adhesion molecule-1
(CD31).
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2.10. Statistical analysis
Analysis of the data was conducted through one-way ANOVA or the
Student’s t-test, with a P value of 0.05 or lower deemed statistically
signicant. The graphical representations displayed statistical outcomes
as the average ±standard deviation. Within the graph legends, “NS”
denotes a result that is not statistically signicant (P greater than 0.05),
while “*”, “**”, and “***” signify P values less than 0.05, 0.01, and
0.001, respectively.
3. Results and discussion
3.1. Preparation and characterization of CFI nanoparticles
The morphologies of the CFI nanoparticles, as seen under SEM and
TEM, are depicted in Fig. 1A and B, respectively. We observed that the
CFI displayed a uniform nanosphere shape devoid of discernible clumps.
Complementing these ndings, DLS analysis (Fig. S1, Supporting In-
formation) suggested that the CFI had an average diameter of 177.4 ±
0.52 nm. Additionally, measurements indicated the CFI’s zeta potential
was −23.1 mV. This characteristic is presumably associated with the
polyphenolic moieties present on the CFI surface, as detailed in Fig. S2 in
the Supporting Information [32].
We undertook a systematic evaluation of CFI’s near-infrared photo-
thermal properties. We exposed various CFI concentrations to NIR ra-
diation (808 nm, 1 W/cm
2
) and monitored the process. Thermal imaging
of CFI during irradiation was acquired using a FLIR infrared camera (as
shown in Fig. 1C). As expected, CFI exhibited a greater temperature
increase compared to PBS. Fig. 1D shows that the temperatures at CFI
nanoparticle concentrations of 2, 1, and 0.5 mg/mL reached 81.6, 72.9,
and 58.5 ◦C, respectively, after 10 min of exposure to the laser. The
efciency of CFI’s photothermal conversion was also inuenced by the
power density of irradiation (Fig. S3, Supporting Information). The
consistent temperature pattern of CFI across four heating and cooling
cycles, depicted in Fig. 1E, indicated its reliable photothermal stability.
Additionally, CFI’s photothermal conversion rate was calculated to be
45.7 % (Figs. S4–S6, Supporting Information). These characteristics
(high photothermal conversion efciency and superior photothermal
stability) make CFI nanoparticles an excellent candidate for photo-
thermal antibacterial applications [33].
In light of the harmful effects of excessive ROS in diabetic wounds,
materials capable of scavenging ROS have attracted signicant interest.
Considering the abundance of phenolic hydroxyl moieties on the CFI
surface, we postulated that CFI might exhibit outstanding capabilities in
scavenging free radicals. In this study, the ROS scavenging performance
of CFI was evaluated through superoxide anion radical (O
2
•−
), ABTS, and
DPPH assays (Fig. 1F–H). As predicted, CFI (1 mg/mL) achieved a
clearance rate exceeding 70 % for various free radicals.
Following this, we examined CFI’s cytoprotective capacity against
oxidative stress induced by H
2
O
2
. After treating RAW 264.7 and
RS1cells with H
2
O
2
and CFI, we assessed the intracellular antioxidant
features by measuring cell viability. For example, after a 6-h exposure to
670
μ
M H
2
O
2
, the cell viability fell to 33.4 % for RAW 264.7 macro-
phages and to 40.5 % for RS1 cells. However, the cell survival rate was
>65 % for CFI nanoparticles greater than 1 mg/mL (Fig. 1I and J).
Moreover, the ow cytometry results, depicted in Fig. 1K and L, further
conrmed that CFI protected RAW 264.7 macrophages and RS1 cells
from oxidative damage, showing similar results. These ndings
demonstrate that CFI displays outstanding photothermal and ROS
scavenging capabilities at concentrations of 1 and 2 mg/mL. Considering
the cost-effectiveness of raw materials, we incorporated 1 mg/mL of the
CFI system into the hydrogel for use as the primary subject in subsequent
experiments.
3.2. Characterization of SCE2 hydrogel
Exploration of functional group transformations pre and post
hydrogel fabrication utilized
1
H NMR and FTIR techniques. Initially,
notable variations in the
1
H NMR spectra of SFMA were evident when
contrasted with SF. In particular, the emergence of distinct signals at
5.60 ppm and 6.04 ppm, linked to the carbon-carbon double bond [34],
was observed in the SFMA’s
1
H NMR data (Fig. 2A), indicating suc-
cessful SFMA synthesis. Furthermore, Fig. 2B presents the
1
H NMR
spectrum for CHS-NB. Assigned peaks at 7.24 ppm and 7.76 ppm cor-
responded to the NB benzene ring, conrming the effective grafting of
the NB group onto CHS [18,35]. Concurrently, SFMA’s unique absorp-
tion bands at 1644 cm
−1
and 1518 cm
−1
, representing the –CH
2
–
–
CH
2
–,
along with CHS-NB (–NB exural vibration at 1564 cm
−1
and 1375
cm
−1
) and CFI (–OH vibration at 3290 cm
−1
), were detected in the FTIR
spectrum of the CFI-integrated matrix, underscoring the successful cre-
ation of the SCE hydrogel (Fig. S7, Supporting Information).
