1. Introduction
Accidents or diseases resulting in skin damage are a commonplace occurrence in everyday life, making wound dressings a critical element of modern healthcare [
1]. Wound dressings are typically porous in nature, with good barrier properties and oxygen permeability, and assist healing by preventing bleeding, absorbing excess exudates, keeping wounds moist, and protecting them from the environment [
2,
3]. However, issues with conventional dressings, such as irritation after prolonged use, poor compatibility with wounds, and a lack of effectiveness in the treatment of chronic wounds, such as severe burns, diabetic wounds, and ulcers, necessitates improved wound-dressing technologies that are biocompatible, actively accelerate healing, and exhibit antibacterial and antifungal activity [
4,
5]. Use of nontoxic and antibacterial biological polymeric nanofibers, such as chitin and bacterial cellulose, in wound-dressing technologies has subsequently received significant attention. Nanofibers are particularly well suited to wound-dressing research due to their high surface-area-to-volume ratio, porosity, pore size distribution, and morphology, which mirror the skin’s natural extracellular matrix, promoting cell adhesion and proliferation [
3,
6]. Chitin, a linear macromolecule composed of
N-acetylglucosamine, and its derivative chitosan are among the most popular nanofibers used in wound-dressing research [
1]. Naturally occurring in arthropod exoskeletons, mollusks, and fungi, chitin is one of the most abundant organic polymers on Earth and is easily extracted from these natural sources by using a mild alkaline treatment, the concentration of which can be increased if desired to produce chitosan, the deacetylated derivative of chitin [
7]. Both chitin and chitosan exhibit properties beneficial to wound-dressing applications, including biocompatibility, biodegradability, hemostatic activity, healing acceleration, nontoxicity, adsorption, and anti-infection properties [
1,
3]. However, significant differences exist in the structure, properties, processing, and associated polymers of animal- and fungi-derived chitin, which have influenced their application in wound-dressing research, respectively. This review aims to investigate the key differences between animal and fungal chitin and the mechanisms through which chitin and chitosan assist wound healing, before examining the historical and current application of these chitin and chitosan variants to wound-dressing research. Advanced wound-dressing technologies utilizing chemically modified chitin and chitosan and nanocomposite architectures are also addressed, as well as the future potential of these chitin types in wound treatment.
2. Differences between Crustacean and Fungal Chitin
Chitin is one of the most abundant organic polymers on Earth, constituting the structural component of arthropod exoskeletons, mollusk radula, cephalopod endoskeletons, fungal cell walls, and fish and lissamphibian scales [
8]. The largest source of chitin globally is suggested to be Zooplankton cuticles, with an estimated 379 million tons of Antarctic krill available worldwide [
9,
10]. However, fishing these tiny organisms is not commercially viable, and, subsequently, shellfish industry wastes, such as shrimp, crab, and lobster shells with chitin contents of 8–40%, are the main source of industrial chitin [
11,
12,
13] (
Table 1). Fungi provide an alternative source of chitin and, despite having lower chitin content than crustaceans (10–26% as a chitin-β-glucan complex), are experiencing increasing academic and commercial interest [
14,
15]. Unlike crustacean chitin, fungal chitin is not limited by seasonal and regional variation and does not require the aggressive acid treatment that crustacean chitin does for purification and demineralization, to remove calcium carbonate and other minerals [
15,
16]. It also supplements the rigid chitin structure with more pliable branched β-glucan, yielding a native nanocomposite architecture that can provide both strong and tough fiber networks when extracted [
15,
17].
Both crustacean and fungal chitin have a similar molecular structure to cellulose, which is the structural component of the primary cell wall of all green plants, algae, and oomycetes. The main difference between cellulose and chitin is the replacement of the C2 hydroxyl group of cellulose with an acetamide group, which can be deacetylated to obtain chitosan (
Figure 1). Two major polymorphic forms of chitin exist, α and β, with α-chitin the most common polymorph for both crustacean and fungal chitin and β-chitin occurring only in squid pen, sea tube worms, and some algae (centric diatom) [
19].
