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\begin{document}

\title{Accelerating the remodeling of collagen in cutaneous full-thickness
wound using FIR soldering technology with bio-targeting nanocomposites
hydrogel}



\author[1]{Kehong Wang}%
\author[1]{Yuxin Chen}%
\author[1]{Xiaopeng Li}%
\author[1]{Mengyin Chen}%
\author[2]{Kexin He}%
\author[1]{Jun Huang}%
\author[3]{Yunfeng Rui}%
\affil[1]{Nanjing University of Science and Technology}%
\affil[2]{Nanjing Medical University}%
\affil[3]{Southeast University Medical College}%


\vspace{-1em}



  
  \date{March 14, 2025}


\begingroup
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\selectlanguage{english}
\begin{abstract}
A novel composite wound dressing hydrogel by incorporating single-walled
carbon nanotubes and indocyanine green into a dual-crosslinked hydrogel
through Schiff base reaction was developed. The objective was to prevent
wound infection and enhance the thermal effect induced by laser energy.
The hydrogel matrix was constructed using oxidized gelatin,
pre-crosslinked with calcium ions, along with carboxymethyl chitosan,
crosslinked via Schiff base reaction. Optimization of the blank
hydrogel's gelation time, swelling index, degradation rate, and
mechanical properties was achieved by adding 0.1\% SWCNT and 0.1\% ICG.
Among them, the SWCNT-loaded hydrogel BCG-SWCNT exhibited superior
performance overall: a gelation time of 102 seconds; a swelling index
above 30 after equilibrium swelling; a degradation rate of 100.5\% on
the seventh day; and a compressive modulus of 8.8 KPa. It displayed
significant inhibition against methicillin-resistant Staphylococcus
aureus infection in wounds. When combined with laser energy usage, the
composite hydrogel demonstrated excellent pro-healing activity in rats.%
\end{abstract}%



\sloppy


Accelerating the remodeling of collagen in cutaneous full-thickness
wound using FIR soldering technology with bio-targeting nanocomposites
hydrogel

Yuxin Chen\textsuperscript{a*}, Kehong Wang\textsuperscript{a*},
Xiaopeng Li\textsuperscript{a}, Mengyin Chen\textsuperscript{a} , Kexin
He\textsuperscript{b}, Jun Huang, Yunfeng Rui\textsuperscript{c}

a Nanjing University of Science and Technology

b Nanjing Medical University

c Nanjing Southeast University

*Corresponding authors: Kehong Wang (\emph{wkh1602@126.com), Yuxin} Chen
(chenyuxin1602@njust.edu.cn)

Abstract: A novel composite wound dressing hydrogel by incorporating
single-walled carbon nanotubes and indocyanine green into a
dual-crosslinked hydrogel through Schiff base reaction was developed.
The objective was to prevent wound infection and enhance the thermal
effect induced by laser energy. The hydrogel matrix was constructed
using oxidized gelatin, pre-crosslinked with calcium ions, along with
carboxymethyl chitosan, crosslinked via Schiff base reaction.
Optimization of the blank hydrogel's gelation time, swelling index,
degradation rate, and mechanical properties was achieved by adding 0.1\%
SWCNT and 0.1\% ICG. Among them, the SWCNT-loaded hydrogel BCG-SWCNT
exhibited superior performance overall: a gelation time of 102 seconds;
a swelling index above 30 after equilibrium swelling; a degradation rate
of 100.5\% on the seventh day; and a compressive modulus of 8.8 KPa. It
displayed significant inhibition against methicillin-resistant
Staphylococcus aureus infection in wounds. When combined with laser
energy usage, the composite hydrogel demonstrated excellent pro-healing
activity in rats.

Keywords: Adhesive hydrogel, Antibacterial ability, Wound healing,
Carboxymethyl chitosan, SWCNTs

Introduction

Collagen makes up 70-80\% of the skin's dry weight, serving as its main
component and giving it strength. During the healing process of skin
wounds, various types of collagens (I, III, V, VII, and XVII) play
important roles. Type I collagen can promote re-epithelialization by
stimulating keratinocyte migration and enhance matrix remodeling by
increasing the expression of matrix metalloproteinases in keratinocytes.
Additionally, type I collagen has immunomodulatory properties that
contribute positively to wound healing. A study published in Nature by
Karin suggests that a three-dimensional environment rich in high-density
fibers of type I collagen can induce immune suppressive functions in M2
macrophages, which may have beneficial effects on diabetic foot ulcer
(DFU) wound healing. However, insufficient deposition of type I collagen
within hypertrophic scar tissue fails to provide adequate
immunomodulation for proper regulation of wound healing. Previous
studies have shown that low-level laser therapy effectively stimulates
fibroblast proliferation within specific energy ranges and promotes
production of both type I and III collagens; however, laser treatment
often fails to regulate the ratio between these two types of collagens
leading to tissue fibrosis directly associated with excessive secretion
of collagen.

