Advancements in seawater immersion wound management: Current treatments and innovations

Abstract

With advancements in naval warfare, the number and severity of seawater injuries have skyrocketed, necessitating effective seawater immersion (SWI) wound management. The unique marine pathogens, salinity, low temperature and alkalinity of seawater are the main environmental factors that can influence SWI wound healing. The current treatment strategy for SWI wounds follows a standard protocol based on terrestrial wound conditions, neglecting seawater conditions. The key requirements for ideal SWI treatment include good adhesion to the wound surface to minimize further exposure to seawater, enhanced wound healing properties to minimize wound healing time and antibacterial properties to prevent infections from marine pathogens. Current SWI wound‐specific treatments range from elaborate techniques like vacuum‐sealed drainage and vacuum‐assisted closure for severe blast injuries to simple application of hydrogels or collagen dressings for minor injuries. This review discusses the current status and development of various treatment modalities for SWI wounds. The development of these treatment strategies and an understanding of their mechanisms of action make us better prepared to manage and treat SWI injuries.

1. INTRODUCTION

Present conflicts between developed and developing nations have raised concerns about the potential for future military engagements. Efficient war weapons and technology have become a top priority for all nations, as they strive to stay ahead in the ever‐evolving world of military advancements. The common battlefield for these wars tends to be oceans, which allow the use of advanced missiles, submarines and explosives. Treating the injuries caused by these weapons in a marine environment can be quite difficult and equally challenging. Factors like the absence of immediate medical help and delays in transportation, especially considering immersion in seawater (SW), add to the challenge. ¹ Due to the substantial differences between the conditions of seawater immersion (SWI) wounds and wounds on land, the conventional approach of treating burns, and blast injuries, is ineffective. ² The epidermal layer of skin becomes defective and more prone to injury with prolonged exposure to saltwater, triggering a higher rate of programmed cell death. ³ Given the impact of SWI on the epidermis, it is evident that these conditions can impede the wound repair process.

In general, the healing of wounds is influenced by both local factors, such as the wound's conditions, infection and oxygenation, and systemic factors, such as the individual's health status, age, stress and obesity. ⁴ The main distinction between terrestrial and SWI wounds is the environment to which they are subjected. Open wounds exposed to the variety of marine pathogens, salinity, low temperature and alkalinity of SW would significantly influence the host's response to injury ² (Figure 1). Thus, these environmental factors in SWI wound cause cellular and molecular changes that can influence the wound repair process (Figure 2).

FIGURE 1.

Different environmental factors affecting terrestrial and seawater immersion (SWI) wound repair.

FIGURE 2.

Comparison of the molecular and cellular environments of terrestrial acute wounds and seawater immersion wounds.

Marine pathogens are unique and rarely reported on land injuries; they are the major player regulating the healing of immersion injuries, for which standard methods of dressing and medication can be ineffective. Skin and soft tissue infections (SSTIs) after exposure of an injury to SW are reported to be due to the abundance of microbial flora in SW (>100 million per litre). These include pathogenic bacteria such as Vibrio species, Pseudomonas and Plesiomonas species, Staphylococcus and Streptococcus species, Escherichia coli and Mycobacterium marinum. ⁵ , ⁶ , ⁷ SW is also considered a reservoir of antibiotic‐resistant E. coli, which can worsen the severity of the wound infection. ⁸ M. marinum‐infected wounds slowly progress to form characteristic ulcerated granulotoma and are treated only with a prolonged course of antibiotics, increasing the risk of developing antibiotic resistance. ⁹ One of the highly virulent among them is Vibrio vulnificus, a halophilic bacterium that can have clinical manifestation ranging from wound infections and gastroenteritis to necrotizing infections and sepsis. ¹⁰ Diagnosis and treatment of these infections mainly depend on early bacterial identification, which is challenging as it requires specific culture media to support growth, followed by prescription of antibiotics and wound care.

