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