Progress in copper‐based materials for wound healing

Chronic wounds have become the leading cause of death, particularly among diabetic patients. Chronic wounds affect ~6.5 million patients each year, according to statistics, and wound care and management incur significant financial costs. The rising prevalence of chronic wounds, combined with the limitations of current treatments, necessitates the development of new and innovative approaches to accelerate wound healing. Copper has been extensively studied for its antibacterial and anti‐inflammatory activities. Copper in its nanoparticle form could have better biological properties and many applications in health care.


| INTRODUCTION
The skin maintains body fluid balance and thermoregulation by acting as a barrier against external microorganisms and a conduit for grease and sweat, and thus plays an important role in the body.When the skin's integrity is compromised, the wound may bleed continuously, allowing large numbers of bacteria to grow in the wound without the protection of the natural skin. 1 Furthermore, the difficulty of healing infected wounds is a major public health issue around the world, putting a significant strain on patients as well as the medical and health systems. 2 Acute wounds usually heal in four different but overlapped stages: haemostasis, inflammation, proliferation, and remodelling. 3Ischaemia, infection, tissue impregnation, or the presence of foreign matter are examples of local factors that can disrupt the normal wound healing process, resulting in chronic wounds.In order to promote wound healing, it is critical to develop wound healing materials with therapeutic properties. 4Copper is an essential metal in the human body.Despite its small size, it is involved in a variety of physiological and metabolic processes.Copper can induce the expression of vascular endothelial growth factor (VEGF) and extracellular skin protein, as well as promote wound healing stability, in a variety of wound healing processes.

| Copper participation constitutes protein
Copper is mostly found in the body in the form of binding, such as combining with protein to form copper protein; copper is also involved in the composition and activation of over 30 enzymes in the body as an enzyme cofactor. 5In mitochondrial aerobic metabolism, as the central metal ion of cytochrome C oxidase.As a cofactor of dopaminehydroxyhydrolase in neurotransmitter biosynthesis and peptidylglycineamidated monooxygenase in neuropeptide biosynthesis.Lysyl oxidase is Abbreviations: Cs, chitosan; CuNPs, copper nanoparticles; CuS NDs, copper sulphide nanodots; EC, endothelial cell; ECM, extracellular matrix; EGF, epidermal growth factor; FGF, fibroblast growth factor; GA, gallic acid; GO/CuO NC, graphene oxide/copper oxide nanocomposites; HIF-1, hypoxia-inducible factor-1; MOF, metal-organic framework; MSCs, mesenchymal stem cells; PVA, polyvinyl alcohol; ROS, reactive oxygen species; SOD, superoxide dismutase; TGF-β, transforming growth factor-β; VEGF, vascular endothelial growth factor.
Wuliang Diao is the first author.required for ECM remodelling and the proliferative phase of healing.
Copper can boost its activity and promote collagen and elastin synthesis.Inadequate copper intake is likely to cause problems with tyrosinase synthesis; if tyrosinase is absent, melanin production will be hampered.SOD (superoxide dismutase) is an antioxidant metalloenzyme that protects body cells from superoxide anion damage.Copper is primarily used for catalysis in the enzyme. 6,7Copper, ironically, is a potent inhibitor of a wide range of cellular enzymes.Copper ions combined with a large number of sulphur, nitrogen, and oxygen-containing protein side chains to inhibit enzyme activity. 6The current research also shows that copper can enhance the secretion of thyrotropin releasing hormone and growth hormone releasing inhibitory hormone and also affects the secretion of catecholamines. 8

| Copper is involved in the regulation of various cytokines and growth factors in all stages of wound healing
During the haemostatic phase, copper stimulates the release of plateletderived growth factor (PDGF). 2 Copper stimulates the release of prostaglandins during the inflammatory phase. 9Copper can up-regulate the expression of VEGF during the proliferation stage by protecting hypoxia-inducible factor-1 (HIF-1) and promoting prostaglandin-1 secretion.It can also stimulate the production of fibroblast growth factor (FGF), nerve growth factor, angiotensin II, and transforming growth factor-β (TGF-β), among other proteins. 10,11Copper can also up-regulate important remodelling proteins like VEGF, FGF, collagen, and elastin during the remodelling stage. 12These cytokines and growth factors can help accelerate wound healing by enhancing early wound repair.