Several cellular mechanisms are compromised in the SWI condition, leading to increased susceptibility to infection and delayed wound closure. One such mechanism is the coagulation pathway, which reduces the excessive blood loss. Zhang et al. ¹¹ observed that the effect of endothelial injury in the SWI condition can ultimately initiate the intrinsic coagulation pathway leading to disseminated intravascular coagulation (DIC). A similar coagulation dysfunction was also observed in a canine model with SWI burn injury, where the circulating endothelial cells were observed to be increasing along with the plasminogen activator inhibitor (PAI‐1) and von Willebrand factor (vWF) activity causing thrombosis. ¹² The effect of SWI after brain injury in a rat model showed mitochondrial dysfunction, a reduction in mitochondrial membrane potential (MMP) and reduced adenosine triphosphate levels along with neural stretch injury. ¹³ Inflammatory and oxidative stress responses were also noticed to be higher in the SWI condition compared to those in freshwater injuries. ² Regrettably, the effects of saltwater immersion on open injuries and delayed wound repair mechanisms have rarely been explored. Recently, various SWI pre‐clinical wound models have been developed to replicate different injuries like burn, blast and subcutaneous and full‐thickness wounds and to analyse the efficiency of advanced treatment modalities (Table 1). Given these conditions, it is evident that providing targeted treatment for SWI wounds is essential for efficient injury care. This review delves into the latest advancements in therapy modalities specifically tailored for SWI wounds, as well as their potential action mechanisms, aimed at improving our understanding and management of immersion wounds.

TABLE 1.

List of various pre‐clinical seawater immersion wound models.

Wound model	Animal	Procedure	References
Seawater‐immersed blast‐injury wounds (SIBIWs)	Miniature pig	Intramuscular anaesthesia was administered An electric detonator was implanted into a skin incision 3 cm long and 1.5 cm deep and triggered with a direct current to mimic a blast scenario The blast wound was immersed in seawater (10°C) for 1 h Complete debridement was done using saline	Shi et al. 14 and Ma et al. 15
Seawater‐immersed sulphur mustard injury (SWI‐MGI)	Miniature pig	Intramuscular anaesthesia was administered A region of 4 ×4 cm on either side of the pig spine was marked and exposed to 5 μL of sulphur mustard on each side for 5 h The wound was immersed in seawater (16°C) for 1 h Complete debridement was done using saline	Zhang et al. 16
Seawater‐immersed subcutaneous wound	Miniature pig	Intramuscular anaesthesia was administered A 4‐cm circular subcutaneous wound was created The wound was immersed in seawater (10°C) for 2 h, with the seawater replaced every 10 min	Cao et al. 17
	Rat	By using a hole puncher, four circular wounds (diameter of 0.6 cm) were created on the rat dorsum The wound was immersed in seawater (32°C) for 4 h	Shen et al. 18
Seawater‐immersed full‐thickness wound	Rat	Four circular excision wounds (diameter of 1.2 cm) on either side of the dorsal trunk were created The wound was immersed in seawater (28°C) for 1 h	Fang et al. 19 and Wang et al. 20

2. PRESENT TREATMENT APPROACHES

Considering the unique conditions of an SWI wound, numerous treatment modalities have been tested for their efficacy. The major features of these modalities include good adhesion to the surface to minimize further exposure to saltwater, faster repair capacity to reduce wound healing time and antibacterial properties to prevent marine pathogen infections. The selection of these modalities mainly depends on the wound type and severity.

2.1. Vacuum sealing drainage

Vacuum sealing drainage (VSD) is a treatment method employed for stimulating skin grafting and healing of both chronic and acute wounds. ²¹ However, its application in SWI wounds is less explored. Shi et al. investigated the application of this technique in SWI blast injuries on a mini pig model and reported a negative pressure of 120 mmHg to be optimum with the highest wound healing rate. Further VSD application was observed to increase macrophage count at the wound site, preventing excessive inflammation and upregulated expression of hyaluronic acid (HA) and its receptor CD44, which are the key components of the extracellular matrix (ECM). ¹⁴ A similar study by Yang et al. ²² focused on application of VSD on immersion blast injury confirmed −120 mmHg to be the ideal pressure and indicated the higher expression of the vascular endothelial growth factor (VEGF) and miRNA‐17‐5p, which are important in promoting angiogenesis.

Mustard gas was commonly used as a chemical weapon until its ban in recent years. A −180‐mmHg pressure exhibited the highest healing rate in treating SWI‐sulphur mustard injury in a pig model with a noticeable reduction in tumour necrosis factor α (TNF‐α) and interleukin (IL)‐6 and increased levels of VEGF reflecting the reduced inflammation in the wound site. ¹⁶ The current research status confirms VSD to be effective in treating SWI blast injury with −120 mmHg by reducing the wound healing time.

