Copper, an Abandoned Player Returning to the Wound Healing Battle

*Gadi Borkow and Eyal Melamed*

## **Abstract**

Copper has two key properties that endow it as an excellent active ingredient to be used in the "wound healing battle". First, copper plays a key role in angiogenesis, dermal fibroblasts proliferation, upregulation of collagen and elastin fibers production by dermal fibroblasts, and it serves as a cofactor of Lysyl oxidase needed for efficient dermal extracellular matrix (ECM) protein cross-linking. Secondly, copper has potent wide-spectrum biocidal properties. Both gram-positive and gramnegative bacteria, including antibiotic resistant bacteria and hard to kill bacterial spores, fungi and viruses, when exposed to high copper concentrations, are killed. Copper has been used as a biocide for centuries by many different civilizations. Impregnation of copper oxide microparticles in wound dressings allows continuous release of copper ions. This results not only in the protection of the wounds and wound dressings from pathogens, but more importantly, enhances wound healing. The article discusses the molecular mechanisms of enhanced wound healing by the copper oxide impregnated dressings, which include in situ upregulation of proangiogenic factors and increased blood vessel formation. It also includes clinical cases showing clearance of infection, induction of granulation and epithelialization of necrotic wounds, reduction of post-operative swelling inflammation and reduction of scar formation, in wounds when they were treated with copper oxide impregnated dressings. We show the positive outcome at all wound healing stages of using the copper impregnated wound dressings, indicating the neglected critical role copper plays in wound healing.

**Keywords:** copper oxide, wound dressings, wound healing, angiogenesis, extracellular matrix, chronic wounds

## **1. Introduction**

Wounds normally heal in finely balanced, efficient, and ordered sequence of repair events distinguished by four distinct, but overlapping, phases: Hemostasis, Inflammation, Proliferation and Remodeling [1, 2]. These coordinated cellular and molecular events involve numerous processes such as cell proliferation, migration and differentiation. All of these processes demand a continued and efficient supply of oxygen and nutrients, due to the increased cellular biosynthetic activities. Following wounding, the altered microenvironment, such as the reduced oxygen supply, initiates the release of factors by epidermal cells,

fibroblasts, macrophages and vascular endothelial cells, all of which stimulate neo-vascularization. The secreted factors include vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor (TGF)-β. VEGF is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. TGF-β, especially TGFβ1, is also a key cytokine that regulates the production and secretion of elastin and collagen [3]. Fibroblasts, which attach to fibrin and integrin cables, produce and secrete collagen and elastin that become cross-linked, forming the dermal extracellular matrix (ECM). This allows the restoration of the structure and function to the injured tissue [4, 5].

## **1.1 Chronic wounds**

Chronic wounds or "hard to heal wounds" are characterized by extensive loss of the integument, clear necrosis, or signs of circulation impairment either localized or more extensive, usually in the limbs, or in pressure areas, leading to extensive loss of substance. Chronic wounds seem to be detained in one or more of the phases of wound healing, and some chronic wounds may never heal or may take years to do so. These wounds have lost the fine balance needed for wound repair, leading to chronic non-healing ulcers, associated with morbidity and mortality due to tissue inflammation and infection [6]. Chronic wounds are usually associated with systemic pathologies [7] that cause ischemia, a restriction in blood supply to tissues. Ischemia occurs in diabetic patients due to atherosclerosis as well as microangiopathic disease [8], in chronic venous ulcers due to chronic venous insufficiency [9], in patients with autoimmune disease or under immunosuppressive drug therapy due to vasculitis [10] and in pressure sores due to necrosis of the integument [11]. Chronic wounds cause patients severe emotional and physical stress and create a significant financial burden on patients and the whole healthcare system. Chronic wounds require special attention and wound care.

### **1.2 Involvement of systemic copper in wound healing**

Copper is an essential mineral involved in many of the physiological processes in all body tissues [12, 13], including the skin and integumentary system [14]. Many of the finely balanced wound healing repair mechanisms are dependent on their interaction with copper (thoroughly reviewed in [6]). This includes, Plateletderived growth factor (PDGF), involved in the hemostasis phase of wound healing, [15, 16]; VEGF and angiogenin, key growth factors that stimulate angiogenesis, an essential process during the Proliferation Phase [17–23]; secretion of collagens (types I, II, and V), HSP-47 and elastin fiber components (elastin, fibrillins) by dermal fibroblasts during the Proliferation and Remodeling Phases [16, 24, 25]; activity of Lysyl oxidase (LOX) needed for efficient extracellular matrix (ECM) protein cross-linking between elastin and collagen [26]; stabilization of the skin ECM once formed [27, 28]; modulation of integrins by differentiated keratinocytes during the Remodeling phase [29], and Matrix metalloproteinases (MMPs, mainly MMP-1, MMP-2, MMP-8, MMP-9) and the serine proteases (human neutrophil elastase, HNE) are the major groups of proteases involved in the wound healing process. It is thus not surprising that copper chelation delays wound closure [30]. Copper is also a cofactor of superoxide dismutase, an antioxidant enzyme found in the skin that inhibits cellular oxidative effects, such as membrane damage and lipid peroxidation and protects against free radicals [19]. Copper is also a cofactor of tyrosinase, a melanin biosynthesis essential enzyme, responsible for skin and hair pigmentation.

## **1.3 Copper and wound infections**

Infections of the wound may delay wound healing, cause wound deterioration and even cause failure of healing [31]. This may occur through several different mechanisms: consistent and high production of inflammatory mediators, metabolic wastes and toxins; tissue hypoxia; causing hemorrhagic and fragile granulation tissue; reducing fibroblast number and total collagen production; and interfering with reepithelization [32, 33]; reducing the available nutrients and oxygen needed by the host cells and causing neutrophils to be in an activated state producing cytolytic enzymes and free oxygen radicals [34]. In chronic wounds bacteria may be covered by biofilm and be protected from the host defenses and develop antibiotic resistance [31]. Thus, reducing the microbial contamination of wounds increases the capacity of the wound to heal.

Copper is also a needed mineral for the normal function of microorganisms [35]. However, the microorganisms need to carefully control the intracellular copper levels. This is since copper under anaerobic condition is found in the highly reactive cuprous form (Cu1+), and as such it can readily react with the microbial proteins, causing disruption of the protein structures by forming thiolate bonds with iron– sulphur clusters [36]. Thus, above an exposure to a certain concentration of copper, microorganisms cannot cope with the excess copper and are killed [37, 38]. Several mechanisms for the potent biocidal activity of copper have been proposed, which include alteration of proteins and inhibition of their biological assembly and activity; plasma membrane permeabilization; and membrane lipid peroxidation [37]. In contrast to the resistant microbes that have evolved to antibiotics in less than 50 years of use, tolerant microbes to copper are extremely rare even though copper has been a part of the earth for millions of years. This lack of resistance to copper may be explained by the capacity of copper to damage in parallel many key factors in micro-organisms [37].

## **2. Cuprous oxide impregnated wound dressing**

## **2.1 General description**

Copper oxide impregnated wound dressings, hereafter called COD, have been cleared for treatment of acute and chronic wounds, including diabetic ulcers, pressure sores, and venous ulcers, by the USA FDA, EU and other regulatory bodies worldwide. The COD are soft, single use wound dressings composed of an absorbent highly absorbent needle punch layer and one or two external non-binding nonwoven orange polypropylene layers. All layers are impregnated with copper oxide microparticles. The orange external layer(s) is intended to be in contact with the wound bed. The wound dressings are provided with or without an adhesive contour, sterile, in a sterilization pouch (**Figure 1**). The non-adhesive wound dressings can be cut, trimmed or fold over according to the size and shape of the wound. The dressings can be used up to 7 days or until they are completely soaked with wound exudate.