| Effects of copper on inflammatory cells
Copper is accumulated during the inflammatory stage and used by inflammatory cells such as macrophages to produce IL-4, IL-10, VEGF, TGF-β, and other anti-inflammatory cell factors that promote fibroblast, keratinocyte, and endothelial cell (EC) proliferation and migration, as well as angiogenesis, extracellular matrix (ECM) deposition, and re-epithelization in wound healing. 13,14DGFs stimulate copper-dependent migration of inflammatory cells like neutrophils, and macrophages. 15Inflammatory cells use PDGF and TGF to clean up damaged wounds and contaminate tissues during the inflammatory phase. 11Schuschke et al. 16 found that mast cells were increased in animals with copper deficiency, which might change the distribution or maturation pattern of white blood cells in tissues.If the human body lacks copper, there will be clinical signs such as neutropenia.In copper deficiency, not only the number of neutrophils in peripheral blood will decrease, but also its functions will be affected.Similarly, macrophage will experience a similar phenomenon in the absence of copper.At injury, if the concentration of copper is low, ATP7A will be inhibited and the migration of VSMCs will also be inhibited.If the concentration of copper ion is too high, it can also inhibit the function of VSMC, but the proliferation of ECs is not affected. 23This indicates that copper-induced angiogenesis is highly cell-type specific.

| Effects of copper on MSCs
Chen et al. 24 proved that adding copper to the standard medium can promote the migration of adipose tissue-derived stromal cells was promoted.This effect is caused by activating the Rac-PAK signalling pathway and enhancing the activity of cytoskeletal proteins.It has also been proved that copper ion can stimulate the migration of rat bone MSCs in complete medium, which is partially mediated by stimulating the HIF-1α-Rho family GTPase 3 pathway. 25In addition, Milewska et al. 26 certify that copper increases the expression of stromal cell-derived factor-1α, which promotes migration and recruitment of human adipose tissue-derived stromal cells.

| Effects of copper on red blood cells
Copper can promote the maturation of red blood cells.When copper is depleted, two haematological manifestations-hypochromic microcytic anaemia and neutropenia-are most common, although neither is specific for copper deficiency. 27But, the study found that copper can promote the absorption and utilisation of iron, activate the formation of haemoglobin, maintain the body red blood cell number in the normal range and ensure the oxygen supply. 28Copper deficiency reduced the activities of ceruloplasmin and cytochrome oxidase, which makes it difficult for the divalent iron to be converted into trivalent iron.Then Fe3+ cannot synthesise haeme with protoporphyrin and generates abnormal red blood cells with short service life.Copper poisoning can occur haemolysis, haemoglobin reduction, and so forth. 73 | Organisation level

| Copper promotes endothelial tissue formation
Copper ions can directly stimulate the migration and proliferation of ECs. 21Copper is considered to be an angiogenic factor and the interaction of copper with the endothelial system stimulates neovascularization in the cornea of avascular rabbits. 29Copper is particularly important for angiogenesis.Copper can participate in the regulation of angiopoietin, a variety of cytokines, and growth factors, and also promote the migration and proliferation of EC to gather at the wound site.Both of them promote angiogenesis, increase the supply of oxygen and nutrients and accelerate wound healing. 12Binding α-actin is a critical step in promoting angiogenesis, because this complex activates the plasminogen activator/plasmin serine protease system, resulting in the production of plasmin. 30Plasmin degrades fibronectin and laminin in the ECM and basement membrane, thus promoting angiogenesis and stimulating EC invasion into the tissue.Copper can promote the production of fibrinolytic enzyme and influence angiogenesis by regulating the interaction between angiotensin and actin. 29

| Copper promotes connective tissue
Copper can enhance the activity of lysyl oxidase and promote the synthesis and maturation of elastin and collagen, thus affecting the integrity of connective tissue. 27Copper helps stabilise the crosslinking of elastin, fibronectin, and (type I, type III) collagen, recruiting fibroblasts through PDGF and TGF-β, stimulating their synthesis into granulation tissue, and promoting the reconstruction of the ECM. 31 A decrease of TGF-β inhibits the formation of keloids and hypertrophic scars.In addition, copper can help angiogenesis by reshaping the ECM and maintain bone strength.The non-specific oxidative potential of copper facilitates the metabolism of ascorbic acid in growing bone. 27

