Hyaluronic Acid-Based Wound Dressing with Antimicrobial Properties for Wound Healing Application

Abstract

Wound healing is a naturally occurring process that can be aided by a wound dressing properly designed to assure an efficient healing process. An infection caused by several microorganisms could interfere with this process, delaying or even impairing wound healing. Hyaluronic acid (HA), a main constituent of the extracellular matrix (ECM) of a vertebrate’s connective tissue, represents a promising biomaterial for wound dressing thanks to its intrinsic biocompatibility, hydrophilicity and bacteriostatic properties. In this review, different and recent types of HA-based wound dressings endowed with intrinsic antimicrobial properties or co-adjuvated by antimicrobial natural or synthetic agents are highlighted.

1. Introduction

Skin represents a primary defence barrier of our body, providing protection against a microorganism’s infiltration and dehydration [1]. A wound is defined as an injury of living tissue that causes a disruption in the continuity of the epithelial lining of the skin or mucosa resulting from a generically external damage (i.e., physical or thermal, typically one in which the skin is cut or broken). Additionally, wounds can be classified as acute following a skin injury that occurs suddenly due to an accident or surgical injury with healing in 8–12 weeks, while chronic wounds occur when they fail to progress through normal stages of healing and cannot be repaired in an orderly and timely manner (e.g., pressure ulcer, leg ulcer, diabetic ulcer and burns). Wound healing is a normal biological process achieved through four precise and highly programmed phases involving haemostasis, inflammation, proliferation and remodelling [2]. During haemostasis, vascular constriction and platelet aggregation, degranulation, and fibrin formation (thrombus) occur. Subsequently, the inflammation event allows neutrophil and monocyte infiltration, the last one differentiating into macrophage. The proliferation phase consists of the re-epithelialization, with cell migration, followed by the angiogenesis and collagen synthesis with new extracellular matrix (ECM) formation. Finally, the remodelling foresees the collagen remodelling and, subsequently, the vascular maturation and regression [3]. Many factors exist that could interfere with one or more of these phases, delaying or even impairing the wound healing process. Among these, infection is seen as one of the most pervasive causes in wound healing failure. If skin protection is compromised, bacteria can colonize the wound, possibly leading to a critical state of interference with the healing process, also called infection. The most common bacteria found in wound chronic infections, and therefore often taken into account to test antibacterial properties, are S. aureus, P. aeruginosa, Enterococcus faecalis and others. The role of infection is then notoriously determined by its ability to delay wound healing by extending the duration of the inflammatory response [4] and worsening tissue damage. Some studies have also attested a role of such bacterial cultures in causing failure in skin re-epithelisation [5]. It follows that an antimicrobial activity is an essential requirement for an appropriate wound dressing. Wound dressing may be defined as a sterile dressing applied to a wound or incision using an aseptic technique with or without medication. Such a dressing must possess certain characteristics in order to be highly effective. An optimal wound dressing has to control the moisture around the wound, possess great transmission of gases, eliminate extra exudates, protect the wound from infections and microorganisms, decrease surface necrosis of the wound, carry out mechanical protection, be easily changed and removed, be biocompatible, biodegradable, elastic and nontoxic, relive the wound pain and, eventually, be cost acceptable [6]. Different synthetic and natural polymers, such as poly(caprolactone) (PCL), poly(vinyl alcohol) (PVA), collagen (COL), chitosan (CS), alginate (ALG) and hyaluronic acid (HA), have been used for the production of different wound dressings [7].