However, key differences exist between crustacean and fungal chitin. Crustacean chitin normally has minimal residual protein and binds with sclerotized proteins and minerals, whereas fungal chitin is associated with other polysaccharides, such as glucan, which can occur in quantities exceeding the chitin content itself [
31]. Crosslinking between chitin and proteins is well established in both crustacean- and insect-derived chitin, although it is still unclear whether the bridging is partially covalent in nature, with the low quantity of residual protein present suggesting that there is little covalent bonding or that the bonds may be cleaved during the chitin extraction process [
32,
33,
34,
35]. On the other hand, the linkages between chitin and glucan in fungi have been proven to be covalent in nature [
36,
37,
38]. Small differences exist between insoluble glucans in mushrooms, yeast, and hyphae. However, most commonly, β-glucan exhibiting a (1→3) backbone and (1→6) branching is associated with chitin [
39]. The location of chitin also varies, being concentrated in the bud scar in yeast and in the cell wall of most other fungi. Notably, in some fungi, both chitin and chitosan are simultaneously co-synthesized, a feature unique to the fungal phylum Zygomycota [
40,
41].
The extraction processes for fungal and crustacean chitin are similar (
Figure 2), with both processes initially requiring the raw material to be washed and homogenized. For fungi, the starting material is mycelial biomass or fruiting bodies, while for crustaceans, it is their shells.
Fungal chitin sources are generally easily homogenized using a kitchen blender [
42], while the harder and more brittle crustacean shells must be crushed. The high mineral content of crustacean shells also requires an acidic demineralization step, typically completed by using 1–2 M hydrochloric acid (HCl) for up to 48 h, although concentrations ranging up to 11 M are possible [
43]. This step is not required when processing fungal chitin. Deproteination is then completed for either fungal or crustacean chitin in mild alkaline conditions, typically 1 M sodium hydroxide (NaOH), before the final material is decolorized, using a bleaching step, if desired. Pure chitin is obtained from crustacean shells as a final product, whereas fungal chitin sources yield a chitin-β-glucan complex following extraction. Pure chitin can be derived from this complex, if desired, using acid treatments to degrade the glucan, yielding X-ray diffraction patterns resembling crustacean chitin [
44].
3. Generation and Properties of Chitosan
Chitosan can be generated from both fungal and crustacean chitin in a simple deacetylation process, whereby the acetyl group of chitin’s acetamide group is cleaved off under strong alkaline conditions (
Figure 3), typically sodium hydroxide (NaOH), with up to 98% yields possible [
45]. Although chitin and chitosan both have useful biomedical properties, including biocompatibility, biodegradability, hemostatic activity, healing acceleration, nontoxicity, adsorption, and anti-infection potential [
3], chitosan generally receives more scientific attention due to its more useful structure, which renders it soluble in aqueous acids. Chitosan’s primary amine group can be protonated under mildly acidic conditions. Conversely, chitin is insoluble in all regular solvents, such as water, organic solvents, and mild acids or bases, due to the hydrogen bonding associated with the acetyl, amino, and hydroxyl groups in its polysaccharide chain [
19]. Protonated chitosan’s charge also makes it a bio adhesive, able to bond to negatively charged surfaces, such as mucous membranes, chelate heavy metal ions, and is biocompatible and biodegradable with superior antibacterial properties to chitin if hydrated or in the form of a hydrogel [
30,
46,
47]. The primary and secondary hydroxyl groups on each repeating unit and the amine group of each deacetylated unit are also reactive and are readily chemically modified to alter the physical and mechanical properties of chitosan [
48]. These advantages provide chitosan with greater processing and biomedical potential than chitin as a component for wound treatment materials. However, its use in biomedical materials is limited by its poor mechanical properties. Chitin is strong, with a nanofibril tensile strength of ~1.6–3.0 GPa [
49], which results from hydrogen bonding between the chains of the macromolecules [
50]. Conversely, the absence of the acetyl group, which contributes to hydrogen bond formation in chitin and stabilizes its crystalline structure, significantly compromises the mechanical properties of chitosan [
51]. This makes chitosan alone mechanically unsuitable for applications that require durability, such as strong films or composites, despite its significant biomedical potential.
