**3. The antibacterial activities of metals**

The antibiotic resistance of microorganisms determines serious complications like infection, and delayed wound healing and great concerns are related to the numbers and types of residing microorganisms and the ability of the host's immune system to control their proliferation [39–41]. Along with the emergence of microorganisms' resistance to multiple antibiotics, the increased healthcare costs and the huge social and economic impact of wound care have increased attention towards the biological mechanisms underlying cutaneous wound complications and have encouraged the researchers towards the development of new bactericide agents [31, 42]. The new frontier in clinical medicine and disease burden is represented by the medical applications of nanotechnology. Antimicrobial nanoparticles (NPs) offer an effective approach against numerous microorganisms where conventional antimicrobial agents fail [43, 44] and, compared with micron-sized particulate matter, have greater potential to enter cells and be more biologically active due to their small size and large surface area [X3]. Endowing ordinary products with new functionalities, consumer products containing engineered nanoparticles, are growing tremendously, and the global nanotechnology industry is becoming a major economic force of the twenty-first century [45, 46]. Some natural antibacterial materials such as zinc, silver and copper possess great antibacterial properties at nanometric size and their way of interaction with bacteria provides unique bactericidal mechanisms [43, 47].

biofilm often involves its physical removal from the wound surface with sharp or surgical

The control of biofilm is a key part of chronic wound management, but the use of antiseptic dressings for preventing and managing biofilm and infection still needs further research involving well-designed, randomized controlled trials [29]. The concept of a bacterial contamination, colonization and biofilm-related infection is now widely accepted in wound care, and the recognition of the biofilm and the evolution of topical antiseptics to control bioburden in wounds are considered strictly related to the concept of TIME (tissue, infection/inflammation, moisture balance and edge of wound) and to its relation with the current best practice [30]. In healthcare, infections lead to longer hospital stays for patients, specifically wound dressings and increased hospital costs [12]. Also worsened by an ageing population and the incidence of diabetes and obesity, the huge economic and social impact of wounds requires higher level of attention and resources to understand biological mechanisms underlying cutaneous wound

Infections of the dermis, including burns, surgical site infections and non-healing diabetic foot ulcers affect over a million people. Individuals with diabetes represent a particularly vulnerable category because many of them develop foot ulceration during the course of their disease and undergo amputation. In addition to diabetics, several other groups of immune-compromised patient populations are plagued by slow-healing and non-healing wounds, such as trauma and burn victims, cancer patients and pressure ulcers in the elderly [32]. The incidence, morbidity, mortality and costs associated with non-healing of chronic skin wounds are dramatic. Chronic wounds cost millions of dollars annually in the healthcare industry of the United States, and biofilm significantly contributes many billions of dollars to the global cost of chronic wounds because of its role in delaying the wound-healing process and extending

Along with the direct medical costs borne by the hospital or insurer, also indirect costs including lost patient productivity and diminished functional status should be considered [36]. The control of bioburden is recognized as an important aspect of wound management, which requires new solutions against microbes and their biofilms. Octenidine dihydrochloride and polyhexanide are effective and tolerated antiseptics used in wound management today, but antiseptics alone may not be able to achieve wound healing without addressing other factors such as the general health of patients or the wound's physical environment [37, 38]. Next generation of wound treatment strategies for non-healing chronic wounds can be achieved by adopting a biofilm-based management approach to wound care, in order to kill

The antibiotic resistance of microorganisms determines serious complications like infection, and delayed wound healing and great concerns are related to the numbers and types of residing microorganisms and the ability of the host's immune system to control their prolif-

debridement [28].

438 Wound Healing - New insights into Ancient Challenges

complications [31].

the inflammatory phase of repair [19, 33–35].

and prevent reattachment of microorganisms [26].

**3. The antibacterial activities of metals**

Zinc is a transitional metal known since ancient time and widely distributed in the human environment. Today, many zinc-containing products are available for topical application in wound management due to the demonstrated improved re-epithelialization, reduced inflammation and bacterial growth. [48, 49]. ZnO has demonstrated to possess both antibacterial and anti-inflammatory properties and to accelerate the healing of both acute and chronic wounds. ZnO-NPs have exhibited antimicrobial capability and effectiveness against Grampositive and Gram-negative bacteria, including pathogens such as *Escherichia coli*, *Salmonella*, *Listeria monocytogenes* and *Staphylococcus aureus* [49]. Several mechanisms have been reported for the antibacterial activity of ZnO-NPs. Some of them involve the interaction with membrane lipids and structure, leading to loss of membrane integrity, malfunction, and finally to bacterial death. ZnO-NPs may also penetrate into bacterial cells, thus resulting in the production of toxic oxygen radicals, which damage DNA, cell membranes or cell proteins [50–52]. The direct interaction between ZnO nanoparticles and cell surfaces affects the permeability of membranes and results in the inhibition of cell growth and cell death. Recent studies have also shown that these nanoparticles have selective toxicity to bacteria but exhibit minimal effects on human cells, thus suggesting their potential as nanomedicine-based antimicrobial agents [53, 54].

The bactericidal effect of metal nanoparticles has been attributed to their small size and high surface to volume ratio, and it is not merely due to the release of metal ions in solutions [55]. Copper ions released subsequently may bind with bacterial DNA molecules and disrupt biochemical processes inside bacterial cells. The exact mechanism behind bactericidal effect of copper nanoparticles is not fully elucidated; however, Cu-NPs were found to cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in *E. coli* [47, 56]. Although the potential use of copper-based nanomaterials in wound healing has recently emerged and also supported by the hypothesis that copper ions regulate the activity and expression of proteins involved in the wound repair process, however, the synthesis of stable metallic Cu-NPs still remains a challenge because of the rapid oxidation to Cu2+ ions in air or aqueous media [47, 57].

In combination with silver, copper nanoparticles may give rise to more complete bactericidal effect against a mixed bacterial population [56]. The broad-spectrum antimicrobial activity of silver has been demonstrated against a wide range of microorganisms, including methicillin resistant bacteria, fungi and viruses [58]. Although the exact antimicrobial mechanism still represents a debated topic, many theories on the action of silver nanoparticles on microbes have been proposed. One of them involves the anchorage and penetration of the nanoparticles into the bacterial cell wall, which cause structural changes in the cell membrane such as permeability and respiration [59–62]. *E. coli* cells treated with silver nanoparticles appear damaged and show the formation of 'pits' in the cell wall of the bacteria, where the silver nanoparticles accumulate [59, 63]. Another antibacterial mechanism involves the release of silver ions and their interaction with the enzymes of the respiratory chain, the cell membrane and the DNA. The binding of silver to the membrane can inhibit the passage of nutrients through the membrane, interfering with normal concentration gradients between the cell and the surrounding environment, so leading to cell death [64, 65]. The formation of free radicals has also the ability to damage the cell membrane and makes it porous, thus causing the death of bacteria [66].

Nanosilver products safety data available in EPA's formal incident reporting database indicates that nanosilver products are safe. Silver nanoparticles can be easily incorporated into matrix materials and have demonstrated a great potential in applications of huge interest in nanotechnology [66]. When incorporated into wound treatment systems, silver nanoparticles can provide clinically relevance in the development of ideal environment for rapid and effective healing. These systems may significantly reduce the time required for the homeostatic equilibrium, while reducing the risk of complications and improving the physical appearance of the scar [67]. Silver nanoparticles induce rapid healing and improved cosmetic appearance in a dose-dependent manner and exert positive effects through their antimicrobial properties, reduction in wound inflammation and modulation of fibrogenic cytokines [68].