Among natural polymers, HA is ubiquitous in connective, epithelial and neural tissue, and is a non-sulphated, linear polysaccharide consisting of alternate N-acetyl-D-glucosamine and D-glucuronic acid linked by β(1→3) and β(1→4) glycoside bonds, belonging to the glycosaminoglycans (GAGs) family [8,9,10] (Figure 1). HA and its derivatives offer long-term safety and a proven ability to reduce bacterial adhesion and biofilm formation. HA is bacteriostatic but not bactericidal, and exhibits dose-dependent effects on different microorganisms in the planktonic phase [11]. HA is widely used in wound dressing applications under different morphologies from films [12] and hydrogels [13,14] to fibres [15,16] and non-woven fabrics [17] and foams [18,19] (Scheme 1). HA-based hydrogel wound dressings stand out due to their ability to be a suitable solution for all four stages of wound healing, as well as being non-irritant and non-reactive with biological tissue and permeable to metabolites. Especially used in ophthalmological applications, HA film-like dressings show desirable properties as they permit the transmission of water vapour, O₂ and CO₂. Moreover, they are thin and elastic, flexible and transparent, and are used generally in combination with hydrogels. However, films are usually not suitable for highly exudating wounds. Being originally used to decrease the severity of abdominal adhesions, by being placed between injured tissues, HA films found fortune as tear film substitutions as a potential remedy for wounds associated with dry eye syndrome [20]. On many occasions, wound healing has to be enhanced through foam-like structures in order to allow an easier spread and make sure little residue is left, making them easily applicable for all body areas, including hair-bearing areas. Generally, HA films compose a hydrophobic outer layer protecting the wound from liquid but allowing gaseous exchange and water vapour. Foam wound dressing characteristics also include the absorption of wound drainage. In this review, an overview of the different HA-based wound dressings having antimicrobial activity for wound healing is provided. In particular, focusing attention to the antimicrobial activity obtained from the bacteriostatic effect of the HA itself or from the incorporation of synthetic or natural antimicrobial agents in the HA-based wound dressing.

2. HA Antimicrobial Properties

As well as serving as a barrier to protect wound tissues from external bacterial infections, substrates for wound dressing should possess an inherent antimicrobial ability to make them even more attractive, in order to prevent wound infections and promote an effective healing process [20]. The ability of HA to inhibit microbial attack during wound healing is associated with its proven ability to reduce bacterial adhesion and biofilm formation in different contexts; not being bactericidal but bacteriostatic, it delays or prevents the growth of bacteria without destroying them [21].

By comparing different biomaterials, both synthetic and natural with bacteriostatic properties, such as collagen, hydroxyapatite, HA and PLGA, it has been proved that HA shows the greatest bacteriostatic effect against S. epidermidis and P. aeruginosa. Therefore, it has been hypothesized that the HA molecules also form a random network of chains that can act as a sieve preventing the spread of bacteria [7]. In particular, a study about the bacteriostatic properties of three different concentrations (0.5, 1.0 and 2.0 mg/mL) of solution of HA at different molecular weights (MW), low (L), medium (M) and high (H) (respectively, 141, 757 and 1300 kDa) concluded that higher MWHA showed better effects in minimizing bacterial contamination when tested on oral and non-oral microorganisms in the planktonic phase [22]. When compared to PLGA, Collagen I and Hydroxyapatite, HA showed the most significant bacteriostatic properties on P. Aeruginosa, S. aureus, S. epidermidis and β-hemolytic Streptococcus [7].

HA/carbon nanofibers composite films have been produced with enhanced mechanical properties. The antimicrobial activity of the HA derivatives was tested against Staphylococcus aureus, a Gram-positive bacterium, and results showed a good antibacterial effect (Figure 2) [11].

A bio-resorbable membrane made of HA and carboxymethylcellulose has been designed. A cellular function investigation has shown how higher concentrations of HA/CMC induced greater bacteriostatic effects on S. aureus and E. coli. Thus, they concluded that HA/CMC might have dose-dependent bacteriostatic effects on S. aureus and E. coli [23].

A Gantrez^®S97/HA crosslinked hydrogel was fabricated and characterized. Synthesized hydrogels showed a significant antimicrobial activity against planktonic bacterial suspensions, indicating they are an ideal substrate for drug delivery devices or biomaterials, such as medicated wound dressings or anti-infective coating [24].

Then, two possible explanations can be considered to justify HA’s bacteriostatic effect. First, due to the macromolecular nature of HA, the presence of long chains give rise to a high viscosity solution in which bacteria’s motion and diffusion is hindered: a proof is that HMWHA-concentrated solutions showed potent bacteriostatic activity. Then, a second possible mechanism acts synergistically with the previous mechanism, so the excess of HA might cause the saturation of the bacterial hyaluronate lyase. Therefore, bacteria seem to lose tissue penetration capability, allowing an efficient immune response by the host [7,25].

To further improve HA’s inherent antibacterial activity, in the literature several modification methods have been proposed. As an example, polyurethane films were coated with a previously modified HA with thiol groups (HA-SH), using polydopamine as the binding agent. Ocatdecyl acrylate (C18) was subsequently used to bind thiol groups in order to attract albumin, allowing the system to selectively bind albumin, a protein responsible for bacterial adhesion, thus granting an effective antimicrobial activity. In order to make sure of this assessment, the antimicrobial susceptibility of the engineered film was tested against S. aureus by evaluating its adhesion. Results showed how the highest amount of adhered bacteria was detected where no C18 was present [26].