| Copper promotes the epithelization process
In that early stage of repair, copper stimulates keratinocyte to repair the epidermal barrier.New tissues generally consist of ECM and collagen.As new tissue is established, the wound contracts with the help of myofibroblasts.Finally, epithelization occurs and epithelial cells reappear.Epithelization is stimulated by epidermal growth factor (EGF) and TGF-β, which can be produced by copper-stimulated macrophages. 13,144 | Individual level

| Copper can maintain the stability of the cardiovascular system
Copper is of great significance for preventing cerebrovascular diseases and vascular rupture.Copper is a component of lysine oxidase, which plays an important role in maintaining the integrity of the vascular wall.When the body is short of copper, the activity of lysine oxidase is reduced, and the synthesis of elastin in the tissue is reduced, thereby enhancing the fragility of blood vessels and tissues and easily causing vessel rupture and haemorrhage. 32Lysine oxidase plays an important role in the formation of atherosclerosis, which is also a risk factor for cerebrovascular disease, so the copper content in patients with cerebrovascular disease is often low. 33SOD can eliminate oxygen free radicals, thus inhibiting or relieving the aging of the body.In addition, diabetic patients are often accompanied by other diseases.
So oxygen free radicals will lead to the accumulation of lipid peroxides, causing diseases such as atherosclerosis.Therefore, superoxide dismutase has a preventive effect on cardiovascular and cerebrovascular diseases caused by hyperlipidaemia. 6

| Copper can maintain the health of the nervous system
In the nervous system, copper excess or deficiency will cause changes in the structure and function of the nervous system. 34Excessive copper promotes expression of bax gene and induces apoptosis of nerve cells. 5tochrome oxidase can promote the formation of myelin sheath and ensure the production of ATP.A prosthetic group of superoxide dismutase that promotes the synthesis of cephalin.When there is a lack of copper in the body, the sensitivity to stimulation from the body will decrease and the nerve development will stagnate.In addition to copper participating in the regulation of nervous system function by affecting the activity of copper-containing enzymes in the nervous system, it also has the function of regulating the expression of neuroreceptors, endocrine, and nervous system-related genes.

| Copper can maintain the body's immune function
Copper plays an important role in the development and maintenance of the immune system.The immune system needs copper to complete many functions, but the mechanism of its direct effect is not clear.
Some studies indicate that copper ions can also stimulate the formation of IL-2 to improve immunity, but also stimulate the proliferation of T cells. 37Neurotrophic factors in human peripheral blood and serum thymic factors are reduced in the presence of severe copper deficiency.At the same time, T-cell and B-cell defects and the function of white blood cells (especially neutrophils) will be significantly weakened, thus damaging the body's immune system. 17The weight of the thymus was significantly lower and the weight of the spleen was significantly higher in the copper deficiency animals than in the control animals, but the antibodies produced by the spleen cells were significantly reduced. 38Copper can improve the specific and nonspecific immune function of human body and also play an antibacterial role by producing reactive oxygen species (ROS) and photothermal effect, even for bacteria that are resistant to antibiotics. 39

| Copper has antioxidant effect
Copper-containing enzymes are always involved in that redox reaction. 27Free copper ions are themselves effective oxidants and play a role in the oxidation resistance.The main copper-containing enzymes in the nervous system are cytochrome oxidase, copper-zinc superoxide dismutase, and ceruloplasmin, all of which are related to the oxidation resistance of the body.Copper had a certain effect of alleviating high concentration of blood glucose in patients with diabetes. 40,41High blood glucose concentrations inhibit angiogenesis by down-regulating the activity of VEGF and HIF-1 (Table 1 and Figure 1).

| COPPER-BASED MATERIAL FOR WOUND SURFACE
Copper is commonly used in chronic wound wounds.Because copper has an excellent ability to promote wound healing and a significant antibacterial effect.In order to accelerate the wound healing, researchers have developed a variety of biopolymers with good biocompatibility, such as chitosan, hydrogel, metal-organic framework (MOF) and other forms for wound healing.These materials provide a good environment for tissue regeneration and specifically promote the inherent characteristics of wound healing.Moreover, they are nontoxic, biocompatible and biodegradable.Each material has its own advantages.