## **2.2 Antimicrobial efficacy**

The COD exert potent wide spectrum antimicrobial efficacy (>4 log reductions), including when the dressings are completely soaked with wound exudate surrogate for 7 days, as demonstrated by us and by independent laboratories using the AATCC Test Method 100. Furthermore, the potent antimicrobial efficacy is maintained even after 7 consecutive microbial inoculations for 7 consecutive days (**Figure 2**).

#### **Figure 1.**

*Copper oxide impregnated wound dressings. The COD are composed of an absorbent layer and one or two external layers. The dressings are provided (a) with or (b) without an adhesive contour. The external layer (c, orange layer) is a non-adherent polypropylene layer placed in contact with the wound bed, which allows the passage of the wound exudates into the internal layer (c, beige layer) that absorbs the wound exudates. COD with two external layers (d) are more appropriate for application in wound cavities and deep wounds. All layers are impregnated with copper oxide microparticles (e, white dots) that endow them with potent biocidal properties.*

The antimicrobial efficacy was demonstrated against the following microorganisms: *Escherichia coli, Enterococcus faecalis, Enterobacter aerogenes, Klebsiella pneumoniae, Staphylococcus epidermis,* Methicillin-resistant *Staphylococcus aureus* (MRSA) and *Candida albicans.* When compared with commercially available silver wound dressings, the antimicrobial efficacy was significantly higher than 9 out of the 12 silver dressings studied, and as good as 3 silver dressings (**Figure 3**, unpublished data).

#### **2.3 Molecular mechanisms of enhanced wound healing**

The capacity of copper to enhance faster closure of full-thickness wounds was demonstrated in several wound animal models, [30, 39, 40], including in diabetic mice [41].

The capacity of the COD to directly enhance repair of chronic wounds by supplying *in situ* essential copper lacking due to poor systemic blood supply (such as in diabetic ulcers), was demonstrated in a murine diabetic model considered to be the

#### **Figure 2.**

*Continuous antimicrobial efficacy of COD even after 7 consecutive bacterial inoculations. Duplicate COD were inoculated with ~1 x 105 Klebsiella pneumonae CFU for 6 consecutive days and incubated at 37 °C. On the seven days the CODs and a Control wound dressings without copper were inoculated with ~4x105 CFU. After additional 24 hours of incubation at 37 °C, the bacteria were recovered and their viability determined. While no bacteria survived on the CODs, 4x105 viable bacteria were recovered from the control dressings.*

*Copper, an Abandoned Player Returning to the Wound Healing Battle DOI: http://dx.doi.org/10.5772/intechopen.96952*

**Figure 3.**

*Antimicrobial efficacy comparison between the COD and commercially available silver dressings. The wound dressings were inoculated with ~106 (left panel) and 4x104 (right panel) MRSA CFU. After 1 hour of incubation at 37 °C the bacteria were recovered from the dressings and their viability was determined.*

most suitable in diabetic wound healing studies [42]. Wounding and subsequent treatments were performed under aseptic conditions, so that the possible effects of the copper dressings would not be related to their biocidal properties.

A phenotype similar to diabetes type 2 in mice is achieved via a homozygous point mutation on the leptin receptor gene (LEPR) in the hypothalamus. Genetically engineered diabetic mice (db/db) show significant wound-healing impairment compared to wild-type mice [42]. Full-thickness single skin wounds were inflicted under sterile conditions on the dorsum of each animal followed by continuous dermal application of either COD or identical dressings without copper on the entire wound test site. Histological analysis of skin specimens taken from the diabetic mice treated with the COD 6, 12 and 17 days after wounding demonstrated a normal woundhealing process, including epidermal regeneration and granulation tissue formation, with numerous new blood vessels, chronic inflammatory infiltrate, generation of new hair follicles and sebaceous glands, and fibroplasia [41].

The very clear increase in angiogenesis in the copper treated mice was confirmed by immunohistochemistry staining using the Von Willebrand Factor that stains capillaries. Analysis of mRNA expression levels in the wound sites of 84 genes using real-time PCR gene-array analysis together with immunohistochemistry staining revealed the upregulation of several angiogenic factors, such as Vascular endothelial growth factor (VEGF). Based on the analysis performed a molecular mechanism was suggested in which a redox between cuprous oxide and cupric oxide generates hypoxia that induces the upregulation of Hypoxia-inducible factor-1alpha (Hif-1α) in the dermal layer, apparently in macrophages [41]. The upregulation of Hif-1α then induces a chain of events, depicted in **Figure 4**, which lead to endothelial cell migration and proliferation, production of new blood capillaries (angiogenesis), immune cell recruitment, fibroblast migration, intense metabolism, increased secretion of extracellular matrix proteins, and increased epithelialization.

### **2.4 Clinical cases showing the enhancement of the COD at all stages of wound healing**

The following cases illustrates the ability of COD to affect infection reduction, angiogenesis and granulation tissue formation, as well as epithelial tissue formation, in hard to heal wounds. In addition, we describe cases of reduction

**Figure 4.**

*Molecular mechanisms of enhanced wound healing by COD. (based on the model published in ref. [41]).*

of post-operative swelling and better post-surgery scar formation. All photos are published in the book with the patients' consent.

## *2.4.1 Clearance of infection, induction of granulation and epithelialization*

Fifty-seven years old male, with history of non-insulin-dependent diabetes mellitus (NIDDM), suffering from ulcers in both feet, mainly on the right side (**Figure 5a**). The etiology was mainly due to vasculitis type reaction (acute leukocytoclastic vasculitis), with minor large arteries involvement (for which angiographic intervention with percutaneous opening of the superficial femoral artery was carried out). The patient was treated with high dose steroids, immunosuppressive medication (Azathioprine, Imuran) and broad-spectrum antibiotic treatment. The patient right foot worsened with development of necrosis mainly in the medial toes, with the infection spreading to involve the tendons and the plantar fascia. Deep ulcers were present over the medial aspect of the heel and the lateral aspects of the foot (**Figures 5a** and **b**).

The patient underwent surgery to debride the wound including 1st and 2nd ray amputation. Cultures taken at surgery yielded *Pseudomonas aeruginosa* resistant to quinolones and the patient was treated with Imipenem. Five days later the patient underwent trans-metatarsal amputation. The wound was partially closed to prevent a loose flap. Nevertheless, necrosis of the edges of the flap was seen few days following surgery (**Figure 5c**). Bedside debridement was carried out. At that time, culture taken from the second surgery revealed that the pseudomonas has now developed resistance to carbapenems and it was decided to stop the antibiotic treatment. The medial heel wound had at least 30% necrotic tissue. The lateral anterior wound had 80–90% necrosis (**Figure 5c** and **d**).

Trans-tibial amputation deemed to be the next step. WBC count was 18,000, which was an improvement from previous higher levels, and the CRP was 3.0 (normal <0.5). However, since the patient's overall condition was stable, it was decided to continue only with local wound care with COD. The dressings were placed deep in the plantar-fascial part of the amputation wound, on the edges of it and on the ulcers (**Figure 5d** and **e**). The dressings were replaced twice a week. Prontosan® irrigation was recommended during dressing change. No supplemental antibiotic was given.