2.2. Vacuum‐assisted closure

Vacuum‐assisted closure (VAC) is often preferred in the case of treating chronic wounds like diabetic foot ulcers and thermal injury. In this technique, sterile foam is applied on the wound surface and sealed; to this, an evacuation tube is connected from which a negative pressure is created ranging from 50 to 125 mmHg. The benefits of the procedure include rapid wound contraction, reduced oedema, improved blood flow and decreased bacterial load. ²³

In vivo studies on a pig model were performed to see if the VAC approach could help with SWI wound repair. When treating subcutaneous SWI injury, researchers discovered that a negative pressure of 180 mmHg was optimal due to the highest fibronectin level, enhanced circulation and deposition of granulation tissue throughout the wound repair process. Further, they also considered the effect of treatment on the bacterial load, which was observed to be redistributing the wound flora from Gram‐negative to Gram‐positive bacteria, which are considered non‐pathogenic, thus reducing the risk of infection. ¹⁷ In contrast, for blast injury, a negative pressure of 120 mmHg was noted to be ideal as faster epithelial cell crawling and active proliferation of peri‐wound cells were observed. Although a rapid reduction in the bacterial index was observed in treatment group compared to that in the control, the bacterial distributions among the two groups were similar. In the early wound stage, the wound was mainly infected by Proteus vulgaris, an SW bacterium, but over a period of time, it was colonized by pig skin parasites, in both groups. Further immunohistochemical analysis of the treatment group (VAC‐120 mmHg) reported higher expression of type III collagen at the early wound healing period compared to that in the control, which is similar to the non‐scar‐healing process observed in embryonic skin. ¹⁵ It appears that both types of SWI wound models can be readily handled by VAC techniques by redistributing the wound flora and promoting wound contraction.

2.3. Human adipose‐derived stem cells

Adipose‐derived stem cells (ADSCs) are type of mesenchymal stem cells that have multiple applications in the field of stem cell therapy due to their capability of differentiating into multiple lineages and minimally invasive extraction procedure. ²⁴ Skin stem cells have the ability to differentiate into specific cell types from the basal layer to the epidermis in the case of deeper wound healing. ²⁵ To examine the efficiency of ADSCs on an SWI wound, nude mice were utilized as a full‐thickness skin defect model. A reduced healing rate was observed in the case of SWI wounds, which was significantly improved in the treatment group with ADSCs (SW + human ADSC (hADSC) group). In vitro assays were performed, confirming the proliferation potential of ADSCs at a low concentration (1.75%) and enhanced migration in comparison to that in the control. The expression of vital proteins like Ki67 and CK19 that are often reduced in the case of SWI was noted to be significantly increased in the presence of hADSCs. Further, the mechanism of action of hADSCs was known to be by regulating epidermal growth factor receptor (EGFR) levels, which in turn influence the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MEK/ERK) pathway in wound repair. ²⁶ Recently, the mechanism behind the proangiogenic effect of hADSCs utilized in SWI wounds was studied based on the differentially expressed genes (DEGs) responsible for the effect using bioinformatic tools. Among 4642 upregulated genes, DCN was prominent, whereas in 5541 downregulated genes, LCN 2 was observed to be significantly reduced. The upregulated DEGs had direct association with angiogenesis, especially in ECM modelling, while the downregulated DEGs were linked to regulation of the cell cycle, known to reduce angiogenesis in tumour; however, in the context of wound healing, their role is yet to be confirmed. Further protein–protein interaction (PPI) network analysis of DEGs identified hub genes that were related at high nodes and their correlation was studied. Most of upregulated hub genes were positively correlated with necroptosis, ferroptosis markers and anti‐inflammatory activities, and most of downregulated hub genes were positively correlated with the necroptosis marker of caspase‐8 and pyroptosis markers but not with anti‐inflammatory markers. Further, the effect of SW and hADSCs on human umbilical vein endothelial cells (HUVECs) was investigated by coculturing, which indicated improved proliferation efficiency when cocultured with hADSCs in simulated SW conditions. ²⁷ These insights on the mechanism of action promise hADSCs' potential in treating SWI wounds.

