1 INTRODUCTION

The skin has multiple layers that protect against harmful external factors such as pathogens, radiation, heat, and wound.1 A wound compromises the structure and function of the skin, and the type of wound is divided into acute (eg, trauma, burns, and surgery) and chronic (eg, diabetic and pressure ulcers).2, 3 The damage of a wound increases by external factors and patient condition producing reactive oxygen species (ROS), which affect the elements involved in skin repair such as in chronic wounds.2, 4

The wound healing process involves a diversity of elements, such as growth factors, cells, signalling pathways, and cytokines, which all work in synergy.5 Growth factors and their signalling pathways coordinate cell responses in the wound healing process.6, 7 The mechanism of these elements helps in developing accurate treatments for impaired healing wounds, wherein growth factors are used in wound healing treatments because they coordinate cell responses in the wound healing phases.

Proliferation, migration, angiogenesis, and inflammation are cellular responses activated by growth factors. Uncontrolled rates of ROS alter the modulation of these cellular responses, resulting in excessive scarring or impaired wounds.6, 7 In this regard, antioxidants have shown regulation of cellular responses activated by growth factors, which aid in the repair of different types of wounds.4, 8, 9 The potential effect of the combination of growth factors and antioxidants could lead the search to find diverse alternatives in cutaneous repair with high quality and reduced time.8 This review aims to present the role of growth factors and antioxidants on skin wound healing-related processes. Furthermore, based on their individual effects, a prospective analysis on their potential combinatorial effect on wound-healing formulations is presented. This information may serve as a guide to envision further studies focused on the confirmation of those effects, allowing the rational design and development of novel skin wound-healing formulations.

2 SKIN WOUND HEALING PHASES: ROLE OF GROWTH FACTORS AND ROS

The wound healing occurs in four overlapped and sequential phases, namely (a) haemostasis, (b) inflammation, (c) proliferation, and (d) remodelling.10 They are synchronised by particular endogenous polypeptides, called growth factors, secreted by six specific cells recruited at the wound site: platelets, macrophages, keratinocytes, fibroblasts, mast cells, and neutrophils.11, 12 These growth factors activate the paracrine and autocrine cell communication by binding to their specific receptors, and also they are essential for the cellular function, namely proliferation, migration, angiogenesis, and inflammation.11, 13 Growth factors that have been demonstrated to have a major role in the wound healing process are platelet-derived growth factor (PDGF), transforming growth factor-beta 1 (TGF-β1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).10 Such growth factors are key elements in wound healing because they are responsible for the cellular communication and regulation of cellular responses that trigger the proliferation, migration, and differentiation of damaged cells and events when the balance of the inflammatory response, neovascularisation, and modulation of extracellular matrix (ECM) occurs.12 The secondary key element in wound healing is ROS.14 ROS produced at controlled levels stimulate haemostasis, pathogen defence, tissue repair, and lymphocyte recruitment in the wound healing process.14 The deficiency of these growth factors and the excess of ROS levels is related to non-healing conditions.12 Nonetheless, growth factor administration is a promising strategy for wound healing management or treatment. Figure 1 shows the effect of the key growth factors and antioxidants involved in each wound healing phase.

Effect of growth factors and antioxidants on each stage of the skin wound-healing process. The four phases involved in the wound healing process are presented. In addition, selected growth factors and antioxidants are added to the phase wherein they have an enhancing effect. (a) Haemostasis phase: platelets produce platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor-β1 (TGF-β1). They act as chemoattractant for inflammatory immune cells. Epigallocatechin gallate (EGCG) binds to PDGF and EGF receptors inhibiting the signalling cascade of them for the inflammatory phase. EGCG also inhibits the expression of EGF receptor (EGFR), which delays the beginning of the epithelialisation. Delphinidin inhibits PDGF signalling after binding to PDGFR, which delays wound repair. (b) Inflammation phase: inflammatory immune cells such as neutrophils, mast cells, and macrophages secrete cytokines, reactive oxygen species (ROS), and growth factors. Microvascularity increases to deliver nutrients, immune cells, and oxygen at the wound site. Monocytes matured to macrophages which produce PDGF, TGF-β1, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) that act as chemoattractant for keratinocyte and fibroblasts. Through this phase, EGCG and delphinidin still inhibit PDGFR. Astaxanthin, β-carotene, and curcumin aid wound healing when balance ROS. (c) Proliferation phase: bFGF, VEGF, PDGF, and TGF-β activate cellular responses that promote angiogenesis, keratinocyte migration and proliferation, fibroblasts migration and differentiation, and collagen synthesis. Astaxanthin stimulates TGF-β1 and VEGF signalling pathways and expression that enhance differentiation of fibroblast, migration of keratinocytes, granulation tissue formation and angiogenesis. Curcumin stimulates TGF-β1 signalling pathways that enhance the expression of VEGF. Delphinidin and β-carotene inhibit VEGF pathways and receptor reducing angiogenesis. EGCG inhibits VEGFR and bFGF expression, which reduce angiogenesis and collagen synthesis, and delays wound closure. (d) Remodelling phase: scar maturation and remodelling of extracellular matrix (ECM) occur when collagen type I (COLI) replaces collagen type III (COLIII), and keratinocytes and fibroblasts organise, apoptosis, and differentiate via bFGF and TGF-β1 signalling pathways and by bFGF and astaxanthin stimulate the expression of bFGF and TGF-β1 modulating collagen production and scar maturation. Curcumin enhance TGF-β and collagen synthesis, which promotes remodelling of ECM. EGCG inhibits TGF-β1 signalling, which reduces fibroblast proliferation, fibroblast differentiation to myofibroblast, and collagen synthesis that modulate scarring. Additionally, EGCG still inhibits the expression of bFGF that aids in scarring control