Moreover, recently, greater attention has been delivered to amine group-related modifications, being tunable functional groups endowed with strong antimicrobial properties. Among o amine-based polymers, particular care has been given to polyethyleneimine (PEI): polyanionic poly(hyaluronic acid) (p(HA)) hydrogel particles were combined with polycationic-modified PEI with different ratios to provide controlled antimicrobial properties. The antimicrobial activity of p(HA)-PEI particles containing different amounts of PEI was tested against E. coli, S. aureus and B. subtilis using the broth microdilution method [27].

An innovative class of chemically modified biomaterials make use of antimicrobial peptides (AMP) to carry out antimicrobial activity, overcoming problems related to toxicity or multi-resisting pathogens. An antimicrobial biopolymer was fabricated covalently grafting nisin, a hydrophobic cationic peptide composed of 34 amino acids, on carboxylic-activated HA. Antimicrobial activity was then tested against S. epidermidis, S. aureus and P. aeruginosa [28].

3. HA-Based Devices Combining Synthetic Antimicrobial Agent

HA possesses antimicrobials effective against different Gram-positive and Gram-negative bacteria [7,11,23]. However, such properties can be increased using antimicrobial agents. Among synthetic substances, the use of silver nanoparticles (AgNPs) is pervasive in the literature as they can be obtained quite easily, in numerous ways, which do not involve the use of potential toxic media.

The effect of AgNPs on wound healing and antimicrobial effects once seeded in H MW HA (HA4) was reported (Table 1). Briefly, HA solutions were prepared by the dissolution of different molecular weights of HA in Milli-Q water at room temperature with continuous stirring; to produce AgNPs-enriched solutions, 1 mL of 10 mM of silver nitrate was added dropwise. When compared to the samples at L MW HA (HA1), M MW HA (HA3) and H MW (HA2) without nanoparticles, HA4 showed a reduction in wound size, improving granulation and inflammatory mediators in impaired older and diabetic wound healing. In particular, as regards the inhibition of pathogenic bacteria, both HA4 and HA2 were found to significantly arrest the number of pathogenic bacteria on wounds throughout the first 24 h in comparison to the control nontreated rats [29] (Figure 3).

A clinical trial involving patients with a vascular ulcer confirmed the safety and wound healing activity of a topical spray powder containing HA sodium salt 0.2% and AgNPs 2.0%, an EC-certified class III medical device. Results showed that such a spray powder is effective not only in reducing wound area, due to the presence of HA, but in keeping the bacterial colonisation under control, showing a reduction in bacterial load (p < 0.025) [30] (Table 1).

Thermosensitive and injectable hydrogels based on HA, corn silk extract (CSE) and AgNPs were prepared and their potential use as a wound care material was investigated. Briefly, hydrogels were fabricated by the addition of HA to a blend of Pluronics F127 and F68 in CSE to obtain concentration 1% w/w, while Ag NPs were biosynthesized by a microwave-assisted green technique using CSE in an organic solvent-free medium. Hydrogels exhibited antibacterial activity against Gram-positive (B. subtilis, S. aureus) and Gram-negative (P. aeruginosa, E. coli) bacteria. A wound healing in vitro assay revealed that the hydrogels allow faster wound closure and repair compared to the controls consisting of the single biomaterials (Table 1) [13].

Moreover, in the literature the use of antibiotics coupled with bio-engineered scaffolds has been widely studied. An example are thin films made up of blended collagen, chitosan and HA fabricated by solvent evaporation after the addition of gentamicin sulphate, a common antibiotic for bacterial infections. In more detail, the HA solution (1% w/w in 0.1 M hydrochloric acid) was added in different ratios to chitosan and collagen mixtures (1% w/w in 0.1 M acetic acid). The antimicrobial activity of such films was tested against S. aureus (Gram-positive bacteria), E. coli and P. aeruginosa (Gram-negative bacteria) by using the diffusion method. Results showed a better efficiency for the samples in which gentamicin sulphate was released from polymeric matrixes, with inhibition zones of approximately 25–30 mm (Table 1) [31].