*Copper, an Abandoned Player Returning to the Wound Healing Battle DOI: http://dx.doi.org/10.5772/intechopen.96952*

#### **Figure 5.**

*Clearance of infection, induction of granulation and epithelialization of necrotic wounds. a. Ulcers colonized with Pseudomonas aeruginosa were present on both feet, mainly on the right foot. b. The ulcers were present over the medial aspects of the heel and the lateral aspect of the foot. c. Two weeks following trans metatarsal amputation necrotic tissue was present on the edges of the partially closed flap. d. The COD dressings started to be used (Day 0) by placing them deep in the plantar-fascial part of the amputation wound and e. by covering the medial and latera ulcers. f. One week later, a reduction in the necrotic tissue and beginning of granulation tissue was observed in all wounds. g and h. After 2 months of COD treatment there was clear epithelialization (white arrows) in the lateral and medial ulcers and granulation tissue formation (yellow arrows) in the lateral and medial ulcers and in the main bulk of the amputation wound that can be seen through the remaining thin necrotic tissue. Cultures from the necrotic tissue were negative for pseudomonas (the resistant original pathogen). i. The granulation tissue seemed to affect the necrotic tissue with autolysis (self-debridement). j and k. After 5 months of COD treatment, the medial and lateral wounds were closed. l. The main wound was partially closed and the rest of the wound was with pink to red granulation tissue.*

The foot condition improved gradually. The superficial semi-necrotic ulcer at the heel and lateral aspect of the foot showed gradual absorption of the necrotic tissue, granulation and epithelization (**Figures 5f**–**i**). The main amputation wound, with large area and volume and inner cavity of 6–7 cm, gradually filled with granulation tissue (**Figure 5g**). The granulation tissue seemed to affect the necrotic tissue with autolysis (self-debridement, **Figure 5i**). New epithelium gradually covered the

#### *Recent Advances in Wound Healing*

healing wounds (**Figure 5g** and **h**). Microbial culture, taken from the necrotic tissue three months after cessation of antibiotic administration, did not yield pseudomonas, although normal non-pathogenic colonizing bacteria were identified.

After 5 months of COD treatment, the medial and lateral wounds were closed (**Figure 5j** and **k**). The main wound was partially closed and the rest of the wound was with pink to red granulation tissue (**Figure 5l**).

## *2.4.2 Increased epithelization*

The powerful ability of COD to promote epithelization is illustrated in the following case of a 71 years old man with NIDDM and diabetic neuropathy. The patient had osteomyelitis of the calcaneus, which necessitated extensive debridement of the heel and the infected calcaneus bone. The wound did not heal and the calcaneus broke through area of weakness due to the missing bone, thus creating a rocker deformity. Repeated surgery with debridement of the soft tissue, correction of the foot alignment and fixation with Steinman pins was carried out (**Figure 6a** and **b**, 1-week

#### **Figure 6.**

*Epithelization of a rocker deformity related plantar deep wound. The patient had resection of infected calcaneal bone with correction and stabilization of ensuing rocker deformity with Steinman pins. COD was applied at surgery. a. one week following surgery the deep calcaneal wound is evident' without signs of infection. b. X-ray showing the inserted Steinman pins. The missing plantar calcaneal bone and the deep soft tissue void underneath can be seen. c. Three weeks post-surgery, following the use of the COD, skin began crawling from the side surface into the depth of the wound. Further coverage of the wound granulation tissue with epidermal tissue is seen after 7 weeks (d), 8 weeks (e) and 3.5 months (f) of COD treatment, resulting in 95% complete closure of the wound at 4.5 months (g). h and i. lateral foot and Axial calcaneal X-rays of the foot 4.5 months after surgery. Although large soft tissue void is prominent, it is filled with practically normal looking skin that crawled in. During all this period the foot was treated solely with COD.*

*Copper, an Abandoned Player Returning to the Wound Healing Battle DOI: http://dx.doi.org/10.5772/intechopen.96952*

post-surgery). At surgery and thereafter the wound was dressed with COD, which was changed weekly. While relatively rapid granulation seemed to fill the depth and walls of the large cavity, normal looking skin began crawling from the side surface into the depth of the wound. **Figure 6c**, taken 3 weeks post-surgery demonstrated epithelization beginning at the superficial wall of the cavity (arrows). This phenomenon is further demonstrated in the photos taken at 7, 8 weeks (**Figure 6d** and **e**), 3.5- and 4.5-months post-surgery (**Figure 6f** and **g**). The corresponding x-rays at 4.5 months are shown in **Figure 6h** and **i**.

## *2.4.3 Reduction of post-operative swelling and inflammation*

62-year-old man suffered from degenerative changes of the 1st metatarsophalangeal joint (Hallux Rigidus) and metatarsalgia. The forefoot deformities included hallux valgus Interphalangeus, subluxed lesser MTPJ's, hammer 2nd toe and Bunionette deformity (**Figure 7a**). Surgery included cheilectomy of the first metatarsal head, Akin-Moberg osteotomy of the base of the proximal phalanx of the big toe, Weil osteotomies of the 2nd and 3rd metatarsals, Chevron osteotomy of the 5th metatarsal and PIPJ arthrodesis to correct the 2nd hammer toe (**Figure 7b**). The later was fixed with Kirschner wires (KW) and so was the 5th metatarsal. The 2nd and 3rd metatarsals were fixed with a screw and the bog toe proximal phalanx osteotomy was secured with absorbable suture. The foot was dressed with COD immediately after surgery with first dressing change after 3 weeks. At that time, surgical wounds were without any sign of infection or inflammation (**Figure 7c**). Despite having 4 metatarsal osteotomies and one toe arthrodesis in any of these sites, there was no swelling to the degree that normal skin wrinkles could be observed (**Figures 7c**-**e**). This is in contrast to the usual significant swelling that is observed for several months after foot osteotomies.

#### **Figure 7.**

*Reduction of swelling after forefoot surgeries and osteotomies. a. Forefoot deformities in a 62-year-old man. Hallux valgus, hammer second toe, subluxed 2nd and 3rd metatarsophalangeal joints and Bunionette deformity are observed. b. X-ray image taken 2 months after surgery demonstrates osteotomy of the base of the proximal phalanx of the big toe, Weil osteotomies of the 2nd and 3rd metatarsals, Chevron osteotomy of the 5th metatarsal and PIPJ arthrodesis of the 2nd hammer toe (arrows). c. Clinical photo of the foot at first dressing change three weeks after surgery (COD dressings were applied during surgery). Surgical wounds are without any sign of inflammation and lack of swelling and skin wrinkles can be seen. The tip of the KW's fixing the 2nd toe and the 5th metatarsal are seen and marked with red arrows (the second toe KW is wrapped with plaster to prevent accidental pullout) d. Oblique view of the same foot at the same visit after stitches removal demonstrates clearly the reduction of swelling. e. Clinical photos taken at 5 weeks post-surgery. Skin wrinkles and no swelling is again evident.*