In the realm of nanomaterials, there has been a considerable amount of
attention directed towards carbon nanotubes (CNTs), owing to their
extensive potential in the field of biomedicine and biotechnology. These
remarkable structures have shown promise in delivering bioactive
substances such as medications, proteins, and nucleic acids;
facilitating targeted therapy for tumors; and enabling biological
imaging. The immense potential of carbon nanotubes stems from their
inherent mechanical, optical, and electrical properties due to their
small size, large surface area, low density, and high stability. One of
the most appealing advantages of carbon nanotubes is their ability to
effectively penetrate biological barriers and even enter the cell
nucleus. However, the application of carbon nanotubes has been severely
limited by their super hydrophobicity and tendency to aggregate in
aqueous media. Previous studies have shown that collagen/single-walled
carbon nanotube composites hold great practical value as scaffolds in
tissue engineering. Collagen has also been used to stabilize silver
nanoparticles in water. Furthermore, functionalizing type I collagen
with single-walled carbon nanotubes (SWCNTs) allows for good dispersion
of SWCNTs in aqueous solutions, making collagen-functionalized SWCNTs
suitable for biomedical and biotechnological applications. Additionally,
research has indicated that under 808 nm laser irradiation, indocyanine
green (ICG) generates a photothermal effect that enables the destruction
of tumor cells by damaging bacterial cells and neighboring cells.

Wound coverings are a form of biomaterial utilized for the purpose of
concealing wounds, ulcers, or other forms of injuries. There exist
primarily three categories of wound dressings developed to facilitate
wound healing \textsuperscript{{[}1-3{]}}. Conventional passive
dressings serve the purpose of passively enveloping the wound, absorbing
exudate, and offering limited safeguarding to the injury. On the other
hand, active dressings engage with the wound in an interactive manner by
effectively absorbing exudate and harmful substances while establishing
an optimal therapeutic environment for gas exchange. The outer layer
structure of the barrier serves as a protective shield, safeguarding the
wound against microbial invasion from its surroundings. This effectively
prevents any contamination and transmission of harmful microorganisms to
the wound. Occlusive dressings not only act as a physical barrier
shielding the wound from external elements but also create an optimal
moist environment that promotes continuous tissue regeneration
throughout the healing process. Moreover, it is crucial for dressings to
adhere appropriately to the wound without causing any secondary harm or
damage \textsuperscript{{[}4{]}}. Recently, there has been extensive
research on the utilization of biodegradable hydrogel molecules in the
pharmaceutical and biomedical fields due to their remarkable properties
including high swelling capacity, biocompatibility, and small
volume\textsuperscript{{[}5-9{]}}. Hydrogels derived from aqueous gels
offer several advantages such as creating a moist environment,
exhibiting excellent biocompatibility, effectively absorbing wound
exudate while minimizing adhesion to injured tissues
\textsuperscript{{[}10{]}}. In addition, the utilization of hydrogel
dressings containing antimicrobial medications can effectively prevent
infection and secondary damage during dressing changes by achieving
sustained release effects. It is important to emphasize that careful
design is necessary for stable hydrogels equipped with outstanding
compressive characteristics so as to reduce potential damage coming from
external forces\textsuperscript{{[}11-13{]}}. Gels are typically formed
through physical or chemical processes that rapidly construct a
three-dimensional network using precursor or monomer
materials\textsuperscript{{[}14-15{]}}. The mechanism of crosslinking
solidification directly affects both the stability of hydrogels and the
state of active substances incorporated within
them\textsuperscript{{[}16{]}}.

Extracellular polysaccharide produced through aerobic fermentation of
Sphingomonas/ Pseudomonas bacteria facilitates covalent crosslinking. It
comprises four distinct monosaccharides as repeating units, including
two d-glucose carbohydrates, one l-rhamnose, and one d-glucuronic
acid\textsuperscript{{[}26-28{]}}. GG has found extensive application in
the formulation of wound dressings, prevention of scar formation, and
inhibition of postoperative adhesions. For example, when GG was combined
with cinnamic acid ester, it resulted in the creation of a
photo-crosslinked polymer known as GG/cinnamate. This novel material
exhibited promising potential for preventing adhesion formation when
tested on rats \textsuperscript{{[}20,29,30{]}}. Numerous investigations
have highlighted the exceptional mechanical properties and wound
dressing capabilities of GG hydrogels. Hydrogel formulations can be
prepared by crosslinking GG using calcium or magnesium
ions\textsuperscript{{[}31-33{]}}. However, the limited stability of
gellan gum in physiological fluids due to monovalent cations hampers its
potential biomedical applications like cell encapsulation. This is
because the exchange of divalent cations leads to a loss of mechanical
stability \textsuperscript{{[}26,34{]}}. To address these issues such as
inadequate mechanical strength, poor physiological stability, and high
gelation temperature, one possible solution is to utilize the oxidation
conversion of cis-diols on GG chains into reactive aldehydes. These
aldehydes can then react with amino groups to form Schiff's base
linkages or ionic crosslinks. For instance, chitosan-GG (OG)
crosslinking can be achieved by reacting the abundant amine groups on
chitosan chains with oxidized aldehyde groups on oxidized gellan gum
(OG), resulting in stable hydrogel formation. Carboxymethyl chitosan
(CMCS), which has improved solubility compared to
chitosan\textsuperscript{{[}15{]}}, is commonly used for constructing
biomedically applicable hydrogels.

In this study, we present the synthesis of biodegradable composite
hydrogels containing microspheres. These hydrogels are formed through
the crosslinking of carboxymethyl chitosan (CMCS) and oxidized cold-set
gelatin (OG) using Schiff's base as a mediator. To enhance their
long-term antibacterial properties, we incorporated single-walled carbon
nanotubes (SWCNTs) and indocyanine green (ICG) nanoparticles, which
possess photothermal effects with antibacterial activity. We conducted
comprehensive investigations to optimize the mechanical properties,
tissue adhesion, cytotoxicity, skin irritation, and in vivo wound
healing performance of these nanoparticle-composite hydrogels for
potential use as wound dressings. Furthermore, we examined the healing
efficacy of these hydrogels on skin tissue wounds under low-energy laser
stimulation.