4. Healing Mechanisms of Chitin and Chitosan
Wound healing is a complex biological process comprising four stages: hemostasis, inflammation, proliferation, and remodeling [
52] (
Figure 4). These stages overlap in time [
53] and follow a specific program, which is introduced and modulated by different cell types. Usually this mechanism works well enough to facilitate rapid repair of damaged skin. However, it does not regenerate the wounded skin completely, with scaring and loss of hair follicles or sweat glands common in healed skin [
54]. Impaired wound healing function and chronic wounds in some patients is also common [
52]. One of the major objectives of wound-healing technologies is to facilitate improved wound healing, tending toward wound regeneration [
53,
54]. Chitin and its derivates have been shown to be useful constituents in wound-dressing materials [
55,
56,
57] and may potentially contribute to the development of skin substitutes facilitating skin regeneration since they appear to influence the wound-healing process on a molecular level.
The hemostasis phase starts immediately upon injury occurrence and is intended to stop hemorrhage by forming a fibrin clot [
52] (
Figure 5), a step which typically depends on platelets [
54]. The formed clot re-establishes a barrier against the outside world and provides an improvised extracellular matrix, which is needed for cell migration [
53]. Chitosan has hemostatic properties, reacting with red blood cells to form a coagulum [
58] (
Table 2), and acting independently from the regular coagulation mechanism, whereas, typically, red blood cells only have a supportive role in the formation of clots [
59]. This improved hemostatic effect was also observed in whole blood, heparinized blood, and defibrinated blood [
58], meaning that chitosan can potentially provide an improved clotting ability and aid hemostasis [
60]. Studies have also been undertaken to assess the effectiveness of chitosan as a hemostatic in surgical settings, with wounds treated using chitosan exhibiting reduced bleeding compared to control wounds [
61,
62]. However, chitosan’s hemostatic effectiveness has not been compared to other hemostatics, and chitin does not seem to have been investigated as a hemostatic.
Once the hemorrhage has been stopped, the degranulating fibrin clot and surrounding tissue cells trigger the next stages of healing by releasing cytokines and growth factors, which attract cells to the wound site [
53]. Chitosan may induce a different clotting mechanism, meaning that the healing process may be altered. This potentially results in a modified healing response, facilitated by the release of fewer growth factors from the platelets [
63]. One of the first cells to respond next are neutrophils, also called polymorphonuclear neutrophils or PMNs. PMNs clean foreign objects, like dirt and bacteria, from the wound and remove damaged cells and are one of the main cell types responsible for inflammation [
53]. Both chitin and chitosan have been shown to have a positive chemotactic effect on canine PMNs [
64,
65], meaning that they attract PMNs. An in vitro study showed that chitin has a stronger effect than chitosan [
65]. However, chitosan may potentially impact the wound to a greater extent, as it degrades more slowly than chitin [
64]. A similar effect has been observed in bovine PMNs [
66]. Studies undertaken in dogs also found increased infiltration of PMNs after 3 days in wounds treated with chitosan, compared to control wounds, and decreased inflammation after 28 days in wounds treated using chitin or chitosan [
67,
68]. This induced increase in PMNs may also improve wound cleansing and shorten the inflammation stage, potentially providing a positive impact on the wound healing process. PMNs have also been observed to release pro-inflammatory cytokines, which may activate surrounding fibroblasts and keratinocytes [
54].