#### **Figure 8.**

*Reduction of scar formation – Case Report 1. a. Bunion surgery that included Chevron type osteotomy of the 1st metatarsal and fixation with two KW's. b. X-Ray of the foot following surgery. c. Surgical incision appearance at two weeks post-surgery. d. Surgical incision appearance at four weeks post-surgery. e. X-Ray of the foot at 7 weeks post-surgery. f. Clinical appearance of the foot at seven weeks post-surgery. g. Surgical incision appearance at seven weeks post-surgery. h. Enlargement of the surgical site at seven weeks post-surgery. Comparison between the scar appearance after 7 weeks (i) and 2 weeks (j) post-surgery shows that most of the scar has disappeared and was not detectable even at high magnification (h).*

*Copper, an Abandoned Player Returning to the Wound Healing Battle DOI: http://dx.doi.org/10.5772/intechopen.96952*

#### **Figure 9.**

*Reduction of scar formation – Case Report 2. a. X-ray of both feet showing 1st and 2nd metatarsal osteotomy due to hallux valgus and metatarsalgia. b. Surgical incision appearance at 2 weeks post-surgery. c. X-ray and d. photographs of both feet one-year post-surgery showing successful foot positioning. e. Surgical incision appearance of right foot one-year post-surgery. f. Surgical incision appearance of right foot one-year postsurgery. g. Hypertrophic scar 25 years post-surgery due to elbow fracture.*

#### *2.4.4 Reduction of scar formation*

Reduction of scar formation may be difficult to prove or demonstrate since the final surgical incision healing is a function of surgical technique as well as the patient own tendency to produce hypertrophic scar or even keloid. We have therefore elected to present the reduced scar formation in two cases of bunion surgery. **Figures 8** and **9** with unexpected rapid healing in one patients and very good healing despite basic tendency to hypertrophic scar in another patient.

#### *2.4.4.1 Case report 1*

The first one is a 20-year-old healthy woman who had bunion surgery which included Chevron type osteotomy of the 1st metatarsal and fixation with KW (**Figure 8a**). The surgical incision and the KW's are seen at two weeks postsurgery (clinical photos and x-rays, (**Figure 8a** and **b**). The KW's were removed at 4 weeks. By that time nice healing of the surgical incision seems to have taken place (**Figure 8c** and **d**). At 7-weeks post-surgery, the osteotomy has healed and clinical appearance is satisfactory (**Figure 8e** and **f**). The surgical scar is very delicate (**Figure 8g**). A comparison between the original incision and its appearance after 7-weeks shows that ~80% of the incision scar is not observed even in high resolution and magnification photography (**Figures 8h**–**j**). This implies that either direct epithelization has occurred or remodeling of the scar took place. The superb cosmetic results at 7-weeks seems to be beyond a "successful case" and we attribute it to the beneficial effect of copper oxide on wound healing.

#### *2.4.4.2 Case report 2*

The second case is of a 49-years-old healthy woman who underwent bilateral hallux valgus surgery, which included distal first metatarsal osteotomy, fixed with KW's and Weil ostetomies of the 2nd (+ 3rd) metatarsals (**Figure 9a**). The feet were dressed with COD. Two weeks post-surgery swelling was minimal and even skin wrinkles could be seen (**Figure 9b**). One year post surgery the foot position is very good (**Figure 9c** and **d**). The dorsal incision scarring is minimal (**Figure 9d**), the medial scar on both feet is hardly visible (**Figure 9e** and **f**). The patient said she has a tendency to create hypertrophic scars, for example a scar following open reduction and internal fixation of elbow fracture 25 year ago (**Figure 9g**).

## **3. Discussion**

Copper is a natural mineral, which is an essential element of nutrition due to its role in many of the physiological processes in all body tissues [12, 13]. We have reviewed the beneficial effect of copper in wound healing based on abundant basic science research as well as our cumulative experience with the use of COD. Copper has been known for its antimicrobial properties including against all common wound pathogens and resistant bacteria. Similar properties are attributed to silver. Indeed, silver-containing wound dressings are widely used in wound treatment to reduce the risk of wound and wound-dressing contamination [43]. It is desirable, of course, to have a wound dressing that also promotes wound healing. Previous research has shown the beneficial effect of copper on skin and integumentary system [14] as well as on wound healing in diabetic mice [41]. The mechanism by which copper exerts its positive roll has been shown to be through up-regulating the level of Hif-1α, which is a key protein in tissue generation, especially in conditions of ischemia, like in hard to heal wounds. In this regard copper differs from silver, which exerts the opposite effect on wound healing, probably by downregulating Hif-1α [44]. Therefore, the usefulness of silver-based dressing in promoting wound healing is questionable, among others due to cellular toxicity [45, 46].

However, since copper has potent biocidal properties [37], but in contrast to silver, is an indispensable trace element extremely well metabolized by the human body [12], we hypothesized that it could substitute silver in wound dressings. This would be justified for the goal of reducing bio-contamination. But, even more importantly, are the key roles copper plays in skin generation and angiogenesis. We further hypothesized that the inability of wounds to heal in individuals with compromised peripheral blood supply (e.g., with vascular diseases or diabetics), is partially due to low levels of copper in the wound site [47]. We suggested that by using a copper oxide-containing wound dressing we would slowly release *in situ* copper ions needed for angiogenesis, skin regeneration and wound healing.

Based on the above, we prepared wound dressings containing copper oxide (COD, **Figure 1**). The COD, which possess potent biocidal properties (**Figures 2** and **3**), were found to be safe in animal studies, showing no skin irritation and no local damage to open wounds or systemic pathological alterations in a porcine

#### *Copper, an Abandoned Player Returning to the Wound Healing Battle DOI: http://dx.doi.org/10.5772/intechopen.96952*

full-thickness wound model (unpublished data). Indeed, the risk of adverse reactions due to dermal contact with copper is extremely low [48, 49]. Furthermore, wounds inflicted in diabetic (db/db) mice under sterile conditions and kept covered throughout the study with sterile wound dressings demonstrated a statistically significant enhancement of wound closure when the dressings contained copper oxide [41]. Enhanced wound healing was nearly that anticipated from wild-type mice, where similar full-thickness dorsal skin wounds reach complete closure 7–10 days earlier than in db/db mice [50]. In contrast, commercially-used silver-containing wound dressings did not accelerate wound healing in this model [41]. Following the clearance of the COD to be used clinically, we have found, as described in some representative cases in the article, the significantly better results obtained with the COD than SOC dressings, including silver-based wound dressings.

The demonstrated cases (**Figures 5**–**9**) show the several effects of COD on different stages and aspects of wounds healing. The effects were reduction of colonized bacteria and superficial infection, as well as increased granulation and epithelization, as demonstrated in **Figure 5**. **Figure 6** shows rapid epithelization of normal looking plantar skin into the cavity underneath the calcaneus. In addition to wound closure, we see in **Figures 7**–**9** improved healing process on primary closed clean surgical incisions, which expresses itself in improved scar formation and reduces swelling.

Due to the effect of COD on the various stages of wound healing, we now often use the COD continuously during the various phases of wound treatment. For example, we apply them on debrided wounds after partial foot amputation due to diabetic foot infection (instead of povidone-iodine or chlorine based dressings), and as healing progresses, we use them to assist in filling the wound with granulation tissue (for example, instead of using Negative Pressure Wound Therapy (NPWT)). Once the wound has filled with new tissue, we use the COD to help epithelization until full wound closure is achieved.

Another advantage of COD is the few dressing changes it needs, usually once or twice weekly. This makes it convenient to the patient and savvy for the health care system. In the hospital, COD may replace chlorine-based dressings (e.g. Eusol or Daikin solutions), which needs changes 2–3 times daily, and thus reduce the workload on the nursing staff as well as diminishing the risk of spreading resistant bacteria and cross contamination in the Ward.