2.4. Collagen wound dressings

Collagen, being a vital component of the ECM, has been proven to enhance wound repair by promoting cell proliferation in cases of chronic wounds. ²⁸ Even though animal‐derived collagen is not preferred due to risk of infection and religious reasons, fish collagen being readily available and a byproduct of seafood processing adds value to the industry. Shark skin collagen sponge (SSCS) is made from the freeze‐drying process of collagen from Prionace glauca, blue sharks, and can be developed into an anti‐SWI dressing by adding polyurethane (PU) film. The physiochemical properties of SSCS include high porosity and good tensile strength and water vapour transmission ratio (WVTR), making this an ideal candidate for SWI wound dressings. The efficacy of this dressing sponge was tested on rats with subcutaneous wounds under SWI conditions, which reported that SSCS alone enhanced the healing rate, with increased CD31 and transforming growth factor β (TGF‐β) expression during the initial stage of wound healing, thereby speeding up the healing process in comparison to chitosan dressings. Meanwhile, SSCS + PU, along with promoting wound healing, was capable of shielding wound from SW exposure for a minimum of 4 h. ¹⁸ Further, to enhance collagen's effectiveness in wound healing, a variety of collagen‐containing systems are also being developed. The composite dressing of chitosan–collagen–alginate provides benefits like biocompatibility, antibacterial and film‐forming properties. To make it suitable for SWI, a PU membrane was added. The effectiveness of this composite dressing was proven by an enhanced wound healing rate, promoting TGF‐β, CD31, fibroblast growth factor (FGF) and basic FGF (bFGF) expression in the early stage of wound healing; low cytotoxicity; and good hemocompatibility along with minimized wound exposure to SW for about 4 h. ²⁹ Both of these studies ¹⁸ , ²⁹ confirm the utility of the combination of collagen and PU membrane in minimizing wound exposure to SW and promoting wound repair.

2.5. Hydrogels

One of the key features of an ideal SWI wound dressing is the ability to adhere to wet surfaces, thereby blocking further contact with water to reduce the risk of infections and to enhance wound healing. This is achievable by using specialized hydrogels, with the wet adhesion value eight times greater than that of fibrin glues. OD/EPL@Fe hydrogel is made of catechol‐modified oxidized HA (OD), ε‐poly‐l‐lysine (EPL) and Fe³⁺, where the lysine residues present can dehydrate the adhesion site, ensuring strong contact. A notable antibacterial effect against Gram‐negative SW bacteria (V. vulnificus and Pseudomonas aeruginosa) was observed as hydrogel components like the amino group of EPL and Fe³⁺ can directly interact with the bacterial membrane and the catechol–Fe³⁺ interaction provides a photothermal effect, which leads to bacterial death. An interesting feature of this includes on‐demand removal of the hydrogel after spraying with deferoxamine mesylate (DFO), a chelating agent for maintaining the hygiene of the wound site. ³⁰

A composite of hydrogel can be beneficial as it combines the benefits of HA, which provides high water retention and biocompatibility, and quaternized chitosan (QCS), which provides antimicrobial activity with good solubility. The composite hydrogel appears promising as it presents good mechanical, antimicrobial and wound healing properties. To test the efficiency of the hydrogel, in vitro studies were conducted on L929 fibroblasts and in vivo studies on an SWI rat wound model. The results reported the good biocompatibility, antimicrobial property and accelerated wound healing effect of the hydrogel. Further immunohistochemical studies observed lower pro‐inflammatory factors (TNF‐α, IL‐1β and IL‐6) and higher anti‐inflammatory factors (TGF‐β1) ideal for rapid wound healing. ¹⁹

The extensive use of antibiotics has led to multi‐drug‐resistant strains of V. vulnificus in SWI wounds that are much difficult to treat; one of the current solutions is to utilize natural predatory bacteria that can kill the resistant strains. Bdellovibrio bacteriovorus acts as a live antibiotic that produces a lipopolysaccharide with neutral lipid A, which can kill infested cells. This B. bacteriovorus‐loaded hydrogel (BG) is a topical formulation with a pore size of 90 mm that releases a live antibiotic when in contact with aqueous solution over a period of 2 h, which specifically targets the Vibrio species, while the host immune system can easily scavenge these bacteria, which limits its penetration into tissues. Apart from Vibrio species, it can be effective against E. coli, P. aeruginosa and meropenem‐resistant P. aeruginosa; thus, it can significantly reduce the dependence on the high antibiotic medications. In vivo experimental studies on an SWI mice wound model reported evenly distributed collagen fibres, reduced pro‐inflammatory cytokines (TNF‐α and IL‐6), enhanced endothelial marker CD31 expression and reduced macrophage maturation and activation marker, which suggest reduced inflammatory response and a better healing trajectory. ³¹