Materials and methods

Materials

The Gellen adhesive (GG) was obtained from Shanghai Macklin Biochemical
Co., Ltd. It has a high molecular weight of 500 kDa and an acetylation
degree exceeding 90\%. Carboxymethyl chitosan (CMCS), with a
carboxymethylation degree surpassing 80\% and exhibiting a viscosity
range of 60-1000 mPa\selectlanguage{ngerman}·s, was sourced from Shanghai Traditional Chinese
Medicine Chemical Reagent Co., Ltd. Bovine serum albumin (BSA) was
acquired from Nanjing Sunshine Biotechnology Co., Ltd. The single-walled
carbon nanotubes referred to as SWCNTs were directly obtained for this
study from Sigma-Aldrich Company in the United States. These SWCNTs have
diameters ranging between 0.7 to 1.3 nm and boast a purity level above
90\%. Indocyanine green (ICG), originating from Shanghai Macklin
Biochemical Co., Ltd., has an approximate molecular weight of 774.96
Daltons.

Laser soldering system

Based on extensive prior research, we have developed a comprehensive
welding system that can meet all the necessary requirements during laser
welding processes, as shown in Figure 1. The skin tissue connection
machine used in this study consists of three components: a laser
operational system, a thermograph, and an overall control system. The
laser operational system includes consecutive fiber lasers with a
wavelength of 1064nm Nd:YAG, along with a workbench and relevant
clamping devices. The thermograph comprises a Fortic near-infrared
thermal imager and its corresponding control subsystem. At the same
time, the overall control system encompasses the temperature output data
processing subsystems of both the laser control system and the thermal
imager. By referring to previous experiments, we have determined
optimized fundamental parameters for the laser such as power level,
scanning path, defocus amount, wavelength selection, and scanning speed
{[}27-28{]}. Through thorough analysis of both macroscopic and
microscopic aspects of skin tissue after performing laser welding
procedures while considering factors like body temperature and thickness
in rats' subjects; we further improved these parameters to achieve
optimal results as presented in Table 1.

Fig.1 Laser soldering system and Ultra-clean operating table

Table.1 Experimental parameters\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
No. & Defocus amount & Power/w & Speed/mm. s\textsuperscript{-1} &
Scanning mode \textsuperscript{{[}26{]}} & Energy
density/J.mm\textsuperscript{-2} & Incident angle/\selectlanguage{ngerman}°\tabularnewline
\midrule
\endhead
1 & -1 & 7 & 100 & Point & 67.543 & 90\tabularnewline
2 & & & & & & 60\tabularnewline
3 & & & & & & 30\tabularnewline
\bottomrule
\end{longtable}

Preparation of BCG-SWCNT and BCG-ICG hydrogel

Initially, oxidized GG (OG) was synthesized by following a slightly
modified procedure \textsuperscript{{[}28{]}}. A solution of 3 g GG in
300 mL distilled water was heated to 90 \selectlanguage{ngerman}°C. Subsequently, with
continuous stirring at room temperature for 24 hours in the absence of
light, 1.5 g NaIO4 was added. To conclude the oxidation reaction, 2 mL
ethylene glycol was introduced after a duration of 30 minutes. The
resulting solution underwent purification through dialysis (MWCO14,000)
against distilled water for a period of three days to eliminate excess
NaIO4 and subsequently lyophilized at -50 °C. The degree of oxidation
determined via iodometric titration amounted to approximately 9.7\%. In
the subsequent step, hydrogel formation involved preparing an aqueous
solution containing OG at a concentration of 35 mg/mL under constant
stirring for half an hour.

The solution was supplemented with CaCl\textsubscript{2} at a
concentration of 1mg/mL. Subsequently, a hydrogel was formed by
thoroughly mixing a CMCS aqueous solution (40 mg/mL) and OG solution in
varying volume ratios at 37 °C overnight. In order to achieve a
homogeneous solution for the preparation of SWCNT and ICG embedded
hydrogel, the pH was adjusted to 6.7. Following a waiting period of
15-30 minutes, the prepared gel received additions of SWCNT homogeneous
dispersant (10mg/mL) and ICG (10mg/ml), resulting in what is referred to
as BCG-SWCNT and BCG-ICG respectively. To ensure sterility during the
fabrication process of the hydrogel, all materials were subjected to UV
or autoclave sterilization procedures within an environment that adhered
to sterile conditions (tissue culture hood).

Morphologies and mechanical properties

The structures of GMs, Gel and GMs/Gel scaffolds were analyzed through
SEM imaging. The samples were subjected to freeze-drying at -50 °C for
24 hours before being coated with gold using a Cressington 108 Auto
(Cressington, Watford UK) for a duration of 90 seconds. A Hitachi SU8010
SEM (Hitachi, Japan) was utilized to observe the morphologies under an
accelerating voltage of 3 kV.

Mechanical properties/Compressive test

The hydrogels were prepared into dimensions of 40 mm ×10 mm × 1 mm and
subjected to tensile testing using a universal testing machine
(CMT-1104, SUST, China) at a crosshead speed of 100 mm/min until
failure. Each group was tested with a minimum of three hydrogel samples.
The Young's modulus was determined by analyzing the stress-strain curve
within a strain range from 0 to 10\%. To assess cyclic tensile
properties, silicone oil was applied on the hydrogel samples to prevent
water loss. The same testing conditions as mentioned earlier were
employed, but with a maximum strain of 100\%. For compression testing,
cylindrical-shaped hydrogel samples measuring 15 mm in diameter and 2 mm
in thickness were compressed at room temperature using the universal
testing machine at a rate of 10\% strain per minute.