Additional studies are needed to further elucidate the exact mechanisms by which copper stimulates wound healing. It is clear, however, that copper directly or indirectly stimulates many factors, some of which are impaired in diabetics and are important for keratinocytes and fibroblasts proliferation, epithelization, collagen synthesis, extracellular matrix remodeling and angiogenesis. Indeed, by utilizing COD dressings on chronic wounds, which had failed to heal or healed slowly with other well-recognized wound care protocols, we found improved wound healing kinetics and wound closure in most patients.

## **4. Conclusions**

As demonstrated by the murine diabetic model [41], the positive effect of the copper oxide-containing dressings is not related solely to its potent biocidal properties, but to the direct stimulation of wound repair. No adverse events were recorded with the use of the copper dressings and all patients showed positive response to its application. Thus, copper dressings appear to hold significant promise in the clinician's ongoing struggle to heal both acute and chronic wounds. Additional randomized, controlled studies should be conducted to further validate the efficacy of topically applied copper oxide-impregnated dressings.

*Recent Advances in Wound Healing*

## **Author details**

Gadi Borkow1 \* and Eyal Melamed2

1 MedCu Technologies Ltd., Herzliya, Israel

2 Foot and Ankle Service, Department of Orthopaedics, Rambam Health Care Campus, Haifa, Israel

\*Address all correspondence to: gadib@medcu.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Copper, an Abandoned Player Returning to the Wound Healing Battle DOI: http://dx.doi.org/10.5772/intechopen.96952*

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[21] Parke A, Bhattacherjee P, Palmer RM, Lazarus NR. Characterization and quantification of copper sulfate-induced vascularization of the rabbit cornea. Am J Pathol. 1988;130(1):173-178.

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[25] Gerard C, Bordeleau LJ, Barralet J, Doillon CJ. The stimulation of angiogenesis and collagen deposition by copper. Biomaterials. 2010;31(5):824-831.

[26] Rucker RB, Kosonen T, Clegg MS, Mitchell AE, Rucker BR, Uriu-Hare JY, Keen CL. Copper, lysyl oxidase, and extracellular matrix protein crosslinking. Am J Clin Nutr. 1998;67(5 Suppl):996S–1002S.

[27] Sajithlal GB, Chithra P, Chandrakasan G. An in vitro study on the role of metal catalyzed oxidation in glycation and crosslinking of collagen. Mol Cell Biochem. 1999;194(1-2):257-263.

[28] Kothapalli CR, Ramamurthi A. Copper nanoparticle cues for biomimetic cellular assembly of crosslinked elastin fibers. Acta Biomater. 2009;5(2):541-553.

[29] Tenaud I, Sainte-Marie I, Jumbou O, Litoux P, Dreno B. In vitro modulation of keratinocyte wound healing integrins by zinc, copper and manganese. Br J Dermatol. 1999;140(1):26-34.

[30] Das A, Sudhahar V, Chen GF, Kim HW, Youn SW, Finney L, Vogt S, Yang J, Kweon J, Surenkhuu B, Ushio-Fukai M, Fukai T. Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo. Sci Rep. 2016;6:33783.

[31] Edwards R, Harding KG. Bacteria and wound healing. Curr Opin Infect Dis. 2004;17(2):91-96.

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[33] Robson MC, Stenberg BD, Heggers JP. Wound healing alterations caused by infection. Clin Plast Surg. 1990;17(3):485-492.

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#### *Copper, an Abandoned Player Returning to the Wound Healing Battle DOI: http://dx.doi.org/10.5772/intechopen.96952*

blood flow in experimental wounds inoculated with Staphylococcus aureus. Eur Surg Res. 1988;20(1):33-38.

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[45] Atiyeh BS, Costagliola M, Hayek SN, Dibo SA. Effect of silver on burn wound infection control and healing: review of the literature. Burns. 2007;33(2):139-148.

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[49] Gorter RW, Butorac M, Cobian EP. Examination of the cutaneous absorption of copper after the use of copper-containing ointments. Am J Ther. 2004;11(6):453-458.

[50] Chan RK, Liu PH, Pietramaggiori G, Ibrahim SI, Hechtman HB, Orgill DP. Effect of recombinant platelet-derived growth factor (Regranex) on wound closure in genetically diabetic mice. J Burn Care Res. 2006;27(2):202-205.

## **Chapter 10**

## Contribution of Topical Agents to Wound Healing

*Tadej Voljč and Danijela Semenič*

## **Abstract**

The process of wound healing is often accompanied by bacterial infection or critical colonization, which leads to an extension of the inflammatory response phase and delayed epithelization. In the review of scientific articles, we found the description and mode of action of topical antiseptic agents, including silver and sodium hypochlorite solution, to control the spread of microorganisms. The value of hyaluronic acid for wound healing is described. Furthermore, a novel treatment option with microspheres is mentioned. Attachment of cells to microspheres establishes a local cytokine response that acts anti-inflammatory, cell attachment results also in morphological and functional cell changes that reactivate healing.

**Keywords:** chronic wounds, wound infections, antisepsis, silver, microspheres, polystyrene microspheres, hyaluronic acid, sodium hypochlorite

## **1. Introduction**

Chronic wounds represent a serious problem for both the patient and the physician.

As chronic wounds are considered venous ulcers, wounds due to peripheral arterial occlusive disease, diabetic neuropathic, diabetic ischemic, diabetic neuroischemic wounds, pressure sores and atypic chronic wounds.

Atypical chronic wounds comprise less than 5% of all chronic wounds [1, 2]. They may present with a clinical picture the clinician has not previously encountered, therefore raising a diagnostic dilemma and challenge. A full range of pathogenic categories, including vascular, autoimmune, inflammatory, infectious, neoplastic, genetic, and drug-related processes, can cause an atypical ulcer [1–3].

Also, every acute wound has a certain potential to become chronic, usually with co-infection or when associated diseases are present.

For the successful treatment of chronic wounds, it is necessary to know and treat the underlying cause and provide the wound appropriate method to optimize wound healing.

## **2. Silver active compounds**

Metallic silver has been used in the treatment of infections from at least the 18th century [4]. More recently products containing silver have been developed for the topical treatment of chronic wounds due to its antiseptic and anti-inflammatory activity [5, 6].

While metallic silver is chemically largely inert, it readily releases an electron in contact with moisture, becoming more reactive and gaining significant biocidal properties. In its ionized form (Ag+ ) silver can interfere with thiol (-SH) groups, promote the production of reactive oxygen species (ROS) and bind to bacterial DNA and RNA. Through these mechanisms it causes structural changes to the bacterial cell wall, intracellular and nuclear membranes, disrupts the production of ATP and inhibits replication, ultimately leading to the loss of function and cell death [4–8]. Silver ion-release products are effective against various bacteria, including methicillin-resistant *S. aureus* (MRSA) and vancomycin resistant enterococcus (VRE) [4], fungi and viral pathogens [9]. Silver containing wound dressings are able to reduce the number of viable bacteria in a matter of minutes [10]. Silver ions were shown to destabilize the biofilm produced by *S. epidermidis* [11]. As such it presents a good adjuvant treatment option to combine with surgical debridement for dealing with bacterial biofilm.