| Pure copper
Copper is one of the few metals that exist in nature as metals and can be used directly. 42Pure copper surface antibacterial effect is significant, even at room temperature only 4 h to kill Escherichia coli. 43though studies have shown that copper is safe for humans, copper is a heavy metal and pure copper is unstable and easily oxidised.In addition to the antibacterial ability, clinical experiments proved that pure copper does not improve the patient's symptoms. 44Thereby limiting its medical application.

| Compounds of copper
Copper compounds are soluble, which is more easily obtained than pure copper, and the copper ion solution with controllable concentration can be obtained. 13Pure copper undergoes a slow oxidation reaction with oxygen to form copper oxide.Common copper oxides include cuprous oxide (I)(Cu 2 O) and copper oxide (II)(CuO). 42Cu 2 O has an excellent antibacterial ability, but Cu + has a high cytotoxicity, which limits its application in the medical field.In contrast, the physical and chemical properties of CuO are stable, and the cell compatibility is good. 45However, the dissolution of copper compounds and the release of copper ions are very fast.The large release of copper ions in a short period of time will produce significant cytotoxicity, leading to red blood cell lysis, and so forth. 46Experiments have proved that mice treated with copper chloride have kidney injury and lung inflammation, and bone MSCs co-cultured with copper chloride have obvious cell death. 20Therefore, using copper ion alone does not facilitate wound healing.

Individual level
The right amount of copper promotes the development of different systems in the body, maintains normal structure and function, and has synergistic effects on immunity and antioxidants

| Copper nanoparticles
There are two main types of nanomaterials used for wound healing: (1) nanophase materials that exhibit their intrinsic properties, including non-metallic nanoparticles, metal nanoparticles, and metal oxide nanoparticles, can treat wound healing; (2) nanophase materials as carriers for drug delivery.Copper nanoparticles (CuNPs) not only have the characteristics of promoting wound healing and anti-bacteria, but also can be used as a carrier to transfer drugs, so they have been widely used. 47CuNPs include pure copper nanoparticles, CuO/Cu 2 O nanoparticles, and copper-containing compound nanoparticles. 42mpared with copper ions, CuNPs have some advantages: the nanoparticles have an orderly and stable structure, and their cytotoxicity is smaller than that of copper ions. 48In addition, CuNPs can be obtained by biosynthesis with plant extracts and are safe for human body and non-immunogenic. 47For example, the water extract of prunellae spica leaf can be used for preparing CuNPs, which have significant antioxidant, antibacterial, and skin wound healing promoting effects. 49ao et al. 39 prepared CuS NDs (copper sulphide nano-dots).Compared with CuS NPs, CuS NDS has the advantages of enhanced stability, slower release of Cu 2+ , higher safety, and more conducive to wound healing.Liu et al. 50prepared ultra-small Cu5.4O nanoparticles (Cu5.4O USNPs).The experimental results showed that Cu5.4O USNPs could promote wound healing.It was verified that Cu5.4O USNPs had no significant toxicity and good biocompatibility through transmission, serum biochemical analysis, and whole blood picture analysis.Even serum examination and histological observation of liver tissue proved that Cu5.4O USNPs had a therapeutic effect on acute liver injury mice.
When the CuNPs are loaded with the active ingredients, the nanoparticles can protect the drug from being degraded by the protease in the wound, thereby remarkably improving the treatment effect in the wound healing process. 51Saddik et al. 47 invented copper nanoparticles loaded with phenytoin (PHT-loaded CuNPs).PHT has the effect of stimulating fibroblast proliferation and enhancing granulation tissue formation.It also inhibits collagenase production, promotes the deposition of collagen and other connective tissue components and reduces the formation of wound exudate. 52PHT and CuNPs cooperated to accelerate the wound healing process.
Although nanoparticles have great potential for application, they still have adverse effects on living cells and potential toxicity.The nanoparticles are 100 times smaller than normal red blood cells, which increases the potential for interaction.At the same time, because the nanoparticles have an active surface, it can easily passes through a biomembrane and destroys cell tissues. 53Cu NPs are prone to rapid oxidation and agglomeration during its use. 54The cytotoxicity was more obvious with the increase of nanoparticle concentration.This may be due to increased ROS production, genotoxicity, and multiple exogenous resistance transporter activities. 55Even at low Copper-based materials act on wounds and promote wound healing by releasing Cu 2+ .PDGF, platelet-derived growth factor; SOD, superoxide dismutase; TGF-β, transforming growth factor-β; VEGF, vascular endothelial growth factor.
concentrations of copper ions, copper ions showed cytotoxicity with the prolongation of culture time. 56Therefore, there is a need to invent a nanomaterial that can inhibit bacteria and control the generation of ROS, which can inhibit bacteria and bacteria without damaging normal tissues. 57