Swelling behaviors

For the swelling experiments, the BCG-SWCNT and BCG-ICG hydrogels were
immersed in PBS (pH 7.4) at a temperature of 37 °C for a duration of 1
hour until they reached equilibrium swelling. The swollen samples were
then periodically removed and weighed (W\textsubscript{t}) after
eliminating any surface adsorbed water using filter paper. To determine
the hydrogel weight, lyophilization was performed at -50 °C followed by
weighing (W\textsubscript{d}). The swelling ratio was calculated using
the following equation:

\begin{equation}
S_{R}=\frac{W_{t}-W_{d}}{W_{d}}\nonumber \\
 \end{equation}

where S is the swelling ratio, and W\textsubscript{t} and
W\textsubscript{d} are the weight of the hydrogel before and after
swelling, respectively.

Rheological properties

Rotational rheometer (MCR308, China) equipped with a 2.5cm flat plate
and a 1 mm gap was used to evaluate rheological properties. A
temporal-scan study took place in a consistent strain level at a
magnitude of 1.0\% with a frequency of 1 cycle per second under ambient
temperature conditions of 25. The combination of the two precursor
solutions occurred through the utilization of a dual-barreled syringe,
followed by injection onto the parallel plate while simultaneously
monitoring the evolution of storage modulus (G' ) and loss modulus (G''
) over a period

Ex vivo adhesion properties

The hydrogels underwent a transformation administered onto the exterior
of pig hides, and rotational force was exerted at the boundary of the
dermis. To assess the sticky substance properties of the hydrogels, a
patch adhesion strength test was conducted. In summary, cuboidal shapes
measuring 20 \selectlanguage{ngerman}× 10 × 4 mm\textsuperscript{3} were cut from porcine skins.
Subsequently, a solution containing BCG (50 \selectlanguage{greek}μ\selectlanguage{english}L) was injected onto those
back skins of pigs. Then, another back skin with either SWCNT or ICG
dispersion liquid (50 \selectlanguage{greek}μ\selectlanguage{english}L) was put on top of the BCG-treated skin
(adhering area: 1*1 cm\textsuperscript{2}). After a period of 30
minutes, a universal testing machine at a temperature of 25 pulled
apart the overlapped skins using a cross-head speed set at 60mm/h until
they separated.

In vitro biodegradation

Before conducting the research, the hydrogels had undergone certain
preparations, weighed and then fully engrossed in a 5 mL solution of
lysozyme (1000 U/mL) in PBS (pH = 7.4) at 37 with continuous stirring
at a speed of 100 rpm for a period of 4 weeks. The solution used for
incubation was refreshed biweekly. The hydrogels were rinsed with ddH2O
and weighed on days 1, 3, 5, 7,14,21 and 28. Equation (3) was utilized
to determine the percentage of degradation.

\(Degradation=\frac{W_{t}}{W_{0}}\times 100\%\) (3)

where W\textsubscript{t} represents the weight at each time interval
after a certain period, and W\textsubscript{0} denotes the initial
weight of the samples

Antibacterial evaluation

\emph{The bacterial strains S. aureus (ATCC25923), E. coli (ATCC25922),
and MRSA (ATCC43300) were grown for an extended period of time at 37 in
a Luria-Bertani medium under constant agitation.} The optical density of
the bacterial suspensions was determined at a wavelength of 600 nm using
a microplate reader, with an OD600 value of 0.1 corresponding to a
concentration of approximately 1 \selectlanguage{ngerman}× 108 colony-forming units per
milliliter. Cylinder-shaped hydrogel samples measuring diameter=10 mm
and height=2 mm was placed into individual wells within a multi-well
plate, followed by incubation with diluted bacterial suspensions
containing approximately106 colony-forming units per milliliter for a
duration of12 hours at37 \selectlanguage{english}. Following the completion of the12-hour
incubation period, the bacterial suspension underwent a thousand-fold
dilution before spreading 100\selectlanguage{greek}μ\selectlanguage{english}L of this diluted solution onto agar
plates. Subsequent cultivation for24 hoursat37  allowed for observation
and analysis of result in bacterial colonies, in order to assess the
antibacterial efficacy of the tested hydrogels.

In-vivo wound healing evaluation

The SD rats were obtained from Changzhou Covens Laboratory Animal co.,
LTD and acclimated to their new surroundings for a duration of one week.
Only male rats weighing between 180-220g were selected for the study,
and they were housed in a specific pathogen-free (SPF) animal facility
with a light-dark cycle of 12 hours each. All surgical procedures
adhered to the approved animal care protocols by the Ethics Committee of
Nanjing Medical University.

The experimental groups consisted of 45 rats, which were assigned
randomly and distributed among three test indexes at four different time
points. Additionally, each time point (3, 7, 14, and 24 days) included
three distinct groups (BCG-con with n = 5, BCG-SWCNT with n = 5, and
BCG-ICG with n = 5), along with one control rat in each group.