Subsequently, a TGA test was conducted to investigate the thermal
stability of the synthesized materials. Fig. 2C shows that the prepared
samples underwent three distinct phases of decomposition. Initially, all
samples demonstrated a gradual weight reduction up to 250 ◦C, mainly
due to the evaporation of loosely bound water within the tested mate-
rials. During the second phase (between 250 and 400 ◦C), the hydrogel’s
mass diminished owing to the degradation of glycosidic links in the
polysaccharide structure. In the nal stage (beyond 400 ◦C), the
decomposition of the SCE hydrogel began, leading to char formation due
to the breakdown of the polymer chains. Furthermore, the total weight
loss of the SCE0 and SCE2 hydrogels at 600 ◦C was 66.10 % and 64.41 %,
respectively. TGA showed that SCE0 and SCE2 hydrogels exhibit com-
parable weight loss trajectories in an air environment, a nding
consistent with their similar chemical makeup. Such results indicate the
SCE hydrogel’s superior thermal stability.
Following this, we utilized SEM to evaluate the surface texture and
microscopic pore structure of the SCE specimens. As displayed in Fig. 2D
and E, the porous mesh architecture of both SCE0 and SCE2 hydrogels
provides an optimal environment for the absorption of exudates and cell
growth. Further inspection through high-resolution SEM imagery indi-
cated that the inner surface of SCE0 was comparatively smooth, whereas
SCE2 exhibited a rougher texture, a result of integrating CFI nano-
particles. In addition, the mean pore size in SCE0 measured 26.82
μ
m
while incorporating CFI into SCE2 resulted in a reduced average pore
size of 20.14
μ
m (Figs. S8 and S9, Supporting Information).
Then, we assessed the hydrogel dressings for their ability to swell and
deswell. As depicted in Fig. 2F, the swelling trajectories of SCE0 and
SCE2 hydrogels revealed a comparable water uptake trend, marked by a
consistent rise and eventual leveling off beyond 200 min. Upon reaching
equilibrium, the SCE0 demonstrated a greater water absorption ratio
(26.92) compared to SCE2 (21.86), suggesting that the hydrogels’ cross-
linking improved upon incorporating CFI [36]. In the water retention
assessment, SCE2 demonstrated slightly superior water retention
compared to SCE0 (Fig. 2G). These results suggest that the fabricated
SCE2 hydrogel exhibits outstanding capabilities in absorbing and
retaining water, enabling rapid absorption of wound exudate and
preservation of a suitably moist environment conducive to wound
healing [37].
3.3. Evaluating the antioxidant capacity of SCE2 hydrogel
An overabundance of ROS at wound locations may impede the pro-
cess of healing. Therefore, herein, we integrated CFI into a SFMA/CHS-
NB matrix and assessed the ability of the prepared nano-doping SFMA/
CHS-NB system to neutralize free radicals, specically O
2
•−
, ABTS, and
DPPH. To begin, we focused on the superoxide anion radical, a prevalent
type of ROS, and used an O
2
•−
producing system, NADH-PMS-NBT, to test
the hydrogels’ neutralizing efcacy. The SCE2 hydrogel outperformed
the SCE0 hydrogel, showing scavenging rates of 40.6 % and 13.5 %,
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Bioactive Materials 39 (2024) 562–581
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Fig. 1. Characterization of CFI nanoparticles. (A) SEM image of CFI. (B) TEM image of CFI. (C) Thermographs of different CFI concentrations over time. (D)
Temperature increase proles of CFI nanoparticles. (E) Photothermal cycling curve of 1 mg/mL CFI. (F–H) The effectiveness of CFI nanoparticles in neutralizing O
2
•−
(F), ABTS (G), and DPPH radicals (H). (I and J) Evaluation of cell survival in RAW 264.7 (I) and RS1 (J) cells exposed to different concentrations of CFI nanoparticles
in a solution with 670
μ
M H
2
O
2
, using the CCK-8 assay. (K and L) Flow cytometric assessment of RAW 264.7 macrophages (K) and RS1 cells (L). Error bars indicate
the average ±standard deviation (n =3). Levels of signicance are denoted as *** for P <0.001 and * for P <0.05.
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respectively, as depicted in Fig. 2H. Further assessments of the hydro-
gels’ antioxidant capabilities were conducted using ABTS (Fig. 2I),
where the results aligned with those from the O
2
•−
trials. Additionally,
the SCE hydrogels underwent a 30-min incubation with DPPH. As ex-
pected, the CFI-enhanced SCE2 hydrogel exhibited more robust scav-
enging abilities than SCE0, as illustrated in Fig. 2J, conrming its
superior antioxidant activity. The impressive radical neutralizing ef-
ciency of SCE2 hydrogel suggests its potential as an antioxidant, which
could be benecial in promoting wound healing.
3.4. Assessment of SCE2 hydrogel’s mechanical properties
Oral mucosa, being a moist and dynamic tissue, necessitates the
creation of patches capable of forming stable, strong adhesion for
treating oral ulcers. Essential features of mucosal hydrogel dressings,
such as mechanical robustness and malleability, enable them to adapt to
constantly varying and intricate wound environments. To assess the
attachment of SCE hydrogels to tissues, macroscopic tests measuring
adhesion were conducted on pig skin. The adhesive capabilities of these
hydrogels were measured through lap-shear (Fig. 3A and B) and tension
Fig. 2. Analyzing the physiochemical characteristics of SCE2 hydrogel. (A and B) Displaying
1
H NMR spectra for SFMA (A) and CHS-NB (B). (C) TGA proles of CFI
and SCE hydrogels. (D and E) Diverse magnications of SEM images featuring SCE0 (D) and SCE2 (E). (F and G) Performance in swelling (F) and deswelling (G) of
SCE hydrogels. (H–J) Examining the radical scavenging abilities of SCE2 hydrogel against O
2
•−
(H), ABTS (I), and DPPH (J). Error bars indicate the average ±
standard deviation (n =3). Signicance levels: *** for P <0.001.