Macrophages, which consume PMNs [
69], become the next dominant leukocyte in the inflammation stage. Macrophages have been shown to be essential for wound healing, as they have a key role in transitioning the wound from the inflammation stage to the cell proliferation stage [
52,
53]. They have many functions, such as phagocytosis of dead or infected cells, attraction of many cells to the wound site, and they also support the formation of granulation tissue, blood vessels, and the extracellular matrix [
53]. Defective wound repair has been observed in animals depleted of macrophages [
53]. The same study, which showed increased PMN infiltration, also showed macrophages increasingly infiltrating wounds treated with chitosan in comparison to control wounds [
67]. A potential reason for this may be chitosan-induced activation of a complement called C5, which attracts macrophages and PMNs [
67]. Chitosan has also been shown to increase the mRNA expression and synthesis of TGF-β1 (transforming growth factor-beta 1) and PDGF (platelet-derived growth factor) in macrophages in vitro [
70]. Both TGF-β1 and PDGF are chemotactic for macrophages and fibroblasts, with TGF-β1 also affecting keratinocytes, which make up the outermost layer of skin, [
54] and PDGF, inducing fibroblast proliferation and collagen production [
70]. It has also been observed that 70% deacylated chitin increases in vitro [
71] and in vivo [
72] secretion of IL-1 in macrophages, which affects fibroblast proliferation [
73] and collagen production [
67]. Chitosan (over 95% deacylated) on the other hand shows no effect in vitro [
71] and lesser effects than the 70% deacylated chitin in vivo [
72].
Fibroblasts, which lay a new skin fundament in the wound, are also a key cell type for wound healing. They produce the extracellular cell matrix [
53], the structure between cells, which consists mainly of collagen [
74]. Collagen plays a key role in scar formation, as excessive collagen deposition can lead to scars [
53]. Insufficient collagen deposition has also been linked to chronic wounds [
63]. Therefore, a balance of collagen production and degradation is necessary to ensure full regeneration. As described above, chitin and chitosan affect the secretion of different cytokines in macrophages, which in turn affect the proliferation of fibroblasts and collagen production. However, the effect of chitin and chitosan on fibroblasts is not only indirect in nature, with chitosan inducing increased IL-8 production in fibroblasts [
66], a strong chemotactic for PMNs and regulator of keratinocyte migration and proliferation [
73]. Another important role of fibroblasts is the production of the extracellular cell matrix. In vitro studies have shown no direct effects of chitosan on the fibroblasts producing the extracellular matrix [
66]. However, indirect effects through microphage stimulation, and therefore fibroblast stimulation, may affect this stage. It has also been hypothesized that chitin and chitosan could be incorporated into the extracellular matrix, through the use of lysozyme [
60], an enzyme capable of degrading chitin and chitosan.
The effects of molecular weight and degree of deacetylation on the wound-healing potential of chitin and chitosan have also been studied, albeit to a lesser extent. An in vivo study on incisions in rats investigated the effect of chitin (300 kDa, <10% deacetylation), chitosan (80 kDa, >80% deacetylation), and their oligomers and monomers on wound break strength and collagenase activity [
75]. The results showed that wounds treated using chitosan (chitosan, oligomer, and monomer) were stronger than those treated using chitin. In both cases, treatments using oligomers were associated with the highest wound break strength, although chitosan had comparable performance, and chitosan monomers were associated with the highest collagenase activity. A further in vitro study on fibroblasts investigated differences in healing based on the molecular weight and degree of deacetylation of chitin and chitosan [
76], utilizing chitin with a degree of deacetylation of 37% and molecular weights of 37 and 197 kDa, and chitosan with degrees of deacetylation of 58% and 89% and molecular weights of 12 kDa/194 kDa and 13 kDa/263 kDa, respectively. The results showed that chitosan with a higher degree of deacetylation had a greater effect on fibroblast proliferation, which was further enhanced at lower molecular weights.
The molecular weights of the chitin and chitosan used in these studies falls in the low-molecular-weight range, as both chitin and chitosan can have molecular weights >1000 kDa [
77]. However, another in vivo study investigating surgical burns in rats used a broader range of molecular weights, ranging from 70 to 750 kDa and peaking at 2000 kDa [
78]. However, the degree of deacetylation of each sample was not varied, with a fixed value of 63%, 75%, and 92%, for each respective molecular weight. The results showed that the 2000 kDa chitosan performed significantly better than the other molecular weights both in wound contraction and collagenase activity, a result that the author attributed to its high molecular weight [
78]. However, since the degree of deacetylation has a greater effect on wound healing than the molecular weight in the low-molecular-weight range [
75,
76], the effects of different degrees of deacetylation at high molecular weights should also be examined.