2.6. Composite irrigation solution

The presence of bacterial load after wound dressing can cause infections, hindering wound healing. Thus, apart from proper dressing, sterilization of the open wound with appropriate irrigation solution is vital in faster wound repair. General irrigation solutions might not be effective in fighting against marine pathogens like Vibrio parahaemolyticus, P. aeruginosa and Vibrio alginolyticus. Thus, a composite irrigation solution (polyhexamethylene biguanide, levofloxacin, chitosan, glacial acetic acid solution) was designed specifically to treat SWI wounds. The antimicrobial efficiency of the solution was tested in vitro, observing complete eradication of the microbial species within 12 h of treatment with the irrigation solution, while an in vivo study on rat full‐thickness wounds presented an average skin irritation response score of 0 mainly due to the low‐irritant reagents used in the solution along with a better wound healing rate. Further histological evaluation showed good neovascularization and neat arrangement of collagen fibres with an intact epidermis compared to that in the control and saline‐treated group. ³²

2.7. Antibiotic‐loaded chitosan gelatin scaffolds

The lack of an antibacterial effect with the current wound dressings has been addressed by using composite scaffolds made up of gelatin microspheres loaded with the fungicide ciprofloxacin hydrochloride (CIP) on chitosan and gelatin interlinked by a genipin matrix; this design ensures the sustained release of the antimicrobial drug and its bioavailability. The composite dressings showed excellent water absorption rates, good stability leading to less frequent changing of wound dressing, satisfactory biocompatibility and antimicrobial activity. In vivo studies on an SWI full‐thickness wound model revealed better wound healing with less scar formation in the treatment group, along with a reduction in serum endotoxins at the wound site, higher collagen production and upregulated expression of anti‐inflammatory factors like TGF‐β1 and downregulated pro‐inflammatory factors such as TNF‐α, IL‐6 and IL‐1β. Thus, along with facilitating wound healing, these scaffolds prevent the risk of bacterial infection of the open wound. ²⁰

3. CONCLUSION

The current status of the treatment modalities for SWI wounds considers the different environmental conditions observed in these cases (Figure 3; Table 2). Each of the treatment modalities caters to the unique conditions of SWI wounds and can be selected based on severity and wound type. VAC and vacuum‐assisted drainage can be better suited for wounds with a larger surface area for wound contraction. hADSCs are also better options for treating slow‐healing wounds using an tissue engineering approach. Collagen and hydrogel‐based treatment provide instant SWI wound‐specific dressing options that not only heal but also prevent further exposure to SW. Composite irrigation solution can be utilized before applying general dressing to prevent residual bacteria on the wound surface. Antibiotic‐loaded chitosan–gelatin scaffolds prevent the bacterial infection that is caused by exposure to marine bacteria. Although most of the experiments were conducted in an SWI setup to recreate SWI conditions, a few of them failed to consider components like lack of SW microorganisms. ²⁶ One of the major limitations of this review is the inclusion of only available English/English‐translated articles as there were many articles in the Chinese language whose translated versions were not available to include in the study. The current treatment modalities appear to be progressing towards addressing all the unique conditions of SWI wounds like marine infections and prolonged SW exposure, with consideration of the convenience of the treatment like self‐administrable patches or elaborate techniques for severe injuries like blast injuries. Further understanding of the interaction between the components of SW and the wound will allow development and design of more sophisticated approaches. Implementing a combination treatment approach will enable cumulating the beneficial features of each technique; for example, combination of the antimicrobial effect and wound healing capacity of different techniques will tackle both of the conditions efficiently, thus providing better treatment outcome.

FIGURE 3.

Current treatment modalities for seawater immersion wound management.

TABLE 2.

Summary of studies on different treatment modalities for seawater immersion (SWI) wounds.