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(Fig. 3C and D) experiments [38]. In comparison to SFMA hydrogel,
which displayed fracture stress values at 27.45 kPa, both SCE0 and SCE2
hydrogels demonstrated superior fracture stress, especially SCE2
hydrogel, peaking at 64.37 kPa. During tension testing, SCE0 hydrogel’s
adhesive strength peaked at 18.13 kPa, and SCE2 hydrogel at 21.28 kPa.
These gures were 2.93 and 3.44 times greater than SFMA hydrogel’s
6.18 kPa, respectively. The heightened adhesion to tissues originates
from the conversion of benzyl alcohol groups into benzaldehyde groups
post-UV light exposure, resulting in the formation of Schiff base struc-
tures with the amino moieties on the tissue surface. Conclusively, SCE
hydrogels showed superior adhesion capabilities compared to SMFA
hydrogel, suggesting their potential as effective bioadhesives in clinical
settings.
Next, we analyzed the rheological properties of SCE hydrogels.
Initially, strain scan tests were employed to investigate the hydrogel
linear viscoelastic region, as shown in Fig. 3E. The gel point of the SCE2
hydrogel was found to be around 600 %, indicating it assumes a
colloidal state at strains beyond this threshold. In these experiments,
both hydrogels displayed phases where the G’ (storage modulus)
consistently surpassed the G’’ (loss modulus) [39]. Then, we assessed
the frequency-dependent rheological characteristics of the hydrogels, as
shown in Fig. 3F. The hydrogels’ self-recovery properties were also
investigated using dynamic amplitude experiments. Fig. 3G demon-
strates the procedure where we initially subjected the hydrogel to a high
strain (1000 %) to cause disintegration, followed by a low strain (1 %) to
evaluate recovery ability. Under a 1000 % strain, there was a notable
drop in G
′
, falling below G
″
, indicating network breakdown. However,
on reverting to a 1 % strain, both G
′
and G
″
of SCE0 and SCE2 hydrogels
Fig. 3. Evaluating the mechanical attributes and adhesive strength of SCE2 hydrogel. (A) Illustration of the hydrogel’s adhesion process with tissue and the cor-
responding lap-shear examination. (B) The hydrogels’ stress−strain prole obtained through lap-shear testing. (C) Illustrative depiction of conducting a tensile
strength assessment. (D) Capturing the stress−strain relationship during the tensile evaluation of the hydrogels. (E–H) Analysis of SCE2 hydrogel’s characteristics
includes strain-responsive rheological behavior (E), frequency-responsive rheological properties (F), evaluations of step-strain (G), and shear-thinning attributes (H).
Displaying SCE2 hydrogel’s ease of injection through representative images. (I) Illustration of SCE2 hydrogel’s injectability within a PBS setting. (J) A sequence of
images showcasing the self-healing capabilities of the SCE2 hydrogel.
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Bioactive Materials 39 (2024) 562–581
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returned to their original values, demonstrating the SCE hydrogels’ swift
recovery capability. The hydrogel demonstrated excellent self-healing
abilities through multiple sequential network breakdown and recovery
cycles [40]. The foundation of SCE2 hydrogel’s remarkable self-healing
capability lies in the Schiff base reaction among polymer chains,
creating dynamic and reversible cross-linking sites. Lastly, a shear test
showed that the SCE2 hydrogel exhibited shear-thinning traits, indi-
cating the hydrogel’s prominent injectability (Fig. 3H) [41].
Additionally, we explored the macroscopic characteristics of the SCE
hydrogels, focusing on their injectability and self-healing properties. We
successfully created continuous hydrogel strips in a glass beaker lled
with PBS using a 2 mL syringe (Fig. 3I). Subsequently, the SCE2 hydrogel
was divided into two halves. Remarkably, these halves reunited within
30 min without external force, appearing almost identical to their
original state (Fig. 3J). These observations support the superior me-
chanical properties of SCE2 hydrogel, implying its potential suitability
for complex wound environments.
3.5. Intracellular antioxidant and biocompatibility abilities of SCE2
hydrogel
Biosafety is critical for using hydrogel materials as versatile dressings
in tissue engineering [42,43]. With this in mind, we rst assessed the
cytocompatibility of the developed SCE hydrogels. To evaluate cell
survival, the CCK-8 assay was used after RAW 264.7 macrophages and
RS1 cells were cultured with extracts from SCE hydrogel for durations of
1, 3, and 5 days. Fig. 4A and B illustrate that cell viability in both SCE0
and SCE2 groups was above 80 %, indicating minimal toxicity of the
developed SCE hydrogels. Fig. 4C shows that the supernatant from
DDW, serving as a positive control, turned bright red, demonstrating red
blood cell disruption [44]. Conversely, the PBS and SCE sets remained
colorless with hemolysis rates below 5 %, demonstrating favorable
hemocompatibility. On the third day of incubation, cells (RAW 264.7,
RS1, and HGF) were stained using a calcein-AM/PI live/dead cell
staining kit [45,46]. The intensity of green uorescence in SCE hydrogel
groups was nearly on par with the control set, and very few dead cells
were detected in these SCE hydrogel groups (as seen in Fig. 4D and E;
Fig. S10 in the Supporting Information). In summary, the developed SCE
hydrogels showed outstanding biocompatibility and hemocompatibility,
making them promising candidates as scaffold materials in biomedical
engineering applications [47].