Bacterial resistance to silver has rarely been reported, likely due to its multiple mechanisms of bactericidal action [12]. However, most reported cases of bacterial resistance to silver stem from burn units, where large amounts of silver salts were used as wound antiseptics. Among the described silver resistant strains were *E. coli*, *Enterobacter cloacae*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Salmonella typhimurium* and *Pseudomonas stutzeri*. The outbreak of a silver resistant strain of Salmonella even caused the closure of a burn unit at the Massachusetts General Hospital after three patients have died of septicemia in 1973 [13, 14].

While ionic silver has the highest therapeutic capacity, it is also rapidly inactivated after being applied to a wound due to its high nonspecific reactivity [7]. Instead, nanoparticles of silver have been used in modern wound dressings, combining a large active surface area and a degree of control over the rate of Ag+ release into the wound [7, 8]. Silver wound dressings have been developed in several ways, some binding nanocrystalline silver to carbon fibers, attaching it to polyurethane foam, attaching it to hydrocellular foam and coating it over polyethylene. Silver can either be presented at the surface of the product facing the wound, diffusing and acting on the wound itself, or be bound inside a foam or mesh acting on the pathogens absorbed into the material [15, 16]. In a favorable environment silver cations can be released into the wound for several days from a single dressing [17], avoiding frequent dressing changes and unnecessary wound manipulation. After an initial lag the rate of Ag<sup>+</sup> release can be constant [7].

The majority of review articles noted the poor quality of the published data on the use of silver in wound care. Many studies in this area are funded or performed by manufacturers of silver-containing wound dressings [12], adding to the risk of bias.

The therapeutic range of Ag<sup>+</sup> concentration is 30–60 ppm, above which a toxic effect on skin keratinocytes becomes increasingly likely. The goal is thus to have a silver wound dressing that can exert a concentration of silver cations in the wound within the therapeutic range (30–60 ppm) for a prolonged period of time (several days) [12, 13].

Traditional preparations of silver in wound care, silver nitrate and silver sulfadiazine, are as such of limited suitability, providing an initial Ag+ concentration very much above the therapeutic range (3176ppm and 3025ppm respectively), with little residual activity [13].

Nanocrystalline silver incorporating wound dressings are able to provide a Ag<sup>+</sup> concentration of 50–100 ppm in a constant manner [12, 18]. As such they are more suited in the treatment of infected wounds compared to traditional silver preparations, albeit still providing a wound silver ion concentration above the target range. The effect of silver ions has been shown to have a synergistic effect with negative

#### *Contribution of Topical Agents to Wound Healing DOI: http://dx.doi.org/10.5772/intechopen.97170*

pressure wound therapy, with silver-coated polyurethane foam providing better results than the use of a polyurethane sponge alone [12]. In this way wound Ag<sup>+</sup> concentration of 20–40 ppm can be achieved, falling in the optimal range. A similar effect can be observed by adding a layer of silver-coated nylon between a polyurethane sponge and the wound [19].

Despite their broad spectrum of antimicrobial action and low incidence of bacterial resistance silver-containing products do not come without limitations. The use of silver sulfadiazine, a once very popular preparation of silver, especially in the treatment of burn wounds, is nowadays widely discouraged due to the comparatively high risk of negative effects on the viable wound tissue and little to no advantages when compared to nanocrystalline silver preparations [12, 13, 20]. Not only traditional preparations of silver but nanocrystalline silver, too, has been shown to have an inhibitory effect on epithelization, albeit to a lower extent [20, 21]. Considering this and other possible side effects of silver-releasing products they are thus not recommended for treating non-infected, clean wounds or closed surgical incisions [12].

Silver-releasing products have been shown to reduce the viability, induce oxidative stress and DNA damage in porcine ex vivo skin cells, as well as promote the production of pro-inflammatory IL-6 by monocytes and reducing the oxidative burst and viability of neutrophils in a dose-dependent manner [20]. It should thus come as no surprise that though an important tool in the fight against wound infections they should be used cautiously. The current state of research suggests they should be used on infected wounds for up to 2 weeks, after which the wound should be evaluated. If there is clear improvement with persistent signs of infection the use can be continued until a total of 4 weeks of silver-assisted therapy is reached. If there is no sign of improvement after two weeks the use should be discontinued immediately. Silver-releasing products should not be used for over 4 weeks without a good clinical rationale [22].

Topical application of silver in a nonadhesive wound dressing can be used not only for the treatment of typical chronic wounds, but also for un-usual wounds for example for the treatment of pyoderma gangrenosum ulcers [23]. Pyoderma gangrenosum (PG) is often associated with autoimmune disease and is a neutrophilic dermatosis, characterized by a wide range of clinical presentations, among which recurrent cutaneous ulcerations are the most characteristic [24]. Ulcers are very painful [23]. In addition to systemic immunosuppressant therapy, topical or intralesional drugs can be used [25].

## **3. Hyaluronic acid**

Hyaluronic acid (HA) is a linear glycosaminoglycan (GAG) molecule composed of disaccharide units of GlcNAc and D-glucuronic acid linked together with β-1,4 and β-1,3 glycosidic bonds. The sequence can be repeated over 20,000 times [26]. Hyaluronic acid was first discovered in the vitreous humor in 1934 by Karl Meyer and John Palmer. They proposed the name "hyaluronic acid" lending from the Greek hyaloid (vitreous) and uronic acid, one of the two repeating monosaccharide units [26].

Most human cells have the ability to synthetize HA at some point in their cell cycle, leading to the presence of HA as a component of the extracellular matrix (ECM) in many tissues throughout the human body [27, 28]. The hygroscopic and viscoelastic properties of HA and its derivatives provide a lubricating environment for cells [29]. Its derivatives are an important role in HA's function. It is involved in various processes, from fertilization and development to cancer [26]. Hyaluronic

acid and its signaling receptors play a role in initiating an inflammatory response, maintaining structural cell integrity, and promoting recovery from tissue injury [30]. Interestingly, high molecular weight HA displays an anti-inflammatory effect whereas low molecular weight HA acts immunostimulatory and pro-inflammatory [31–33]. High molecular weight HA has been shown to exhibit a cytoprotective effect [34].

HA stimulates the development of fibrin, phagocytic activity, neutrophil and macrophage mobility, assists in cellular infiltration and in the mobilization of proinflammatory cytokines [29, 35]. Hyaluronic acid plays a role in all stages of wound healing [35]. In the early granulation stage HA, abundant in the ECM, facilitates cell proliferation and migration into the temporary wound matrix, and helps with the organization of the granulation tissue matrix. At a later stage HA helps to stabilize the matrix by scavenging free radicals. In the proliferation stage HA plays a role in supporting and regulating basal keratinocytes [35]. A lower HA content has been observed in hypertrophic scars and in the keloid compared to ordinary scars [32].

Due to its many regulatory functions hyaluronic acid has seen significant use as a topical agent in wound treatment. Without yet a clear literature consensus on its efficacy there does seem to be an overall positive effect of HA on the healing of chronic wound ulcers of various etiologies, burns and epithelial surgical wounds no matter the form in which HA is applied (e.g., pad, cream or substrate) [36]. However, the low number of high-quality studies in this area limits any systematic review trying to determine HA's effects in clinical use. To illustrate, a marked increase in the healing rate of diabetic foot ulcers was described in a paper, even when compared to other forms of ulcers among chronic wounds [36, 37], while another systematic review found no advantage whatsoever in the HA group when compared to paraffin gauze [29]. Both systematic reviews were only able to include two papers studying the topic.