After anaesthetization (1.5--3 vol.\% Isoflurane), the dorsal surface
hairs were shaved and full-thickness wounds measuring 12 mm in diameter
were induced on the dorsal area of the diabetic mouse, and subsequently
covered with hydrogel disks of varying dimensions (9 mm in diameter and
1 mm thick). Then we use the 1064nm laser to scanning the wounds in the
0 day and 3 days of wounds modeling respectively. For the control group,
the wound site was left without any treatment. The wound healing rate
was calculated using the following equation:

\(Healing\ Percentage(\%)=\left(\frac{\text{WA}_{0}-\text{WA}_{t}}{\text{WA}_{0}}\right)\times 100\)\% (1)

W\textsubscript{A0} represents the initial wound area on day of injury
(day 0), while W\textsubscript{At} denotes the subsequent wound areas on
corresponding days

Statical analysis

The data displays the averaged quantities along with their associated
statistical uncertainty, represented as average \selectlanguage{ngerman}± variability. A p-value
lower than 0.05 signifies a statistically meaningful differentiation.

Results and discussion

Fabrication and characterization of hydrogels

The composite hydrogels were formed through the process of crosslinking
CMCS with pre-crosslinked OG containing calcium ions, as illustrated in
the schematic diagrams (Fig. 2(a)). The gelation primarily occurs
through a Schiff-base reaction involving the amino groups in CMCS and
aldehyde groups in OG (Fig. 2(b)). The aldehyde group was formed by
oxidizing the cis-o-diol present in GG with sodium periodate, resulting
in an estimated 9.7\% degree of oxidation determined by iodometric
titration. Throughout the entire reaction process, prior to that, OG
underwent physical crosslinking treatment with Ca\textsuperscript{2+}.
The temperature was maintained below T\textsubscript{gelation} to
facilitate the formation of a double helix conformation for
OG\textsuperscript{{[}40{]}}. After being combined with CMCS, GG
undergoes ionic crosslinking and reinforcement of chemical crosslinking
is achieved by the formation of Schiff base, resulting in the
establishment of a dual network structure. \textsuperscript{{[}28{]}}.
The presence of -CHO facilitates the crosslinking between CMCS and OG,
enabling gel formation. To fabricate drug-loaded composite gel scaffolds
(Fig.2(c)), various drugs such as BSA, SWCNT, and ICG are dissolved or
dispersed in the GG solution at specific concentrations. These three
composite hydrogels contain different pigment groups that can regulate
the laser's energy absorptivity. As a result, the laser treatment on
wounds can be modified and varied under these hydrogels, as depicted in
fig.2(d).

Figure.2. Preparation and characterization of the hydrogels. (a)
Representation of the molecular structure of Gellan Gum (GG), Gellan gum
with oxidation (OG), and chitosan carboxymethylated (CMCS).
(b)(c)Schematic illustration of the preparation of BCG-Con/SWCNT/ICG
hydrogels through Schiff base reaction. (d) Schematic illustration of
BCG-Con/SWCNT/ICG hydrogels in the treatment of wounds in conjunction
with the 1064nm consecutive low energy laser. (e)(f)(g) The hydrogel
morphologies were examined using SEM, with scale bars measuring 250 \selectlanguage{greek}μ\selectlanguage{english}m,
50 \selectlanguage{greek}μ\selectlanguage{english}m, and 10 \selectlanguage{greek}μ\selectlanguage{english}m correspondingly. (h) Pore size, (i) swelling ratio,
(j)density, and (k) Thermal Gravimetric Analysis of the hydrogels. Data
are presented as mean \selectlanguage{ngerman}± SD (\emph{n} = 3); *\emph{p \textless{}} 0.05,
**\emph{p \textless{}} 0.01, ***\emph{p \textless{}} 0.001.

The SEM images in Fig. 2 e-g display the lyophilized hydrogels created
using different types of nanoparticles derived from CMCS and OG
composite solution. It is evident that the hydrogels formed by BSA,
CMCS, and OG possess an uninterrupted porous structure with three
dimensions, which is a result of ice crystals during freeze-drying. The
existence of this three-dimensional porous structure enhances the uptake
of interstitial fluid and blood, thereby encouraging beneficial
hemostasis.\textsuperscript{{[}34{]}}. It can be observed that the
hydrogels created using CMCS and OG possessed a continuous porous
structure in three dimensions, which was formed through the freezing
process involving ice crystals. The microstructure of the three
different hydrogels prepared with various core components did not
exhibit any significant disparities. The walls of the pores exhibited a
consistent texture, while the voids displayed a compact appearance.
These porous formations promote cellular nourishment and oxygen
provision during the process of wound healing. High-resolution SEM
images demonstrated a slight reduction in pore size for the hydrogels in
the following order: BCG-Con, BCG-ICG, and BCG-SWCNT (as depicted in
Fig.2(h)). It is important to note that there was an increase in surface
roughness observed for BCG-SWCNTs (inset of Fig.2.(g)), potentially
enhancing RBC adhesion and blood clot
formation\textsuperscript{{[}35{]}}.

The OG/CMCS composite gel was utilized to encapsulate the SWCNTs due to
their negative surface charges, which enable them to form an
electrostatic bond with the cationic OG polymer. As a result, the
BCG-SWCNTs hydrogels exhibited reduced swelling ratios and increased
density (as shown in Fig.2. (i), (j)).

Furthermore, the thermal stability of hydrogels was investigated using
thermal gravimetric analysis (TGA), as depicted in Figure.2(h). The
weight reduction observed in BCG-SWCNTs within the temperature range of
450-550°C can be attributed to the dihydroxylation process occurring in
NHCH2-COOH groups present in its structure. Notably, when compared to
pure BCG hydrogel, it was found that incorporating SWCNTs into the
hydrogel resulted in a decreased final weight loss. This observation
suggests that SWCNTs have a positive impact on enhancing the thermal
stability of the hydrogel.