2.1 Haemostasis phase

After an injury occurs, the first stage is vasoconstriction, also known as haemostasis.12 During this phase, platelets make contact with fibronectin and collagen forming a fibrin clot that stops the bleeding and blocks the entry of pathogens.15 The generation of early ROS from platelets reduces the blood flow allowing to promote the vasoconstriction.14 Along with the monocytes, platelets secrete PDGF, EGF, and TGF-β1, which act as chemoattractants of inflammatory cells and promote the adaptive immune response of the inflammatory phase.12, 15, 16 Table 1 describes each role and different pathways regulated by growth factors during haemostasis.

TABLE 1. Function and signalling pathway of selected growth factors during wound healing Growth factor Function Signalling pathway References PDGF Stimulate cell proliferation and migration Ras/Erk1/2/MAPK 81 Up-regulation of IGF-1 and VEGF expression PLCγ PI3K/AKT Stimulate angiogenesis PI3K/AKT/eNOS 82 Stimulate migration and cytoskeletal remodelling Rho GTPase 83 PI3K/AKT Regulate proinflammatory cytokines PI3K/AKT/NF-κβ 74 VEGF Activate proliferation of endothelial cells in angiogenesis PLCγ/ PKC/ Ras/Raf/ MEK/ ERK 84 Stimulate cell migration of keratinocyte and endothelial cells FAK 84, 85 p38/MAPK Stimulate permeability and vasodilation PI3K/AKT/eNOS 85 EGF Activate migration and proliferation of keratinocyte Erk1/2/MAPK 81, 86 Activate production of type I collagen PI3K/AKT 86 Induce migration and formation of vascular tubes in endothelial cells (angiogenesis) PI3K 81 MAPK Induce production of MMP and cell proliferation in fibroblasts PI3K 81 Rac ERK Inhibition of infiltration of inflammatory cells RANTES 86 MCP-1 Suppress secretion of IL-1α, IL-8, and TNF-α in inflammatory cells NF-κβ 87 bFGF Stimulate fibroblast and endothelial cells proliferation, migration, and differentiation Ras/Raf/MEK/MAPK 88 PI3K/AKT Activate production of ROS to induce fibroblast migration and collagen production PI3K/AKT/Rac1/JNK/NOX 89 FAK/Paxillin Stimulate and collagenase production PLCγ/ IP3-Ca2+/ DAG/PKC 10, 90 Activate inflammatory response NF-κβ/JNK 91 Regulate scar formation activating TGF-β signalling. Wnt/β-catenin 19, 91 Activate angiogenesis producing ROS Wnt/β-catenin TGF-β1 Fibroblast proliferation, migration, and differentiation Wnt/β-catenin 78 Regulate differentiation of fibroblast to myofibroblast Smad/Erk 78 Enhance collagen deposit TGF-β/Smad 78, 92 β-catenin Stimulate collagen synthesis in fibroblast JNK/ET-1/c-Jun 93 Note: For each of the five main growth factors involved in wound healing their functions (related to one or several healing stages) and signalling pathway are presented. Abbreviations: AKT, protein kinase B; bFGF, fibroblast growth factor; DAG, diacylglycerol; EGF, epithelial growth factor; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; JNK, c-Jun N-terminal kinase; FAK, focal adhesion kinase; IP3, inositol trisphosphate; MCP-1, monocyte chemoattractant protein-1; NF-κβ, nuclear factor kappa beta; NOX, NADPH oxidase; PI3K, phosphatidylinositol 3-kinase; PDGF, platelet-derived growth factor; Rac1, Ras-related C3 botulinum toxin substrate 1; RANTES, regulated on activation, normal T cell expressed and secreted; Smad, small mothers against decapentaplegic; TGF-β, transforming growth factor; VEGF, vascular endothelial growth factor; Wnt, wingless-related integration site. 2.2 Inflammatory phase