The hydrogel’s antioxidant capability was veried at the cellular
level. To begin with, a DCFH-DA uorescent probe was employed for
evaluating intracellular ROS levels, while also investigating the safe-
guarding impacts of SCE hydrogels within oxidative stress conditions.
Results (Fig. 4F and G; Figs. S11–S14, Supporting Information) revealed
that RAW 264.7, RS1, and HGF cells exposed to the H
2
O
2
group
exhibited heightened uorescence, indicating elevated ROS levels.
Contrastingly, cells in the SCE2 hydrogel group showed markedly
reduced uorescence, indicative of lower ROS levels. These ndings
align with ow cytometry results (Fig. 4H and I), which revealed
increased green uorescence in the H
2
O
2
group and diminished DCFH-
DA green uorescence in the SCE hydrogel-treated set. Additionally,
Fig. 4J and K indicated a signicant reduction in cell growth due to
oxidative damage following H
2
O
2
exposure. However, cell survival rates
notably improved in the SCE hydrogel-treated sets. For instance, RAW
264.7 and RS1 cells co-cultured with 670
μ
M H
2
O
2
and SCE2 for 6 h
exhibited cell survival rates of 61.16 % and 69.15 %, higher than the
H
2
O
2
and SCE0 hydrogel groups. These ndings further substantiate
SCE2 hydrogel’s potent antioxidant capability in shielding cells against
oxidative stress, highlighting its potential in wound healing
applications.
3.6. Investigation of migration and angiogenesis of SCE2 hydrogel in vitro
To explore cell proliferation in oxidative stress conditions more
deeply, we conducted in vitro scratch assays. Using a pipette tip, we
scratched the plate bottom containing RS1 cells and then treated with
H
2
O
2
and hydrogel. After 12 h and 24 h, we monitored the changes in
the scratch area. As illustrated in Fig. 4L, the control group cells
exhibited normal migration and proliferation with time, signicantly
reducing the migration distance after 24 h [48]. The scratch area in the
H
2
O
2
group remained largely unchanged, indicating minimal cell
movement toward the scratches. On the other hand, the SCE2 group cells
showcased better migration capabilities compared to the H
2
O
2
group,
leading to a gradual decrease in the scratch area. The leftover scratch
area was a mere 34.7 % of the original area, implying that the formu-
lated SCE2 could enhance cells’ antioxidant capability by neutralizing
exogenous oxidants (Fig. S15, Supporting Information).
Additionally, we used HUVECs to assess the inuence of the newly
developed SCE2 hydrogel on angiogenesis, especially under conditions
of oxidative stress [49]. After incubation, HUVECs were arranged on
Matrigel (Fig. 4M; Fig. S16, Supporting Information). The cells were
then subjected to H
2
O
2
and hydrogel treatment to monitor the devel-
opment of HUVEC lumens in each group. After 8 h, HUVECs in the
control group formed an interconnected tubular network with an in-
crease in blood vessels and enhanced structural integrity [50]. In stark
contrast, H
2
O
2
signicantly disrupted angiogenesis in HUVECs, with a
complete lack of angiogenic activity. However, cells treated with SCE2
hydrogel showed close clustering and managed to establish a tubular
network, implying that SCE2 hydrogel has the potential to mitigate
ROS’s harmful effects and maintain the angiogenic capability of
HUVECs.
3.7. Assessing the photothermal antibacterial ability of SCE2 hydrogel
After evaluating the cellular level properties of the SCE2 hydrogels,
we focused on assessing their photothermal antibacterial capabilities.
Fig. 5A illustrates that the temperature of the SCE hydrogels rose more
swiftly as the concentration of CFI increased. Specically, a hydrogel
with 0.5 mg/mL of CFI nanoparticles witnessed a solution temperature
increase to 68.2 ◦C. When the CFI concentration was elevated to 2 mg/
mL, the temperatures climbed to 87.2 ◦C. In a similar vein, escalating
laser power densities led to higher temperatures when irradiating the
SCE2 at a constant concentration (Fig. 5B). The hydrogel’s thermal
imagery, captured after 10 min of exposure at 808 nm, conrmed the
observed temperature increase (Fig. S17, Supporting Information). To
assess the photostability of the SCE hydrogels, we allowed them to cool
down naturally after 10 min of light exposure. Fig. 5C reveals that there
was no notable reduction in the temperature increase across four cycles
of activating and deactivating the laser, indicating the hydrogel’s su-
perior photothermal stability. Moreover, the
η
of SCE2, a crucial metric
reecting the material’s photothermal capability, was recorded at 46.3
% (Fig. 5D; Fig. S18, Supporting Information). This further indicates the
exceptional photothermal capabilities of the designed SCE2 hydrogel.
Infection is a major factor hindering the process of wound healing
[51]. In our study, we employed a plate counting method to measure the
antibacterial effect of the SCE2 hydrogel, performing antibacterial ex-
periments for both gram-negative (MRPA) and gram-positive (MRSA)
bacteria in either the absence or presence of 808 nm NIR (Fig. 5E). The
SCE hydrogels had little to no antimicrobial effect (Fig. S19, Supporting
Information). As anticipated, the SCE2 +NIR group demonstrated an
antibacterial effect close to 100 %, indicating that incorporating 808 nm
NIR (1 W/cm
2
for 10 min) signicantly enhanced the sterilizing efcacy
of the SCE2.