Gelation time, biocompatibility, and biodegradation of the hydrogels

The solidification time of hydrogels plays a crucial role in the
biomedical field when it comes to hemostasis applications. The time at
which gelation occurs can impact how well the hydrogel can be injected
during clinical use. Rapid gelation is not ideal for removing air
bubbles from the hydrogel, as these bubbles can create flaws in the
scaffolds and affect overall performance. In our experiments with gels,
we noticed a notable rise in the quantity of amine groups relative to
aldehyde groups within OG as the proportionate mass of CMCS escalated.

The disparity in the quantity of amino groups and aldehyde groups led to
an increased gap, resulting in a longer duration for gel formation. To
investigate the gelation time of BCG-Con/SWCNT/ICG hydrogels, we
separately loaded CMCS/OG and BSA/SWCNT/ICG solutions into three cap
tubes each (as shown in the inset of Fig. 2(a)). The time at which
gelation occurs was then documented after mixing these solutions
together in their respective cap tubes. Throughout this study, the
gelation time for all three volume ratios remained consistent at
approximately 90-120s. Figure .2(e) illustrates that BCG-Con exhibited
the shortest gelation time, taking only 86s, while BCG-SWCNTs had the
longest duration at 120s±15s.

In addition, we performed a study on the bonding capacity of the
hydrogel utilizing swine dermis which exhibits physical attributes of
living organisms akin to those seen within the dermal layer of
humans.\textsuperscript{{[}36{]}}. The BCG-SWCNTs/ICG hydrogels were
applied onto the outer layer of the porcine dermis. Skin was subjected
to torsional strain; it was observed that the hydrogels adhered firmly
and did not detach (inset of Fig. 2(b)(c)(d)). To investigate the
adhesive characteristics, shear lap testing was conducted using a
universal testing machine (inset of Fig. 2(f)(g)). The strength of
adhesion values for the BCG-Con, BCG-SWCNTs, and BCG-ICG hydrogels were
measured as 58.69±6.11KPa, 55.62±4.89KPa, and 49.78±5.61KPa
respectively. The strong adherence between the skin surface and
BCG-SWCNTs/ICG hydrogels can be ascribed to interactions involving
covalent bonds and hydrogen bonding that occur due to chemical reactions
between specific components in both materials\textsuperscript{{[}37{]}}.
Specifically, the hydrogels consist of ODEX that contains a high
concentration of --CHO groups. These groups have the ability to react
with --NH\textsubscript{2} groups in porcine skin and form a covalent
bond known as --C=N. Additionally, there is hydrogen bonding between
--CONH and --OH groups present in porcine skin, as well as -OH (from
ICG) and -CHO (from OG) groups within the structure of the hydrogel.
Therefore, by incorporating an appropriate amount of SWCNTs and ICG into
hydrogels, their adhesive properties can be enhanced.

Good hydrogels should degrade once or before wound closure has been
achieved. Our work analyzed the degradation properties of the hydrogels
in vitro by observing their weight reduce when exposed to a solution
containing lysozyme. (as shown in the inset of Figure 2(h)). It was
observed that all the hydrogels experienced relatively rapid initial
degradation within 7 to 8 days, the weights of these entities experience
a gradual reduction. The BCG-SWCNTs hydrogel exhibited approximately
95\% degradation after 4 weeks, while the BCG-ICG and BCG-Con hydrogels
displayed a slower rate of degradation compared to the BCG-SWCNTs
variant. An in vivo biodegradation study involving subcutaneous
implantation into rat models demonstrated a similar trend: around
80\%--90\% of hydrogel degradation occurred within 2 weeks
post-implantation (as depicted in the inset of Figure 2(i)). Our
findings regarding the degradation behavior align with prior research,
this is for the susceptibility of Schiff base bond to
hydrolysis\textsuperscript{{[}38{]}}.

Figure.3. Characteristics related to gel formation, bonding ability, and
breakdown properties were investigated for these hydrogel samples.
(a)The capillary tubes were initially filled with solutions containing
CMCS/OG and BSA/SWCNT/ICG before undergoing gelation processes. The
adhesive behavior was evaluated by subjecting these hydrogels to
torsional stress when applied onto porcine skin surfaces as shown in
figures (b), (c), (d) respectively. The time required for complete gel
formation was also measured as depicted in figure \textbackslash{}euro.
A schematic diagram illustrating how adhesion strength was tested is
provided in figure f while figure g presents data on adhesion strength
under torsional stress conditions on skin surfaces. Degradation studies
were conducted both in vitro(h)and in vivo(i). Experimental results are
expressed as mean values ± standard deviation based on three
replicates(n=3).*Statistical significance at p\textless{}0 .05.

Mechanical properties

Angular frequency scanning experiments can be utilized in rheological
measurements to investigate the network structure stability of hydrogels
when subjected to cyclic shear forces. The G' values for BCG-Con,
BCG-SWCNT, and BCG-ICG reach a stable state at approximately 1750 Pa,
2260 Pa, and 2110 Pa respectively. Both BCG-Con and BCG-ICG gels display
fluctuations in G'' at higher frequencies. As depicted in Figure .4.(a),
the incorporation of SWCNT into the composite hydrogel enhances its G'
value and ensures stability across a frequency range of 0.01
\textasciitilde{} 100 Hz, suggesting that the composite hydrogel
exhibits robust elasticity. Specifically, the G' value for BCG-SWCNT gel
remains consistently high at 2630 Pa, while the G'' value for BCG-ICG
gel exhibits fluctuations that are not conducive to maintaining
elasticity in the composite hydrogel. Moreover, throughout this study,
G' consistently surpasses G'' by one order of magnitude, indicating
exceptional elastic performance of the composite hydrogel that plays a
crucial role in promoting wound healing.