#### **3.1 Combination of different active substances (Ag<sup>+</sup> , chlorhexidine, hyaluronic acid)**

Modern wound healing products combine different active substances in a single product, better adjusting the finished product to the clinical requirements of a certain wound type. An example is a combination of Ag<sup>+</sup> and chlorhexidine, both

**Figure 1.**

*Chronic wound before treatment with a combination of Ag+, chlorhexidine and hyaluronic acid in a spray.*

*Contribution of Topical Agents to Wound Healing DOI: http://dx.doi.org/10.5772/intechopen.97170*

#### **Figure 2.**

*The result of treatment with spray that contains Ag+, chlorhexidine and hyaluronic acid after 6 weeks.*

antiseptic agents bound to silicon dioxide, with added hyaluronic acid to promote the healing process.

**Figure 1** shows an example of a chronic wound treated in our institution. Written consent for publication by the patient was obtained.

Presented is a 73-year-old male patient, with diabetes, venous insufficiency, peripheral arterial disease, and heart failure, with consequent bilateral lower leg edema. The treatment was carried out with a combination of Ag<sup>+</sup> , chlorhexidine and hyaluronic acid in a spray, every 2 days, covered by a non-adhesive modern dressing. The result of treatment is visible after 6 weeks (**Figure 2**).

Promising results for this combination have also been reported regarding the incontinence associated dermatitis related wound regression rate, moisture control and pain reduction in a study [38].

## **4. Sodium hypochlorite**

Sodium hypochlorite is a strong oxidizing agent and was first discovered in 1787 in Paris by Berthollet. During World War I it was used by Alexis Carrel and Henry D. Dakin as an effective antiseptic agent for combat wounds, sparking its popularity as a wound antiseptic between the two world wars. The popularity of such use later declined with the rise of antibiotics, but it remained a popular household product – sodium hypochlorite is the active ingredient in bleach [39].

With an increasing awareness of the limitations of antibiotic drugs it is again being investigated as a viable option for the prevention and treatment of wound infections. Today a 0.5% sodium hypochlorite solution or more diluted preparations are used. Dakin's solution has been known to have a bactericidal effect against *S. aureus* (MRSA and non methicillin-resistant), *Pseudomonas aeruginosa*, *Escherichia coli*, *Proteus mirabilis*, *Serratia marcescens*, *Enterobacter cloacae*, group D enterococci, *Bacteroides fragilis*, *Streptococcus mitis*, *Staphylococcus epidermidis*, and a fungicidal effect on *Candida albicans* among others [39].

However, as a strong oxidizing agent it is certainly able to exert an important cytotoxic effect on healthy human cells, too. While lower concentrations of sodium hypochlorite have been shown to retain their bactericidal effect, it seems that the cytotoxic effect on human cells sees a stronger reduction with dilution [39]. This area is notably lacking in detailed research.

While sodium hypochlorite is currently not widely used as a wound antiseptic some institutions are reporting positive results with its use. It has been reported to be effective in the treatment of infected and open wounds [39]. In patients undergoing coronary artery bypass surgery it has been shown to be more effective as an irrigator for the prevention of post-sternotomy wound infections when compared to povidone-iodine [40]. It has also been investigated as an irrigator for drain tubes after breast and axillary operations, helping provide much lower rates of positive drain bulb cultures and a lower bacterial load when combined with a chlorhexidine disc at the drain exit site and compared to the standard of drain care (cleansing with alcohol swabs) [41].

## **5. Polystyrene microspheres**

The microspheres are round in shape, located in a suspension of sterile watersoluble solution at a concentration of 0.025%, consist of polystyrene and have a diameter of 5 micrometers.

They are intended for topical application on the wound bed, applied in the form of drops (1–2 drops/cm2 of the wound bed). The mechanism of action is explained by both preclinical in vitro and in vivo studies [42], where the presence of microspheres shows increased cell proliferation, increased cell migration and also increased activity of membrane-bound enzyme proteolytic complexes on cells [43].

The property of the size and surface of the microspheres offers a supportive microenvironment on the surface of the chronic wound, as it serves as an additional surface to which epithelial, endothelial, and even inflammatory cells can attach and migrate [44, 45]. Microspheres have a negative charge on their surface, which accelerates the secretion of growth factors such as growth factor beta-1 [45], and an excess of proteolytic enzymes, which inhibit normal healing, metalloproteinases and human neutrophil elastase, also bind to their surface [42].

The purpose of treatment with microspheres is, in a way, to "de-chronicize" a chronic wound into an acute wound condition, by inducing changes in the microenvironment that would allow the best possible conditions for healing [46].

In a study where microspheres were administered to 54 patients with chronic wounds of various etiologies over a period of 4.5 weeks [42], an effective reduction in wound size and area was described. 39% of the wounds completely healed, and in the remaining cases, effective growth of granulation tissue of the wound bed was observed, which covered up to 75% of the surface of the wound bottom [42].

Accelerated growth of granulation tissue is an indicator that the wound is in the proliferative phase of healing. Granulation tissue is the basis for later reepithelialization or surgical closure of the wound with suturing, skin graft or flap [47], and the healing indicator is a reduction in wound size, which means that epithelialization began from the edges of the wound [42]. Wounds where bone or tendon are exposed in depth are less responsive to healing [48, 49], but according to the results described in the literature, such wounds may also respond effectively to microsphere treatment [42].

From the possible side effects in the literature, pain and itching are the most common symptoms [42].

In a multicenter randomized double-blind study treating 66 patients with a chronic wound of various etiologies, complete wound healing was found in 20% of patients, in 80% of patients the wound bed was 3/4 covered with granulations after 12 weeks of treatment [46].

Treatment with the application of microspheres potentially accelerates the growth of granulation tissue and epithelialization. We also notice that after the *Contribution of Topical Agents to Wound Healing DOI: http://dx.doi.org/10.5772/intechopen.97170*

**Figure 3.** *Chronic wound before treatment with polystirene microspheres.*

**Figure 4.**

*Effective epithelization and scar formation after 12 weeks of treatment with polystirene microspheres.*

application of microspheres, fibrin plaques are easier to remove from the surface of the wound bottom. Microsphere therapy is suitable for the treatment of both inpatient and outpatient patients, and application by the community health service is also possible. The secondary coating covering the microspheres must be nonadhesive and as non-absorbent as possible.

An example where the effective treatment with microspheres is presented is a 44-year-old male patient with a chronic wound on the skin of the abdominal wall after a hepatectomy, after hernioplasty and subsequent resection of the infected hernia mesh. Drops with microspheres were applied on the wound bed every 2 days, over which a non-adhesive modern wound dressing with a silicone contact layer and polyurethane foam was applied. The condition of the wound before treatment (**Figure 3**) and the condition after 12 weeks of treatment (**Figure 4**) with effective epithelization and scar formation [50].

## **6. Activated charcoal**

With its biocompatibility and large surface area, activated charcoal acts as a useful adsorbent of fatty molecules. The most important raw materials for its production are rice, coconut shell and different types of wood. Activated charcoal is obtained by heating the material to around 1000°C in an oxygen-free environment and the subsequent breakdown of carbon-rich compounds. Through this process the material becomes porous, greatly increasing its surface area and its adsorbent capacity [51].