To elucidate the potential antibacterial mechanism behind SCE
hydrogels, we used SEM to inspect the detailed morphological alter-
ations in bacteria. In the control group, MRSA and MRPA maintained
their morphology with smooth bacterial cell membranes. The bacteria
subjected to the SCE2 hydrogel +NIR laser experienced more extensive
damage, such as ruptures or perforations in the cell membrane, afrm-
ing that the SCE2 hydrogel, aided by the photothermal effect of NIR
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light, can enhance bacterial eradication.
Following that, a live/dead bacterial staining test was conducted to
examine the bacterial cell membrane integrity, as seen in Fig. S20 in the
Supporting Information. Compared to the control set, the group treated
with SCE2 hydrogel had only a little red uorescence, indicating
essentially devoid bactericidal activity of SCE2 hydrogel. The strongest
red PI uorescence was detected in the group treated with SCE2 +NIR,
indicating that PI penetrated the compromised bacterial membranes,
coloring the bacteria red. Hence, SCE2 hydrogel showcases superior
photothermal antibacterial capability and could serve as a robust anti-
bacterial platform.
Furthermore, the architectural changes in bacteria underwent ex-
amination using TEM (Fig. 5F). Upon exposure to NIR light, MRSA and
MRPA bacterial samples were gathered to assess morphological and
structural characteristics via TEM. Contrary to the unexposed bacteria,
which maintained a regular bacilliform shape with uniform nucleoplasm
and a noticeable agellum, the group treated with SCE2 +NIR exhibited
signicant alterations. Their cell walls thinned or vanished entirely,
membranes wrinkled, and structural integrity was compromised. Addi-
tionally, bacterial swelling or shrinkage occurred, accompanied by
membrane distortions, ruptures, and subsequent release of cellular
contents.
3.8. Analysis of SCE2 hydrogel’s therapeutic effectiveness via RNA
sequencing
To delve deeper into the SCE2 hydrogel’s inammatory mechanism,
we subjected the RAW 264.7macrophage cell line to IFN-γ and LPS
stimulation, subsequently treating them with SCE2 hydrogel for gene
expression alterations analysis through RNA sequencing. The resulting
Venn diagram (Fig. 6A) revealed exclusivity in gene presence, with the
M1 group having 3 unique genes, the SCE0 group showing 404 singular
genes, and the SCE2 group presenting 447 individual genes. Further,
sample integrity was assessed using Principal Component Analysis
(PCA), which highlighted distinct distributions between the M1 and
SCE2 groups, suggesting varied total gene expression proles (Fig. 6B).
Volcano diagrams indicated that 519 genes were markedly and differ-
entially expressed; among them, 163 genes were heightened in expres-
sion, and 356 were suppressed (Fig. 6C). Analysis of these DEGs through
Gene ontology (GO) revealed enriched biological processes such as
cellular reaction to tumor necrosis factor and various immune and in-
ammatory responses (Fig. 6D).
To shed light on the cellular signaling mechanisms of SCE2 hydrogel
in inammatory therapy, both the Kyoto Encyclopedia of Genes and
Genomes (KEGG) and Gene Ontology (GO) databases were employed for
gene set enrichment analysis. The signaling pathway enrichment dia-
gram from the KEGG database indicated changes in gene expression
related to TNF, NF-kB, and IF-17 signaling pathways in the SCE2 group,
as shown in Fig. 6E. Previous research has noted the function of ROS
within the IL-17 signaling pathway. Activation of this pathway markedly
increases the secretion of chemokines and pro-inammatory cytokines
like CCL2, TNF-
α
, and IL-1β. It also shifts macrophages towards the pro-
inammatory M1 subtype and stimulates neutrophil recruitment and
activation. Remarkably, SCE2 hydrogel mitigates inammation by
neutralizing ROS and suppressing IL-17 signaling pathway activities.
Gene expression disparities between the M1 and SCE2 groups were
depicted in a heatmap (Fig. 6F). Post-treatment with SCE2 hydrogel, a
notable upregulation was observed in several critical pro-inammatory
genes, including CCL5, IL-6, CCL3, S100A8, and NOS2. As displayed in
Fig. 6G–a chord diagram displayed the diminished gene expression and
linked GO biological processes, including the control of inammation,
angiogenesis, immune responses, and improved regulation of apoptosis.
To unravel the specic pathways inuenced by SCE2, further investi-
gation was conducted through gene set enrichment analysis (GSEA), as
displayed in Fig. 6H. These ndings indicated a reduction in TNF and
NOD-like receptor signaling pathways in macrophages after SCE2
hydrogel application. Collectively, the data robustly suggest that the
administration of SCE2 hydrogel signicantly promotes wound healing
by attenuating oxidative stress and curbing inammatory responses.