The gel's compressive performance was evaluated at 37°C using a
universal mechanical testing system equipped with a 500 N load cell. The
specimen underwent compression without any restrictions at a speed of 1
mm per minute until it reached the point of yielding. The compression
characteristic curve of the hydrogel is illustrated in Figure 4. (b)(c).
In BCG hydrogel, an elevated proportion of CMCS leads to a gradual
reduction in the compression modulus, suggesting that hydrogels
fabricated with a greater abundance of amine and aldehyde groups
demonstrate enhanced resistance against compression. The incorporation
of SWCNTs significantly enhances the mechanical characteristics of the
composite hydrogel. However, excessive quantities may result in
system-wide aggregation, leading to complete collapse under certain
stress levels {[}42{]}. Notably, the BCG-ICG composite hydrogel exhibits
remarkable antioxidant properties and achieves an optimal compressive
performance with a compression modulus measuring 19.3 KPa.

As depicted in figure 4 (d), Fourier transform infrared spectroscopy was
employed in this study to examine the alterations in functional groups
within hydrogels. A peak is observed at 1732 cm\textsuperscript{-1},
indicating the occurrence of oxidation in the -CHO bond during gelation.
The presence of peaks at 1630 cm\textsuperscript{-1} for BCG-SWCNT and
BCG-ICG hydrogels confirms the Schiff base reaction between -CHO and
-NH\textsubscript{2}. Additionally, a peak at 3622
cm\textsuperscript{-1} corresponds to the -OH bond following CMCS and OG
reactions. Moreover, there is a shift in the stretching vibration of -OH
from 3622 cm\textsuperscript{-1} to 3615 cm\textsuperscript{-1}, which
can be attributed to the conversion of -OH present in BCG into both -OH
and -COOH within BCG-ICG/SWCNT samples.

In Figure 4 (e), X-ray diffraction patterns were acquired using a Rigaku
X-ray diffractometer from Japan, covering the range of 10°-80°. The
diffraction peaks observed at angles of 11.9°, 20°, and 25° corresponded
to the crystallographic planes (001), (020,110), and (020) of HNTs
respectively. These distinctive peaks exhibited a gradual increase in
intensity when transitioning from BCG-ICG to BCG-SWCNT to BCG-Con
samples, indicating uniform dispersion and loading of ICG and SWCNTs
onto CMCS/OG hydrogels.

In vitro antibacterial ability of the hydrogels

Hydrogels are anticipated to facilitate wound repair following
hemostasis. To assess the in vitro wound healing efficacy of the
hydrogels, we conducted a wound scratch assay. The control groups
consisted of BCG-Con dressings containing BSA and CMCS, as well as OG.
As depicted in Figure 5(a), the BCG-SWCNT groups exhibited the highest
rate of cell migration at 67.25\% among all tested groups. Subsequently,
RT-qPCR analysis was performed to investigate the expression levels of
genes associated with wound healing in HUVEC cells. CD31 and VEGF
represent typical angiogenesis genes involved in the process of wound
healing \textsuperscript{{[}39{]}}.

As depicted in Figure. 5(b), the expression levels of CD31 and VEGF in
HUVECs were significantly increased by BCG-SWCNT/ICG, particularly
BCG-SWCNT, compared to BCG-ICG and BCG-Con groups. Likewise, both Cola1
and Col3a1 levels were notably elevated in HUVECs treated with BCG-ICG.
Notably, among all groups, BCG-SWCNT exhibited the highest COL1a1
expression while displaying the lowest level of COL3a1. It is worth
mentioning that although COL1 plays a crucial role in wound healing, the
combination of high COL1a1 expression along with low COL3a1 expression
may contribute to enhanced fibrosis around the wound area. These
findings suggest that utilizing hydrogels composed of BCG-SWCNT/ICG
could stimulate vessel fibrillar collagen formation and facilitate
tissue regeneration through upregulation of genes related to
vascularization and collagen production.

Figure.5. (a) Live/dead staining was used to visualize cell migration
and healing rate of HUVECs treated with different hydrogels after 24
hours. Representative fluorescence images were captured, with a scale
bar of 250 \selectlanguage{greek}μ\selectlanguage{english}m. (b) The levels of CD31, VEGF, Col1a1, and Col3a1 genes
related to wound healing were analyzed in HUVECs cocultured with the
hydrogels. (c) Hydrogel treatments were tested for their ability to
inhibit the growth of S. aureus, E. coli, and MRSA over time using
dynamic growth curves. A clinically used BCG-Con wound dressing served
as a positive control group. (d) After 12 hours of treatment, bacterial
colonies were photographed for analysis. Results are presented as mean \selectlanguage{ngerman}±
SD (n = 3); *p \textless{} 0.05, **p \textless{} 0.01, ***p \textless{}
0.001.

