Accelerating
the remodeling of collagen in cutaneous full-thickness wound using FIR
soldering technology with bio-targeting nanocomposites hydrogel
Yuxin Chena*, Kehong Wanga*,
Xiaopeng Lia, Mengyin Chena , Kexin
Heb, Jun Huang, Yunfeng Ruic
a Nanjing University of Science and Technology
b Nanjing Medical University
c Nanjing Southeast University
*Corresponding authors: Kehong Wang
(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 [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 [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[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 [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[11-13]. Gels are typically formed through
physical or chemical processes that rapidly construct a
three-dimensional network using precursor or monomer materials[14-15]. The mechanism of crosslinking
solidification directly affects both the stability of hydrogels and the
state of active substances incorporated within them[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[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 [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[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 [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[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·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