3.9. In vivo healing of MRSA-infected diabetic back skin wounds by SCE2
hydrogel
Following the acquisition of in vitro results, our investigation shifted
to analyzing the in vivo back skin wound healing performance of the
SCE2 hydrogel. Initially, we focused on determining the hemostatic ef-
cacy of SCE2 hydrogels. In the event of a wound, swift hemostasis plays
a crucial role in ensuring a repair of superior quality. To facilitate this,
we incorporated NB groups with photo-triggered adhesive features into
the hydrogel’s matrix, with the objective of enabling the dressings to
promptly control bleeding. The SCE2 hydrogel’s hemostatic effective-
ness was appraised using both a tail model and a liver model. Within the
liver hemostasis model (Fig. S21, Supporting Information), groups
treated with the hydrogel exhibited the most pronounced hemostatic
effect, displaying almost no apparent bleeding in contrast to the
noticeable hemorrhage observed in other groups. This conclusion was
echoed in the tail hemostasis model, further substantiating SCE2
hydrogel’s remarkable capacity for in vivo bleeding cessation. Quanti-
tative analysis revealed that blood loss in the untreated control (139.53
mg) and gauze-applied (98.73 mg) groups were signicantly higher
compared to the SCE2 hydrogel group (34.7 mg) (Figs. S22 and S23,
Supporting Information).
Following that, we explored the SCE2 hydrogel’s capabilities in
photothermal heating and sterilization. After a 3-min exposure to the
NIR laser, the temperature at the rat wound sites treated with PBS and
SCE0 remained relatively unchanged (Fig. S24, Supporting Informa-
tion). In contrast, for the SCE2 +NIR group, the temperature notably
rose from 36.8 ◦C to 60.7 ◦C. We then collected and quantied bacteria
from the wound sites post-treatment using an agar plate method
(Fig. S25, Supporting Information). Aligning with the in vitro ndings of
hydrogel’s antimicrobial efcacy, the control and 3 M groups exhibited
the highest bacterial counts. Meanwhile, the SCE0 and SCE2 groups
showed moderate bacterial presence, and notably, the SCE2 +NIR
group recorded the lowest bacterial count. These observations collec-
tively indicate that the SCE2 hydrogel, when aided by NIR, demon-
strated superior antibacterial effectiveness.
Subsequently, the healing properties of the developed SCE2 hydrogel
were evaluated using a MRSA-infected diabetic rat back skin wound
model. Wound progression was documented through photographs taken
on days 0, 3, 7, and 14, as presented in Fig. 7A. The wound closure in
diabetic rats treated with SCE0, SCE2, and SCE2 +NIR occurred more
rapidly compared to those receiving PBS and 3 M treatments (Fig. 7B).
Notably, after 14 days of treatment, a signicant wound remained in the
3 M hydrogel-treated group (wound area =31.3 %), similar to the PBS-
Fig. 4. In vitro cellular and hemolytic assays with SCE2 hydrogel. (A and B) Viability of RAW 264.7 macrophages (A) and RS1 cells (B) following exposure to PBS and
SCE groups for durations of 1, 3, and 5 days. (C) Hemolysis assessment and images of SCE hydrogels post 2-h exposure to erythrocytes at 37 ◦C. (D and E) Fluo-
rescence imaging of calcein-AM/PI staining in RAW 264.7 (D) and RS1 cells (E) after 3 days of varied treatments (scale bar: 100
μ
m). (F) Assessment of intracellular
ROS neutralization by SCE hydrogels in RAW 264.7 cells employing DCFH-DA (scale bar: 20
μ
m). (G) Evaluation of ROS neutralization in RS1 cells by SCE hydrogels,
indicated by DCFH-DA (scale bar: 50
μ
m). (H and I) Flow cytometric analysis of ROS in RAW 264.7 (H) and RS1 cells (I) using DCFH-DA as the detecting agent. (J and
K) Survival rates of RAW 264.7 (J) and RS1 (K) cells post-incubation in SCE and H
2
O
2
solution. (L) Images showing the migration of RS1 cells at intervals of 0, 12, and
24 h (scale bar: 300
μ
m). (M) Visuals captured through the uorescence of HUVECs forming vessels (scale bar: 100
μ
m). Error bars represent the average ±standard
deviation (n =3). Levels of signicance are denoted as *** for P <0.001, ** for P <0.01, * for P <0.05, and NS for P >0.05.
Y. Xiang et al.
Bioactive Materials 39 (2024) 562–581
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Fig. 5. Examining the SCE2 hydrogel’s in vitro photothermal antibacterial properties (A) Analyzing the temperature rise patterns among various hydrogels. (B)
Studying the thermal increase in SCE2 hydrogel under different NIR light exposures. (C) Investigating the photothermal resilience of SCE2 hydrogel through four
alternating heating and cooling cycles. (D) Analyzing the heating and cooling phases of SCE2 hydrogel’s photothermal process. (E) Assessing SCE2 hydrogel’s
antibacterial effectiveness against MRSA and MRPA through methods like agar plate counting, SEM, and bacterial live-dead staining. (F) Presenting TEM imagery of
bacteria pre and post exposure to SCE2 hydrogel combined with NIR treatment.
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Bioactive Materials 39 (2024) 562–581
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Fig. 6. RNA sequencing evaluation of SCE2 hydrogel’s therapeutic efcacy. (A) Differential gene count comparison in a Venn diagram for M1, SCE0, and SCE2
categories. (B) Three-group PCA plot illustrating proteomic data with three biological replicates per group. (C) Volcano chart presenting the downregulated and
upregulated gene expressions in the SCE2 versus M1 gene expressions. (D) Analysis of GO pathways enriched among detected DEGs. (E) Dot chart depicting selected
DEGs’ KEGG enrichment analysis outcomes between M1 and SCE2 groups. (F) Heat map representing downregulated genes in both M1 and SCE2 groups. (G) Chord
diagram illustrating GO enrichment terms linked to specic downregulated genes associated with oxidative stress. (H) GSEA results indicating cytosolic TNF and NF-
kB signaling pathways’ gene set enrichment in cells treated with SCE2 hydrogel.