| Biopolymers
Desirable materials for wound healing should provide suitable conditions for promoting wound healing, protect the wound from bacterial invasion, not adhere to the wound, allow gas and fluid exchange, be no cytotoxicity, as well as have good biocompatibility and biodegradability. 58Biopolymers (such as hydrogels and chitosan) provide a good environment for tissue regeneration and have the inherent characteristics of promoting wound healing.The incorporation of nanoparticles into natural polymers not only improves the physicochemical, mechanical, and biological properties of the polymer, but also improves the performance and stability of the nanoparticles. 55Compared with pure NPs, the release rate of copper ions in the composite material is much slower, the cytotoxicity is greatly reduced and the composite material shows better capability of promoting wound healing and higher safety. 59

| Hydrogels
Hydrogels have good water retention and provide a long-term moist environment for wound healing. 60Hydrogels have interconnected pores that facilitate the absorption of wound exudate and ensure the survival and metabolism of skin cells.At the same time, the hydrogel provides a higher surface area to volume ratio, which facilitates the integration of drugs, nanomaterials, or bioactive materials. 61In addition, hydrogel wound dressings act as a barrier against microbial invasion, but allow oxygen to infiltrate and inhibit the growth of anaerobes, effectively protecting open wounds in the early stages of wound healing. 62Zhou et al. 2 produced an injectable hydrogel of hyaluronic acid mixed with copper sulphide nanoparticles that could be injected into a wound area.In the initial stage of wound healing, it can effectively up-regulate the VEGF expression in the wound area and promote the formation of new blood vessels, as well as increase collagen deposition, and has good photothermal effect, which helps to promote wound healing.

| Metal-organic framework
MOF is a compound composed of metal nodes and organic ligands, with the outstanding characteristics of ultra-high porosity and large specific surface area. 63But traditional MOF material have poor biocompatibility. 64In contrast, copper-based organic frameworks synthesised from copper showed better biological effects for promoting tissue repair. 65Xiao et al. 66 modified copper-based MOF nanoparticles (Cu-MOF NP) with folic acid not only improved the stability of MOF, but also resulted in the slow release of copper ions and reduced toxicity.In addition, Cu-MOF NP could also stimulate EC production, promote collagen deposition and reepithelization and increase wound closure rate, significantly accelerating wound healing in diabetic mice. 66Xiao et al. 67 successfully prepared a novel hydrogen-HKUST-1 (H-HKUST-1) composite with similar effects.

| Dressings
The antibacterial wound dress can effectively avoid that residues of the antibacterial nanomaterial for injection in the body, reduce the biological toxicity of the antibacterial nanomaterial to normal cells and tissues, form a protective layer on the surface of the wound surface at the same time, prevent external bacterial infection and promote the application of the antibacterial wound dressing in the medical field. 57tibacterial wound dressings impregnated with copper oxide microparticles are useful for stimulating direct healing of diabetic feet or wounds that are difficult to heal.

| Bioactive glass
Bioactive glass particles used for wound healing have a disadvantage that they do not have mechanical support and are not easily treated.
In addition, because of the sharp shape of bioactive glass particles, it may lead to potential inflammatory reaction when directly applied to wounds. 71The addition of copper to bioactive glass does not adversely affect that bioactivity of the glass but improve the thermal stability of the bioactive glass. 72Copper-containing bioactive glass has excellent antibacterial ability, promote tissue regeneration ability and biocompatibility. 31Zhao et al. 73 prepared bioactive glass containing copper.The experimental results showed that it can promote cell migration, collagen deposition, vascular reconstruction and upregulate the expression of angiogenesis-related genes.