Sl. no.	Treatment type	Wound type	Animal model	Seawater immersion condition	Microbial load observed	Biomarkers assessed and result	Limitations	References
1	Vacuum sealing drainage	Blast injury (electronic detonator)	Mini pig	10°C seawater for 1 h	Not considered (intramuscular penicillin administered)	Upregulated CD44 and hyaluronic acid (HA) expression	Lack of consideration of marine microbial infection	Shi et al. 14
		Blast injury	Miniature pig	10°C seawater for 1 h	Not considered	Upregulated miR‐17‐5p and VEGF		Yang et al. 22
		Sulphur mustard injury	Pig	Seawater for 1 h	Not considered	Upregulated VEGF Reduced IL‐6 and TNF‐α expression		Zhang et al. 16
2	Vacuum‐assisted closure	Subcutaneous wound (diameter of 4 cm)	Miniature pig	10°C seawater for 2 h	Gram‐positive Staphylococcus xylosus and Staphylococcus saccharolyticus Gram‐negative Pseudomonas alcaligenes and Pseudomonas aeruginosa	Upregulated fibronectin synthesis	Low sample size (n = 4 and 5) Limited surface area in the miniature pig model	Cao et al. 17
		Blast injury	Pig	Seawater for 1 h	Proteus vulgaris, Escherichia coli, Pseudomonas fluorescens, Klebsiella oxytoca and Vibrio parahaemolyticus	Upregulated type III collagen and reduced type I collagen in the early wound healing stage Higher expression of TNF‐α in the early wound healing stage		Ma et al. 15
3	Collagen wound dressing	Subcutaneous wound (diameter of 0.6 cm)	Rat	32°C seawater for 4 h	Not considered	Upregulated TGF‐β and CD31 expression	Lack of consideration of marine microbial infection	Shen et al. 18
		Full‐thickness wound (diameter of 0.8 cm)	Rat	28°C seawater for 4 h	Not considered	Upregulated TGF‐β, CD31, FGF and bFGF expression		Xie et al. 29
4	Adipose‐derived stem cells (ADSCs)	Full‐thickness wound (diameter of 0.8 cm)	Mice	Seawater for 1 h	Not considered	Upregulated Ki67, CK19 and EGF	Lack of consideration of marine microbial infection	Xiong et al. 26
5	Hydrogel	Full‐thickness wound (diameter of 1 cm)	Rat	20°C seawater for 1 h	P. aeruginosa and Vibrio vulnificus	Upregulated CD31, VEGF, collagen I and IL‐10 expression Reduced IL‐6 and TNF‐α expression		Lv et al. 30
		Full‐thickness wound (diameter of 1 cm)	Rat	28°C seawater for 1 h	E. coli, Staphylococcus aureus and P. aeruginosa	Upregulated TGF‐β1 expression Reduction in TNF‐α, IL‐1β and IL‐6		Wang et al. 19
		Full‐thickness wound (diameter of 1 cm)	Mice	Seawater‐soaked sterile gauze for 1 h	V. vulnificus	Upregulated CD31 expression Reduced TNF‐α and IL‐6 levels and F4/80 marker (macrophage activation marker)		Liu et al. 31
6	Composite irrigation solution	Full‐thickness wounds (diameter of 1 cm)	Rat	‐	S. aureus, E. coli, V. parahaemolyticus, Vibrio alginolyticus, P. aeruginosa and Candida albicans	Uniform and even distributed collagen fibres	Lack of saline conditions maintained as the bacterial suspension was directly inoculated	Wang et al. 32
7	Antibiotic‐loaded chitosan–gelatin scaffolds	Full‐thickness wound (diameter of 1.2 cm)	Rat	28°C seawater for 1 h	E. coli, S. aureus and P. aeruginosa	Upregulated TGF‐β1 expression Downregulated pro‐inflammatory factors (TNF‐α, IL‐6 and IL‐1β)		Fang et al. 20

Abbreviations: bFGF, basic FGF; FGF, fibroblast growth factor; TNF‐α, tumour necrosis factor α; TGF‐β, transforming growth factor β; VEGF, vascular endothelial growth factor.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interests.

Rao D, Kumar P, Prabhu V. Advancements in seawater immersion wound management: Current treatments and innovations. Int Wound J. 2024;21(10):e70070. doi: 10.1111/iwj.70070

DATA AVAILABILITY STATEMENT

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.