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Fig. 7. Assessment of SCE2 hydrogel’s therapeutic efcacy on back skin wounds in diabetic rats. (A) Depictions of skin injury progression in PBS, 3 M, SCE0, SCE2,
and SCE2 +NIR. (B) Visual representation of healing stages in wounds. (C) Comparative measurement of wound sizes in all groups. (D–F) Staining for histology in rat
skin sections from the wound region: H&E (D), Masson’s trichrome (E), and markers TNF-
α
, MPO, CD31, VEGF, CD86, and CD206 (F). Error bars represent the
average ±standard deviation (n =3).
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treated group (wound area =33.1 %), suggesting that 3 M hydrogel is
less effective in healing bacterially infected wounds. In contrast, the
SCE0 and SCE2 hydrogels exhibited considerable decreases in the
wound area, reaching 24.6 % and 14.8 % after 14 days, demonstrating
their efcacy in expediting wound healing. Remarkably, the SCE2 +NIR
group accomplished near-total wound healing, exhibiting an average
wound area of merely 8.1 % at the conclusion of the monitoring period,
as elaborated in Fig. 7C.
Moreover, fourteen days post-surgery, we collected the wounds for
histological examination, as depicted in Fig. 7D. The SCE2 +NIR group
exhibited markedly better healing than the PBS group. The newly re-
generated skin in this group showcased complete epithelial and dermal
structures, alongside a notable increase in new blood vessels and hair
follicle formation. We then used Masson staining to assess collagen
regeneration [52]. In the SCE2 +NIR group, there was a noticeable
presence of dense and orderly aligned collagen bers, as depicted in
Fig. 7E, indicative of enhanced collagen production [53]. Furthermore,
TNF-
α
staining was conducted. Fig. 7F shows pronounced inammation
in the control set, while the SCE2 +NIR set displayed lower TNF-
α
levels
compared to both SCE0 and SCE2 hydrogel groups [54]. This observa-
tion gains additional support from MPO staining of neutrophils,
demonstrating the minimal inammatory response in the SCE2 +NIR
group, as evidenced in Figs. S26A and S26B in the Supporting
Information.
Furthermore, we employed CD31 and VEGF staining to assess neo-
vascularization. Fig. 7F shows that the SCE2 +NIR group had signi-
cantly more new blood vessel proliferation than the PBS group, as
further detailed in Figs. S27A and S27B in the Supporting Information
[55]. Consistent with results from the rat back skin wound experiments,
the SCE2 +NIR set possessed the least CD86 expression and the highest
CD206 expression, as evidenced in Fig. 7F and S28 in the Supporting
Information. The SCE2 hydrogel enhanced with NIR contributed to a
reduction in M1 macrophages and an increase in M2 macrophages, thus
promoting the production of proteins related to anti-inammatory and
angiogenic cellular factors. This contributed signicantly to the accel-
erated healing of MRSA-infected back skin trauma.
3.10. In vivo healing of MRSA-infected diabetic oral ulcer wounds by
SCE2 hydrogel
After conrming the SCE2 hydrogel’s ability to repair wounds on
dorsal skin, a model for oral ulcers was subsequently developed to
further evaluate its healing efcacy (Fig. 8A and B). The detailed process
for constructing the model is shown in Fig. 8A. When a chemical reac-
tion was induced in the rat mucosa using glacial acetic acid, an imme-
diate reaction occurred at the application site, marked by swift
whitening of the mucosa. Besides the sets receiving hydrogel treatments,
the negative control was subjected to PBS administration, and the pos-
itive control involved treatment with a glucocorticoid solution con-
taining dexamethasone (DEX). Fig. 8B shows a clearly dened, hardened
central ulcer in the oral cavity of each rat immediately following sur-
gery. By the second day, this ulcer became increasingly noticeable, with
the adjacent mucosa appearing swollen, reddened, and coated by a
yellowish pseudomembrane. Two days following the commencement of
hydrogel therapy, we noted a reduction in both the thickness of the
pseudomembrane and the size of the ulcer area. After four days, a small
section of the pseudomembrane remained within the PBS group.
Conversely, the DEX and SCE2 sets showed decreased swelling and
redness in the surrounding mucosal area, with the ulcer size being
notably smaller in the SCE2 +NIR set. By day ve, the oral ulcers in the
SCE2 +NIR set had virtually healed.
Subsequently, on the fth day, samples of wounds from the oral re-
gion were collected for comprehensive histological and morphological
analysis. Fig. 8C reveals that the epidermis in both PBS and DEX groups
showed signs of necrosis and peeling, with signicant inltration of
inammatory cells. In contrast, the SCE2 set exhibited decreased
inltration of such cells along with a modest enhancement in broblast
proliferation. Furthermore, the SCE2 +NIR set displayed a fully intact
epithelial layer with barely any connective tissue presence in the sub-
mucosal area. Regarding Masson’s trichrome staining (Fig. 8D), the
SCE2 +NIR group displayed the most prominent collagen deposition,
characterized by dense, orderly bers. Immunohistochemical analysis
was then employed to assess mucosal inammation in each group. TNF-
α
expression was notably higher in both the SCE0 hydrogel and DEX sets,
comparatively lower in the SCE2 set, and least in the SCE2 +NIR set.