The inflammatory phase begins with the activation of the adaptive immune response, and the migration of inflammatory cells, such as macrophages, T cells, monocytes, mast cells, and neutrophils, to control pathogens, regulate ROS, and degrade foreign material.16, 17 They balance inflammatory responses secreting the growth factors and cytokines, also producing ROS, that regulate this process.16, 18

The inflammatory balance is mediated by pro-inflammatory and anti-inflammatory agents.16 The pro-inflammatory agents promote ROS production in the inflammatory microenvironment. Neutrophils act as pro-inflammatory agents because they can generate ROS that function as pathogen inhibitors,16, 18 and secrete chemoattractants, such as VEGF, and cytokines especially IL-6, TNF-α, and IL-1.12 Macrophages, maturated from monocytes, are the key agents in the inflammatory phase because they release pro-inflammatory cytokines, such as IL-1 and TNF-α, along with growth factors, such as bFGF, PDGF, and VEGF, that promote proliferation of fibroblasts, keratinocytes, and epithelial cells through MAPK and PI3K-AKT pathways; also PI3K-Akt-eNOS, NF-kB, and FAK-ERK-MCP1 pathways of VEGF and PDGF produce ROS.16, 17, 19 The later function of these growth factors is the attraction of more inflammatory cells to further stimulate its secretion.16, 18

As new cells form the new tissue by the activation of growth factor signalling, macrophages and T cells secrete anti-inflammatory cytokines and growth factors, such as IL-10 and TGF-β1, to suppress the pro-inflammatory response and balance the inflammatory microenvironment at the site.16 Chronic and excessive scarring wounds have uncontrolled inflammatory agents and ROS excess that induces a prolonged inflammation phase.18 On the contrary, when a proper inflammatory balance is achieved in acute wounds, the wound healing process proceeds into the following stage. Table 1 presents the role of different growth factors during the inflammatory phase.

2.3 Proliferative phase

This phase consists of four processes that occur simultaneously and depend on each other, being the angiogenesis, granulation tissue formation, re-epithelialisation, and wound contraction.15, 18 All these phenomena are modulated by VEGF, PDGF, bFGF, and TGF-β1 (Figure 1), and diverse signalling pathways are involved.

Angiogenesis, the formation of vascularity, provides oxygen and growth factors to induce the formation of granulation tissue.18 Angiogenesis is stimulated by bFGF, VEGF, and TGF-β signalling pathways (Table 1). VEGF is the mainly responsible for endothelial proliferation and migration, and blood vessel maturation promoted via MAPK and PI3K-Akt-eNOS, and the later signalling pathway produces ROS.20, 21 At the same time, the low generation of ROS stimulates the proliferation and migration of fibroblast enhancing collagen production to prepare granulation tissue formation and wound closure.20 Granulation tissue formation and type III collagen are promoted principally by bFGF and TGF-β and provide the structure for fibroblast and keratinocyte migration and vascular formation.10, 18

Re-epithelialisation, known by the proliferation and migration of keratinocytes, promotes the closure of wounds mainly stimulated by signalling pathways in Table 1, such as MAPK, FAK-paxillin, PI3K-Akt-mTOR pathways of VEGF, EGF, bFGF, TGF-β, and ROS.18, 19, 22 Dysfunction of angiogenesis is present in diabetic foot ulcers and burns,16 and this highlights the relevance of this event in non-healing conditions.11

2.4 Remodelling phase

The remodelling or maturation phase is where the scar is formed, the fibroblast matures to myofibroblasts and collagen structure is remodelled.18 The TGF-β1 and bFGF stay at last to enhance ECM maturing or known as replacement and degradation of type III collagen by type I collagen by the action of collagenases, metalloproteinases, and fibroblasts (MMP).2, 4 In this process, ROS has an active role in enhancing bFGF expression, modulating the production of collagen, and remodelling the ECM.14, 20 The principal activated signalling pathways in this phase are MAPK, Smad, and β-catenin pathways (Table 1). The complications associated with this phase are the overexpression of MMP and collagenases that constantly destruct ECM structure in chronic wounds, and the underexpression of the later enzymes and elevated synthesis of type III collagen in excessive scarring wounds such as hypertrophic wounds, burns, and infected wounds.4

Signalling pathways are the mediators of the cellular responses in which redox signalling is also a critical point in all the wound healing phases.20 Therefore, ROS at low or controlled concentration function as pathogen controller and help to activate proliferation, migration, inflammation, and angiogenesis cell responses. Nonetheless, ROS in excess or without control induce a chronic inflammatory response at the inflammation phase occurring in an impaired wound.14, 20 In this regard, antioxidants play a key role in the efficiency and speed of the wound healing process.