4. HA-Based Devices Combining Natural Antimicrobial Agents

Among natural substances acting as antimicrobial agents, antimicrobial peptides (AMP) have recently been appreciated as a potential new class of antimicrobial drugs, being expected to solve the problem of antibiotic drug resistance. They exert their activity in a quite unique way, permeabilizing bacteria cell membranes: pores or structural defects are formed, even at low concentrations. A specific AMP called Tet213 combined with substrates of alginate, HA and collagen showed how such substrates’ dressings exhibited antimicrobial activity against E. coli, MRSA and S. aureus, inhibiting or killing bacteria in infected wounds. Briefly, a solution of three polymers was prepared in deionised water with concentrations being 1.2%, 0.5%, and 0.6% (w/v), respectively, and then Tet213 was added. Thus, samples were lyophilized to obtain the wound dressing. Therefore, those wound dressings granted an effective healing, enhancing collagen deposition and improving angiogenesis thanks to the fast release of AMP [32] (

Table 1. Different types of HA-based devices combining both synthetic and natural antimicrobial agents with proven antimicrobial activity.

Antimicrobial Agent Combination	Type of HA Wound Dressing	Tested Pathogens	Reference
Silver Nanoparticles (AgNPs)	Wound healing ability of different MW of hyaluronan; in vivo study. Hydrogel	-	[29]
	HA and metallic silver treatment of chronic wounds: healing rate and bacterial load control; clinical trial. Spray powder	-	[30]
	Antibacterial thermosensitive hydrogels based on corn silk extract, HA and nanosilver improving wound healing with antimicrobial properties. Hydrogel		[13]
		B. subtilis
		S. aureus
		P. aeruginosa
		E. coli
Gentamicin Sulphate	Antimicrobial activity of a blend of collagen/chitosan/HA with gentamicin sulphate. Film		[31]
		S. aureus
		E. coli
		P. aeruginosa
Antimicrobial Peptides (AMPs)	Biofunction of antimicrobial peptide-conjugated alginate/HA/collagen resulting in significant inhibition of bacteria in infected wounds with rapid healing Scaffold		[32]
		E. coli
		Methicillin-resistant Staphylococcus aureus (MRSA)
		S. aureus
Curcumin	Wound healing ability of curcumin conjugated to HA; in vitro and in vivo evaluation. Scaffold	MRSA	[34]
	Curcumin-grafted HA-modified pullulan polymers as a functional wound dressing material. Film	S. aureus E. coli	[37]
ε-polylysine	Dual crosslinked hyaluronic acid/ε-polylysine hydrogel with self-healing and antibacterial properties for wound healing polymers; in vivo study. Hydrogel	S. aureus	[38]
		E. coli
Quaternized chitosan	Antimicrobial hyaluronic acid/quaternized chitosan hydrogels for the promotion of seawater-immersion wound healing. Hydrogel	S. aureus	[39]

).

Similarly, curcumin is a natural phenolic substance extracted from the rhizome of Curcuma longa and is claimed to possess antibacterial properties. Moreover, curcumin exhibits photodynamic properties, which have been exploited in order to enhance its antimicrobial efficacy on infected wounds [33]. Therefore, the effects in vivo and in vitro of curcumin-modified HA (250 kDa) substrates on antimicrobial and wound healing properties have been studied. For such a study, HA-cur conjugate was prepared by stirring for 1 h at room temperature a solution of 1% (w/v) of HA solubilized in 1:1 water/DMSO with N-Dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). The bactericidal efficacy of HA conjugate was evaluated in the dark and after exposure to blue light [34] (Table 1). They explained the bactericidal effect of curcumin by asserting that it inhibits cellular proteins, FtsZ and sortase A, both involved in bacterial cytokinesis and cell adhesion [35,36].

The potential of a wound healing biomaterial consisting of a curcumin-grafted HA-modified pullulan was evaluated. To produce this biomaterial, pullulan was dissolved in 10 mL DMSO and then was stirred at 50 °C with succinic anhydride and DMAP. After precipitation in anhydrous ethanol, precipitation was dissolved in ethanol, then dialyzed and lyophilized. The antibacterial effect was tested on S. aureus and E. coli by the agar diffusion method and it was shown how the antimicrobial activity of curcumin-grafted polymers on Gram-positive bacteria is better than that of Gram-negative bacteria (Figure 4) [37] (Table 1).

Another antimicrobial bioadhesive hydrogel for wound dressings with inherent antibacterial properties was fabricated by mixing modified HA and ε-polylysine (EPL). Hydrogels were produced using the aldehyde form of HA (HA-CHO), by mixing 0.5% (w/w) HA solution to an equal molar NaIO₄ solution dropwise. After 2 h reaction, it was terminated by adding 1 mL ethylene glycol into the system for another 1 h. Instead, EPL and levodopa were dissolved into an enriched ethanesulfonic acid (MES) solution. Antimicrobial properties were tested through haematoxylin and eosin staining, Masson staining, and α-SMA and CD31 staining. Results showed how such hydrogels were able to kill both Gram-positive and Gram-negative bacteria thanks to their high-density positive charge [38] (Table 1).