It is however clear that chitosan with a higher degree of deacetylation exhibits higher biological activity than that with a lower degree of deacetylation [
75,
76]. The effect of molecular weight on wound healing in the low-molecular-weight range studied (<300 kDa) also seems to be enhanced at lower molecular weights (<100 kDa). However, since chitin and chitosan can exhibit molecular weights that fall well outside this range, further studies for higher molecular weights (>300 kDa) are required, especially when considering the correlation between the hemostatic activity of chitosan and higher molecular weights [
79].
5. Fungi-Derived Chitin and Chitosan Wound Dressings
Medical applications of fungi date back to ancient times, where it was used as a styptic to stop bleeding and as a crude precursor to modern antibiotics in the treatment of infections [
80,
81,
82,
83]. However, true academic interest in medical materials derived from fungi did not begin until the 1970s, when the mycelia of several fungal species were investigated as wound-healing accelerants. Prudden et al. [
84] studied the topical application of powdered mycelium from
Phycomycetes mucor,
Penicillium notatum, and
Aspergillus niger on rat wounds to confirm the healing properties of glucosamine (polymer units of chitosan), which was thought to be responsible for the healing potential of the cartilage material historically used in wound treatment [
84]. Wounded rat skin treated with powdered mycelium was found to have a higher tensile strength than untreated or cartilage powder treated wounds as they healed, with
P. mucor associated with the highest skin tensile strengths (
Figure 6). In further investigations, a chitin-β-glucan powder was also produced through NaOH and HCl treatment of the same mycelium and compared to purified crustacean chitin, again as a topically applied healing accelerant on rat wounds. Lobster and king crab chitin outperformed the purified mycelium powder; however, all fungal species except
A. niger provided higher skin tensile strength than the cartilage and shrimp chitin healing agents [
85].
Following these successes, chitin quickly gained popularity academically as a wound-healing accelerant, with most subsequent work focusing on the healing potential of crustacean chitin. However, the lower costs and simpler purification of fungal chitin did attract research interest, with further studies utilizing NaOH and acetic acid purified
Aspergillus oryzae,
Mucor mucedo, and
Phycomyces blakesleeanus mycelium demonstrating increased cell proliferation in fibroblasts at low concentrations. This cell-proliferating effect facilitated through the use of the fungal material was found to correlate with the chitin or chitosan content of the material, with
P. blakesleeanus showing the highest proliferation at 0.01% and 0.5% w/v. Additionally cell attractant properties were found in
P. blakesleeanus and
M. mucedo, which were suggested to assist in wound healing [
86].
Commercialization of fungi-derived wound treatment materials occurred in 1997, with a research group from Taiwan extracting a chitin-polysaccharide mixture from
Ganoderma tsugae, comprising β-1-3-glucan (~60%) and
N-acetylglucosamine (~40%), and creating a weavable skin substitute called Sacchachitin (
Figure 7). This novel wound dressing was tested on rats [
56] and Guinea pigs [
87], before being tested in a preliminary clinical trial on two human patients with chronic wounds, in 2005 [
55]. Animal studies showed that Sacchachitin improved wound healing significantly compared to conventional gauze and had comparable performance to Beschitin, a commercially available wound dressing from crustacean chitin, developed in 1988 [
87]. Improvements in healing were also observed in human trials for chronic wounds open for seven months or longer, with the underlying healing mechanisms being fibroblast and keratinocyte proliferation and the activity of matrix metalloproteinases (MMPs) (human cells) [
55]. A Sacchachitin nanogel derivative was also produced for the treatment of corneal burns in rabbits, demonstrating promise with significant increases in cornea cell proliferation and wound closure stimulation, in addition to enhanced corneal wound healing due to the inhibition of protein breakdown [
88].