The hydrogels were tested for their antibacterial properties against S.
aureus, E. coli, and MRSA. Initially, the number of bacteria was
monitored by measuring the OD values at 600 nm in bacterial suspensions.
Interestingly, all the hydrogels exhibited comparable antibacterial
activity to the BCG group when it came to S. aureus and E. coli (see
Fig. 5(c) inset). Additionally, we observed that BCG-SWCNT hydrogels
displayed a more potent inhibitory effect on MRSA compared to BCG-ICG.
To provide a visual representation of this observation, agar plates were
used to culture bacterial suspensions after incubation for 12 hours (see
Fig. 5(c) inset).

Based on the growth curve of bacteria, both the BCG hydrogels and
BCG-SWCNT/ICG groups effectively eliminated nearly all bacteria.
However, the effectiveness of BCG-ICG in inhibiting MRSA growth was
comparatively lower. The antibacterial properties of the hydrogels are
attributed to CMCS and OG. CMCS has the ability to engage in
interactions with teichoic acid and lipopolysaccharide present on the
surface of bacteria \textsuperscript{{[}40{]}}. Additionally, OG's -CHO
groups have the potential to undergo a reaction with
-NH\textsubscript{2} groups found in the wall of a bacterial
cell\textsuperscript{{[}41{]}}, resulting in damage to the bacterial
plasma membrane. These in vitro findings collectively suggest that
BCG-SWCNT hydrogels not only facilitate wound healing but also impede
bacterial proliferation.

In vivo wound healing and antibacterial properties

To assess the effectiveness of hydrogel dressings in promoting wound
healing and combating bacterial infection, we created a model of
infected wounds by removing full-thickness cutaneous wounds and exposing
them to MRSA. Subsequently, hydrogel dressings were applied above the
wound area along with laser scanning at 1064nm and 6.97J/cm2 on day 0
and day 3 after infection (as shown in the inset of Figure 6(a)). Images
depicting the wounds observed during a 14-day period are presented in
Figure 6(b). On day 3, serious infection and secretion were observed in
both the BCG-Con group and BCG-SWCNTs group. Additionally, the BCG group
exhibited only a wound contraction rate of 29.13\%, while it was
slightly higher at 32.02\% for the BCG-ICG group (as indicated in the
inset of Figure 6(c)(d)). Notably, compared to the control group and
BCG-ICG group, significant acceleration in wound healing was observed
with the use of BCG-SWCNTs hydrogels. These findings regarding in vivo
wound healing outcomes aligned well with our earlier observations from
an in vitro study.

On the 7\textsuperscript{th} day, the BCG-SWCNTs group exhibited the
most effective therapeutic outcome with over 50\% healing of wounds.
Remarkably, after 13 days of treatment, these wounds showed nearly
complete healing (99.28\%). By day 14, the wounds treated with BCG-ICG
hydrogel also demonstrated significant improvement in healing, while the
BCG-Con group achieved a healing rate of 95.48\%.

Figure.6. The therapeutic impact of hydrogels on in vivo MRSA-infected
wounds was evaluated through an experimental setup illustrated
schematically in Figure (a). Full-thickness skin injuries were inflicted
and subsequently exposed to MRSA bacteria for a period of twenty-four
hours before being treated with hydrogel injections followed by exposure
to a laser emitting at a wavelength of 1064nm directly above the wounded
area. Photographic evidence showcasing various treatment outcomes is
displayed in Figure (b) across multiple time points including days zero
through fourteen. The dynamic healing ratios for distinct treatment
groups are depicted graphically in Figures (c) and (d) at intervals
spanning from day four today fourteen post-treatment initiation

Histological examination of the skin samples involved staining with H\&E
and Masson's trichrome after treatment (inset of Fig. 7). The results
from H\&E staining revealed a significant presence of inflammatory cells
infiltrating the control groups even after 14 days of infection, as
indicated by the black dotted box. Additionally, both the control and
BCG-ICG groups still exhibited skin defects, suggesting that bacterial
infection hindered wound healing progress. In contrast, complete repair
was observed in wounds treated with BCG-SWCNTs hydrogels without any
signs of inflammation. This confirms the effective inhibition of
bacterial infection and promotion of wound healing by the hydrogel.
Furthermore, histological analysis demonstrated that wounds in the
BCG-SWCNTs group displayed similar characteristics to adjacent normal
tissues.

Masson's staining revealed that the BCG-SWCNTs hydrogel groups exhibited
enhanced blue staining areas, indicating a significant increase in
collagen deposition within treated wounds. This suggests that the
hydrogels possess great potential as reliable and effective materials
for promoting healing in infected wounds.

Figure.7. H\&E and Masson's trichrome staining were performed on wound
tissues 14 days after various treatments. Inflammatory tissues are
indicated by blue dotted circles. The right panel displays magnified
views of the areas outlined by black dotted boxes in the left panel. The
scale bars represent measurements of 500\selectlanguage{greek}μ\selectlanguage{english}m and 200\selectlanguage{greek}μ\selectlanguage{english}m, respectively.

Conclusions

To conclude, we have achieved successful development of a versatile
hydrogel consisting of BCG-SWCNTs that exhibits improved adhesion,
antibacterial properties, and potential for wound healing. Our findings
demonstrate that the hydrogels can undergo in situ crosslinking via the
Schiff base reaction to rapidly cover wounds and prevent further
infection. Additionally, these hydrogels exhibit excellent
biodegradability both in laboratory settings and in living organisms.
Moreover, when combined with 1064nm laser scanning, the BCG-SWCNTs
hydrogels promote wound healing while effectively inhibiting bacterial
infections caused by highly pathogenic MRSA. These results suggest that
our hydrogel formulation holds promise as an emergency trauma management
solution and as an efficient gel for accelerating wound healing with
laser assistance.

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