Novel hydrogels based on the combination of natural polymers have been designed for the treatment of various wounds. A composite hydrogel made up of HA and quaternized chitosan (QCS) has been fabricated in order to find a solution for wound healing in the presence of seawater, containing high quantities of sodium and potentially dangerous bacteria. Hydrogels were produced and dialyzed using the HA-CHO, as conducted in the previous example. To produce hydrazide-modified HA, HA was dissolved in distilled water and then ADH was added. To this solution a DMSO/H₂O solution (volume ratio 1:1) with EDC and HoBt was subsequently added. The reaction was terminated by adding NaOH solution. HA–ADH solution was then precipitated in ethanol and dialyzed after redissolution in a dialysis sack. The antimicrobial activity of QCS is given both by chitosan and by the addition of quaternary ammonium. In particular, chitosan exerts its antimicrobial action thanks to the positive charge of the amino group at the C2 position, after protonation at acid pH, which can interact with the surface of bacteria and cause bacterial death. The addition of quaternary amino groups, with intrinsic antimicrobial activity, combines with polyatomic amino groups to form double antimicrobial active groups. Antimicrobial activity was tested on S. aureus by measuring the number of bacteria on the surface of hydrogels. Results showed that the addition of quaternized chitosan enhanced the mechanical properties of the hydrogels, which exhibited an excellent antibacterial property compared to the previous reported antibacterial hydrogels in vitro and in vivo [39] (Table 1).

5. Conclusions and Future Perspectives

HA plays crucial roles in the wound healing process, being a major constituent of all vertebrates’ connective tissue ECM, promoting the formation of a fibrin clot and the production of interleukins and proinflammatory cytokines [40]. Thus, thanks to its hydrophilicity, biocompatibility and easily tuneable chemical properties, HA-based wound dressings have been produced so far in different compositions and shapes (scaffolds, films, fibers, non-woven fabrics, etc.). Moreover, HA shows strong bacteriostatic activity as it helps to reduce bacterial adhesion and biofilm formation [21], especially higher molecular weight HA [22]. It is a desirable property when dealing with wound healing since bacterial infections can delay healing processes. As a matter of fact, several surgical procedures dealing with regenerative medicine showed that the reduction in bacterial burden at the wound site thanks to bacteriostatic substance may improve the clinical outcomes [41]. Several papers give evidence that HA can show bacteriostatic effects on staphylococci and streptococci, depending on HA concentration and molecular weight [22,42], while P. aeruginosa seems not to be affected by HA [7,43]. However, HA intrinsic properties can be enhanced using synthetic and natural antimicrobial agents. Among synthetic dressings, AgNPs show a pervasive use in the literature, as they can be easily obtained with different approaches, also avoiding the use of organic toxic solvent [44]. Studies show effective antimicrobial activity in vitro, as well as in vivo and in clinical trials. Antibiotics can be regarded as antimicrobial synthetic agents. As an example, gentamicin sulphate can be coupled with bio-engineered substrates for wound healing. Alternatively, natural agents such as ε-polylysine and quaternized chitosan can be used as antimicrobial activity enhancers. One of the most promising solutions is curcumin, a natural phenolic substance proven to possess strong antibacterial properties. Moreover, AMP, a new class of antimicrobial drugs, is expected to potentially solve the problem of toxicity, as well as of antibiotic drug resistance.

In the near future, some aspects involving the production of substrates for wound healing need to be further addressed. Particular attention should be given to a solvent-free fabrication in order to avoid the use of toxic organic solvents. In parallel, the production of more morphologically complex substrates should be addressed, focusing, as an example, on non-woven fabrics’ substrates, whose properties seem to be interestingly adjusted by acting on composition, fiber density and orientation, or order of layering. Furthermore, to allow their successful commercialization, a reproducible process to produce HA derivatives is required, and their pharmacokinetic/pharmacodynamic properties must be optimized. In addition, the biological activity of HA-based wound dressings could be improved through the incorporation of other biomolecules such as adhesive proteins (e.g., fibronectin, laminin, fibrinogen) and/or stem cells for accomplishing an improved healing process.