Commercial activated charcoal containing wound dressings contain between 85 and 98% active carbon, with the main difference being the material used to cover the charcoal cloth. Such materials include viscose rayon, alginate, polyethylene, polyamide and nylon [52]. *E.coli* was among the first cultures whose adherence to activated charcoal has been studied [53]. Gram-negative bacteria have been found to adhere stronger and wash out less easily from activated charcoal than Grampositive bacteria [54].

## **7. Polyhexanide**

Polyhexanide, also known as polyhexamethylene biguanide (PHMB), is a synthetic compound with a broad antimicrobial spectrum, including various types of Gram-positive and Gram-negative bacteria and some fungi (*Candida* spp., *Aspergillus* spp.). It acts as a strong base, binding to negatively charged phospholipid molecules in cell membranes of microorganisms, disturbing their integrity and leading to loss of viability. Its effect on neutral phospholipids of human cells is supposed to be negligible [55].

Betaine, with its amphoteric properties, acts as a surfactant and can be used in the cleaning of wounds. Often polyhexanide and betaine are used together in order to reduce the microbial burden and promote wound healing. As a combination they are available in a number of commercial products and were shown to be effective in reducing the number of viable bacteria in a formed biofilm produced by MRSA [55, 56]. Products containing both polyhexanide and betaine are currently often used in the management of pressure ulcers, venous ulcers and other chronic wounds, while further indications are under investigation [55].

## **8. Povidone iodine**

Povidone iodine's broad spectrum of activity, ability to penetrate biofilms, lack of associated resistance, anti-inflammatory properties, low cytotoxicity and good

tolerability have been cited as important factors, and no negative effect on wound healing has been observed in clinical practice [57].

The efficacy of povidone iodine on wound healing in the presence of biofilms has been reviewev [57, 58]. Studies have confirmed the *in vitro* efficacy of povidone iodine against *S. epidermidis* and *S. aureus* growth, as well as the inhibition of staphylococcal biofilm formation at sub-inhibitory concentrations [57–59].

## **9. Other topical agents**

Different clinical practices are used according to different medical institutions in the world. However, several other compounds are also part a wound-care specialist's daily routine. According to the conclusion of infectious disease specialists and surgical infection specialist at the University Medical Center Ljubljana, antibiotic ointments for chronic wounds are not recommended, with purpose, to prevent possible acquired microbial resistance. Topical antibiotic ointment is occasionally used only for impetiginous skin lesions and dermatological indications, not for the treatment of chronic wounds.

The use of topical antibiotics should be discouraged if appropriate antiseptics are available [57, 60].

While the general principles of action remain similar across different substances, a chapter on topical wound healing agents would not be complete without a brief overview of other important agents.

### **9.1 Mafenide acetate**

Mafenidine acetate has been developed as a topical Sulphonamide in 1966. It is effective in reducing the wound bacterial load through the inhibition of nucleoside synthesis and became especially popular in the treatment of burn wounds. First marketed as a 10% cream formulation it did not come without important side effects – both local (neoeschar formation, pain) and systemic (metabolic acidosis) [61, 62]. Its concentration has later been reduced to 5%, with a reduction in the incidence of side effects, but even with preparations used today inhibitory effects on skin DNA and protein synthesis remain a concern [61–63]. This and the high cost of mafenide therapy led to recent research interests on whether the mafenide concentration could further be reduced. While the 5% mafenide acetate cream remains an important agent in burn treatment, novel research indicates that a 2,5% mafenide cream could be equally efficacious as its 5% counterpart [61, 62].

### **9.2 Bacitracin**

Bacitran is derived from *Bacillus Subtilis* and the *licheniformis* group of bacteria and is one of the most widely used topical antibiotics. Its bactericidal activity spans against several Gram-positive and Gram-negative organisms [64]. Bacitracin can provide a 90% reduction of bacterial viability in 1 hour after application and an important reduction of bacterial adherence, without important systemic effects being associated with topical use [65]. There is some evidence to support its use in the prevention of surgical wound infections, however, the use of bacitracin for superficial clean wounds is discouraged [66–69]. While having important antibiotic properties it has also been reported to cause allergic contact dermatitis in a range from 7.7 to 9.2% in a patch-test. As with many antibiotic agents topical products have been associated with an increase in microbial antibiotic resistance. The recommended alternative to bacitracin and other topical

antibiotics for the treatment of superficial clean wounds is white petrolatum, with comparable wound infection and wound healing rates [68, 69].

## **9.3 Neomycin**

With a largely similar profile of side effects to bacitracin, is another highly popular topical antibiotic agent [68]. It is classified as an aminoglycoside and inhibits bacterial protein synthesis by binding to ribosomal RNA. It is effective against Gram-negative bacteria, with the exception of *Pseudomonas aeruginosa*, against some Gram-positive bacteria, including staphylococci, but not against streptococci and anaerobes. Its use extends across the spectrum of the prevention and treatment of chronic and non-chronic wound infections, superinfections and burns [64]. The incidence of allergic contact dermatitis is higher compared to bacitracin (7–13% in a patch-test) [69]. Plasmid-mediated resistance to neomycin has been reported in several bacterial strains, including staphylococci, *E. coli, Klebsiella spp.* and *Proteus spp.* [64].

## **10. Conclusions**

Hyaluronic acid, activated charcoal, chlorhexidine, sodium hypochlorite, polyhexanide and polystyrene microspheres serve as good examples of different already well established and potential up-and-coming topical treatment solutions. The advantage of these active ingredients is that no acquired microbial resistance, has been known so far.

By ensuring the optimal microenvironment of the wound, the transition from the inflammatory phase to the proliferative phase of healing is enabled.

The choice of treatment method must be both clinically and cost-effectively.

A short review chapter discusses the possibilities for managing the bacterial load in the wound bed, the advantages and disadvantages of different topical agents and their mode of action.

As with many already established formulations, new topical agents should be put through testing in the form of blinded randomized controlled trials, in order to provide valid support for the formulation's efficacy and safety. Only through this process can we achieve important and much needed evidence-based advances in regard to the treatment of wounds with novel and ever developing topical agents.

## **Conflict of interest**

The authors declare no conflict of interest.

*Contribution of Topical Agents to Wound Healing DOI: http://dx.doi.org/10.5772/intechopen.97170*

## **Author details**

Tadej Voljč1 and Danijela Semenič1,2\*

1 University Medical Center Ljubljana, Ljubljana, Slovenia

2 Medical Faculty, University of Ljubljana, Slovenia

\*Address all correspondence to: danijela.semenic@kclj.si

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## *Edited by Shahin Aghaei*

The human wound-healing process could be divided into four discrete phases, which have also been indicated as the hemostasis, the inflammatory, the proliferation, and the remodeling phase. For a wound to be healed efficaciously, all four phases must sequentially happen at an expected time setting. Numerous aspects can hinder one or more stages of this procedure, thus can cause inappropriate or diminished wound healing. This book reviews the recent literature on the most significant factors that affect wound healing and the potential cellular and/or molecular mechanisms involved. The factors discussed include physiology of wound healing, interferon, stem cells and photobiomodulation, chronic venous ulcer, chronic fistula, bionanomaterials, topical antiseptic agents, including silver and sodium hypochlorite solution, diabetic ulcers, and nutritional supplements such as copper.

Published in London, UK © 2022 IntechOpen © akwitps / iStock

Recent Advances in Wound Healing

Recent Advances in

Wound Healing

*Edited by Shahin Aghaei*