| Chitosan
Chitosan (Cs) is a natural polymer with unique haemostatic ability.
Besides, Cs can enhance the function of inflammatory cells, kill most pathogenic microorganisms, control inflammation, and promote wound healing. 74Both chitosan and its degradation products can stimulate the proliferation of fibroblasts to help the regeneration of the wound site. 75The invention relates to a composite material consisting of chitosan and nano copper, which can stabilise nano copper under the action of chitosan so that the nano copper is not easy to oxidise and agglomerate and can enhance the capability of the nano copper to promote wound healing.

| Polyvinyl alcohol
Polyvinyl alcohol (PVA) is a hydrophilic synthetic polymer, due to its biocompatibility, swelling, non-carcinogenicity, flexibility, non-toxic and biodegradable properties.Venkataprasanna et al. 59 invent a graphene oxide/copper oxide nanocomposites (GO/CuO NC).In combination with PVA and Cs, GO has antibacterial ability and enhanced cell adhesion.CuO is involved in re-epithelization and matrix remodelling, while Cs is involved in cell proliferation and granulation, improves cell survival rate and promotes wound healing.

| Gallic acid
Gallic acid (GA) is a natural polyphenol with antioxidant properties, which can reduce inflammation and oxidative stress damage. 76,77Animal experiments showed that the antibacterial dressing added with CS Cu GA NCs could effectively promote wound healing caused by staphylococcus aureus infection and had no damage to normal tissues. 57

| Glucan
Glucan has only hydroxyl groups, so it can be used as scaffold material.Paunica-Panda et al. 78 made a new composite material based on glucan and collagen as biopolymers, which can be mixed with copper for wound healing and has great potential.Ma et al. 79 added nanoparticles into dextran hydrogel, which had good biocompatibility and biodegradability.

| Alginate
Due to its biological characteristics of biocompatibility, non-toxicity, biodegradability, non-immunogenicity, easy access, and strong absorption capacity, alginate is a biopolymer that has been effectively used for wound healing in diabetes 80 (Table 2).

| PROSPECT
This review is a summary of some copper-based materials that can be used for wound healing.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
T A B L E 2 Copper-based material for wound surface.

2. 4 . 5 |
Copper has an important role in the regulation of blood glucose Impaired glucose tolerance after copper deficiency.Copper helps stimulate the activities of cytochrome c oxidase, catalase, and succinate dehydrogenase in the human body, and promotes the metabolism of sugars and lipids, providing the energy needed by the body.

T A B L E 1
The role of copper in the human body.composition and activation of a variety of enzymes in the form of enzyme cofactors, and can play an inhibitory role in some cellular enzymes Promotes the secretion of hormones in the body Regulates the production of various factors at various stages of wound healingCell levelThe right amount of copper promotes the maturation of erythrocytes and ensures oxygenation, which promotes the value-addition of inflammatory cells, the production of cytokines, as well as the valueaddition and migration of fibroblasts, endothelial cells, vascular smooth muscle cells, and mesenchymal stem cellsOrganisation levelIt promotes the formation of different tissues by enhancing the activity of a variety of enzymes in the body, regulating a variety of factors, and promoting the value adding, activation, and migration of cells Copper-based nanomaterials have potential applications in wound dressings, and innovative methods based on controlling the release of copper ions are used as potential therapeutic tools for wound healing.This kind of material can not only increase the local transmission of copper ions, but also exert excellent antibacterial effect.Therapeutic products developed now often combine multiple drugs and materials, combining the inherent characteristics of each substance to synergistically accelerate wound healing.These products are considered ideal materials for promoting wound healing and are convenient to use due to their high biocompatibility, low toxicity, and biodegradability.Further studies should focus on the effect of copper homeostasis on wound healing.Keep the concentration of copper ions within a certain range in tissues and cells, and find out the best concentration range of copper ions for different tissues and cells.
19,182.2.2 | Effects of copper on fibroblasts Alizadeh et al.19studied the effect of copper on cell migration and believed that copper could promote the migration and proliferation of human skin fibroblasts. And,  speed of migration and proliferation varies with the concentration of copper.Studies have shown that cop- 70,69Cui et al.70prepared a gelatin sponge dressing loaded with CuO 2 , which had pH-controllable OH release and effective antibacterial activity. In th acidic microenvironment where bacteria infected the wound, it could stimulate the pHsensitive dressing to release Cu2+ and H 2 O 2 , promote wound healing and sterilise.