3 ANTIOXIDANTS IN WOUND HEALING

ROS, and the respective pro-inflammatory cell signalling, have a key role in wound healing.23, 24 When enzymatic endogenous antioxidants in cell are not capable to overcome the high rate of oxidative stress, the administration of exogenous antioxidants allows the balance of ROS and inhibition of inflammatory signalling pathways20 enhancing wound healing.25, 26

Cutaneous antioxidants are mainly classified as non-enzymatic and enzymatic.27 The enzymatic antioxidants are endogenous molecules found in oxidative cell mechanism, with catalase, glutathione peroxidase, and superoxide dismutase being some of the examples.27 The non-enzymatic kinds are both endogenous and exogenous molecules, mainly obtained from plants and found in a wide variety, classified as carotenoids and polyphenols.26, 27

Carotenoids and polyphenols with anti-inflammatory, antioxidant, and antibacterial properties are used in cancer and wound healing therapies.4, 28, 29 The mechanisms of oxidative stress control and NF-κB inflammatory signalling in the wound healing phases are leading to the discovery of therapies for non-healing and aberrant scarring wounds.4, 20 Scientific literature concerning exogenous supplementation of antioxidants for wound healing enhancement focuses on carotenoids and polyphenols.1, 4 This makes sense as these two bioactive families are among the most characterised in terms of antioxidant activity, given their availability in natural generally recognised as safe (GRAS) sources.29-32 Both carotenoids and polyphenols have been reported to play a key role during the inflammation, proliferation, migration, and angiogenesis stages in wound healing. Figure 1 presents the effect of selected antioxidants in wound healing. It is important to remark that, as part of such a role, antioxidants may have a direct effect on the expression and activity of different growth factors. This opens the opportunity of harnessing such interactions to develop wound healing formulations with enhanced effectiveness. In this section, the reported effects of selected, well-characterised antioxidants in wound healing are presented.

3.1 Carotenoids

Carotenoids are present in vegetables, fruits, marine animals, and microalgae, characterised by its red and orange-coloured pigment.33 Carotenoids have a lipophilic structure and are classified in (a) carotenes (non-polar), and (b) xanthophylls (amphipathic, given their terminal hydroxyl groups).33 They are strong scavengers of ROS resulting in well controllers of oxidative stress in wound healing.34 Table 2 shows the reported effect of β-carotene and astaxanthin, two well-characterised carotenoids, over the wound healing process.

TABLE 2. Target signalling pathway and response of selected antioxidants applied in wound healing Antioxidant Response Target signalling pathway References Astaxanthin Regulate collagen through inhibition MMP-1 and production of collagen TIMP1 33 Inhibit inflammatory signalling NF-κβ/eNOS 33 Promote angiogenesis Wnt/β-catenin 94 Promote cell migration in keratinocytes Rac1/Cdc42/RhoA 39 β-carotene Inhibit angiogenesis VEGF/NF-κβ 62 CREB Inhibit metalloproteinases: MMP-1, MMP-10, MMP-2, and MMP-9 ROS-induced MAPK 33, 95 Inhibit inflammatory signalling NF-κβ/PI3K/eNOS 62 Curcumin Act as anti-inflammatory, antioxidant, anti-infectious, and antiangiogenesis IKKα/β/NF-κβ 4, 96 NF-κβ/VEGF Inhibit angiogenesis and granulation tissue formation in fibroblasts VEGFR2/Erk-1/2 66 AKT Inhibit synthesis of collagen, proliferation, and differentiation of fibroblasts TGF-β/Smad2 97 Inhibit proliferation of fibroblast and synthesis of collagen in hypertrophic scarring Wnt/ β-catenin 97 Delphinidin Inhibit inflammatory responses and ROS NF-κβ 47 Act as anti-angiogenesis effector VEGFR 98 Anti-proliferative VEGFR/Erk-1/2 45, 98 PLCγ EGCG Regulate inflammatory response and ROS NF-κβ 99 Inhibit proliferation of fibroblast and synthesis of collagen in hypertrophic scarring Wnt/β-catenin 97 Act as antitumour an antiangiogenesis effector VEGFR 68 EGFR PI3K/AKT Erk1/2/p-p38/MAPK Abbreviations: AKT, protein kinase B; Cdc42, cell division cycle 42; CREB, cAMP-response element-binding protein; EGCG, epigallocatechin gallate; EGFR, EGF receptor; eNOS, endothelial nitric oxide synthase; Erk-1/2, extracellular signal-regulated kinase-1/2; JNK, c-Jun N-terminal kinase; NF-κβ, nuclear factor kappa beta; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; p-p38, phosphorylated p38; PLCγ, phospholipase C gamma; Rac1, Ras-related C3 botulinum toxin substrate 1; RhoA, Ras homologue family member A; ROS, reactive oxygen species; Smad, small mothers against decapentaplegic; TGF-β, transf