More recently, with the rise of research interest into the wound-treatment potential of purer crustacean chitin derivatives, academic interest in the medical applications of fungal material has shifted to the investigation of fungal exopolysaccharides (EPS). EPS are not components of the fungal intercellular matrix or cell wall, where chitin is typically found, but rather occur on the cell surface or in the extracellular matrix [
89]. Compared to other fungal polysaccharides, they are mass producible in a short time and easily isolated and purified [
89]. Thai studies investigating the EPS of 16 different native fungal strains identified three strains that were biocompatible with Vero cells, which are primate cells resembling fibroblasts, and increased interleukin-8 (IL-8) production in fibroblasts, improving wound healing [
90]. EPS have also been combined with traditional antibacterial agents, such as ciprofloxacin, to create active-agent-loaded fungi-derived wound dressings [
91]. Fungal β-glucans, such as lentinan from
L. edodes (shiitake), schizophyllan from
S. commune (split gill), zymosan from
S. cereviase (baker’s yeast), pleuran from
P. ostreatus (oyster), and ganoderan from
G. lucidum (reishii), have also been extensively studied due to the human immune system’s ability to recognize them, promoting immune stimulation, antibacterial, antitumor, anticancer, and antioxidant properties [
92,
93,
94,
95]. These findings, coupled with the varying chitin, chitosan, and polysaccharide profiles of the over 5.1 million species of fungi in existence [
96] and recent advances in fungal material technology [
7,
14,
17,
97,
98,
99,
100,
101], suggest that fungi-derived wound treatments warrant further investigation. In particular, the native chitin-β-glucan composite architecture of fungal chitin could be utilized to achieve scaffolds exceeding the mechanical performance of crustacean chitin [
17] and novel antibacterial properties resulting from composite dressings incorporating naturally generated complexes of fungal chitin, chitosan, β-glucans, and exopolysaccharides could pave the way for new low-cost, natural, and mass-producible dressing technologies.
7. Human Clinical Trials Utilizing Chitin and Chitosan for Wound Treatment
Commercialization of chitin and chitosan-based wound treatment products, such as Axiostat
®, Beschitin
® W, Bexident
® Post, Celox
®, Chitohem
®, HemCon
®, Medisorb
® R, Surgi shield
®, and SEQUA
® San Chitosan, in addition to custom-prepared treatments, have prompted several human clinical trials of chitin and chitosan for wound-healing applications. These studies predominantly focus on chitosan and chitosan derivatives for use as wound dressings, gels, powders, films, membranes, and even mouthwash for oral, nasal, ear, and skin applications, in addition to treatment of ulcers and serious hemorrhages (
Table 3).
These wound treatments have experienced significant success in oral clinical trials where they have been used to treat aphthous stomatitis (mouth ulcers), postoperative and tooth extraction wounds. Chitosan mouthwash (0.5%) reduced aphthous stomatitis pain and ulcer size, exhibiting comparable performance to Triamcinolone, a corticosteroid product [
165]. Similar results could also be achieved by using a mucoadhesive film [
166]. Dental surgery clinical trials also found chitosan-based wound treatments to significantly shorten bleeding time and improve wound healing for patients undergoing minor surgery and tooth extractions [
167,
168]. Clinical trials examining the effect of chitosan on a range of other wounds also exhibited very promising results, with improved cell adherence, hemostasis and re-epithelialization [
164,
171], less itching and sensitivity [
171], less exudate and odor [
175], and reductions in wound healing time [
170,
173], post-hemodialysis puncture site bleeding [
173], and rebleeding [
174] in diabetic, puncture, superficial, skin graft, and sinus postoperative wounds. Chitosan has also been utilized in clinical trials for treatment of ulcers with notable reductions in exudate, pain on dressing removal, wound area and depth when compared to traditional Vaseline gauze dressings [
176,
177]. Additionally, military-grade chitosan-based wound-treatment products, such as Celox
®, have been used as lower-cost alternatives to oxytocin, prostaglandin, and uterine balloons in the treatment of life-threatening obstetric hemorrhages, completely stopping uncontrolled bleeding within seconds to minutes. Although chitosan was used in most clinical trials, it should also be noted that chitin membranes have been used to assist in the closure of chronic tympanic membrane perforations in the ear [
162].