This pattern suggests that treatment with SCE2 +NIR effectively
reduced inammation in the ulcerated area, as shown in Fig. S29A of the
Supporting Information. Additionally, MPO staining for neutrophils
corroborated the minimal inammatory response in the SCE2 +NIR
group, as detailed in Fig. S29B in the Supporting Information.
Additionally, CD31 and VEGF stains were employed to assess new
blood vessel growth. Fig. 8E demonstrated a signicantly higher growth
of new vessels in the SCE2 +NIR compared to others (Fig. S30, Sup-
porting Information). Neovascularization plays a vital role in providing
nutrients and oxygen to wounds with high metabolic demands, thus
aiding in the development of granulation tissue. Similar to ndings in rat
back skin injuries, therapy with SCE2 +NIR demonstrated the least
expression of CD86 and the most pronounced expression of CD206
(Fig. S31, Supporting Information). As M1 macrophages decreased and
M2 macrophages increased, SCE2 hydrogel enhanced by NIR encour-
aged the production of proteins related to anti-inammatory and
angiogenic cells, leading to the accelerated healing of oral sores.
Ultimately, we examined the biosafety of the developed SCE2
hydrogel dressings, a crucial factor in determining their suitability for
wound care. We began by performing blood biochemical tests following
different treatments. The results showed no notable differences between
subjects treated with the SCE2 hydrogel and the control group, indi-
cating that the SCE2 hydrogel is biocompatible with blood (Fig. S32,
Supporting Information). Furthermore, H&E methods were employed to
assess the SCE2 hydrogel’s potential organ toxicity in rats. Relative to
healthy, untreated tissues, organs from rats that received hydrogel
treatment showed no signicant inltration of inammatory cells
(Fig. S33, Supporting Information). Such ndings suggest that the SCE2
hydrogel exhibits minimal biological toxicity toward healthy in vivo
tissues. Together, these ndings emphasize the SCE2 hydrogel’s favor-
able safety prole, making it a suitable candidate for clinical applica-
tions in treating oral wounds.
4. Conclusions
In summary, our research presents the approach of using SCE2
hydrogel for developing an immunomodulatory patch that attaches to
the oral mucosa. This patch is composed of methacrylate silk broin,
nitrobenzyl-modied chondroitin sulfate, and nanoparticles derived
from cuttlesh ink, designed to improve both the duration of adhesion
and healing efciency under wet conditions. As expected, exposure to
ultraviolet light leads to the formation of a sticky hydrogel layer over
mucosal injuries. Subsequently, the SCE2’s orderly release of bioactive
substances provides antibacterial, anti-inammatory, and antioxidative
benets, signicantly enhancing treatment outcomes for oral mucosal
defects. Additionally, the SCE2 patch is entirely absorbable by the body
and exhibits negligible toxicity after serving its intended purpose. Using
a streptozotocin-induced diabetic rat model, the SCE2 hydrogel’s ability
to facilitate the repair of dorsal skin injuries and oral ulcers was vali-
dated. We foresee that the SCE2 platform presented in this study will
advance the development of tissue adhesives and motivate the creation
of wound dressings using naturally derived bioactive substances.
Ethics approval and consent to participate
The research involving animals was conducted in strict compliance
with the guidelines and standards established by the Wenzhou Medical
Y. Xiang et al.
Bioactive Materials 39 (2024) 562–581
579
Fig. 8. Assessment of SCE2 hydrogel’s therapeutic efcacy on oral ulcer wounds in type 1 diabetic rats. (A) Diagram illustrating the creation and treatment approach
for oral ulcers. (B) Digital images showing oral ulcer healing in PBS, DEX, SCE2, and SCE2 +NIR sets throughout 1–5 days. (C–E) Stained sections of oral ulcer
wounds: H&E (C), Masson’s trichrome (D), and markers TNF-
α
, MPO, CD31, VEGF, CD86, and CD206 (E).
Y. Xiang et al.
Bioactive Materials 39 (2024) 562–581
580
University’s Institutional Animal Care and Use Committee, which
approved the authorization number Wydw7019-0134.
Declaration of interests statement
Jianliang Shen is an editorial board member for Bioactive Materials
and was not involved in the editorial review or the decision to publish
this article. All authors declare that there are no competing interests.
CRediT authorship contribution statement
Yajing Xiang: Writing – original draft, Software, Methodology,
Investigation, Data curation. Zhuge Pan: Formal analysis. Xiaoliang
Qi: Writing – review & editing, Conceptualization. XinXin Ge: Software.
Junbo Xiang: Methodology. Hangbin Xu: Data curation. Erya Cai:
Validation. Yulong Lan: Data curation. Xiaojing Chen: Data curation.
Ying Li: Software. Yizuo Shi: Validation. Jianliang Shen: Supervision.
Jinsong Liu: Visualization.
Acknowledgements
The nancial backing for this research comes from the National
Natural Science Foundation of China (82371016, 21977081,
82071170), the Zhejiang Provincial Natural Science Foundation for
Distinguished Young Scholar (LR23C100001), the Zhejiang Provincial
Science and Technology Project for Public Welfare (LGF21H140004),
the Natural Science Foundation of Zhejiang Province (LQ22E030011),
and the Wenzhou Municipal Science and Technology Project
(ZY2019009).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioactmat.2024.04.022.
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