Wound Healing, Tissue Repair, and Regeneration in Diabetes

By Debasis Bagchi

Wound Healing, Tissue Repair and Regeneration in Diabetes explores a wide range of topics related to wound healing, tissue repair and regeneration, putting a special focus on diabetes and obesity. The book addresses the molecular and cellular pathways involved in the process of wound repair and regeneration. Other sections explore a wide spectrum of nutritional supplements and novel therapeutic approaches, provide a comprehensive overview, present various types of clinical aspects related to diabetic wounds, including infection, neuropathy, and vasculopathy, provide an exhaustive review of various foods, minerals, supplements and phytochemicals that have been proven beneficial, and assess future directions.

This book is sure to be a welcome resource for nutritionists, practitioners, surgeons, nurses, wound researchers and other health professionals.

• Explains diabetic wounds and their complications
• Assesses the role of nutraceuticals, herbal supplements and other modalities for use in treating diabetic wounds
• Provides protocols for diabetic wound management

• Language: English
• Publisher: Academic Press
• Release date: Apr 18, 2020
• ISBN: 9780128164143

Book preview

assistance.

Part 1

Background and overview

Chapter 1

The diabetic foot

Shomita S. Mathew-Steiner; Dolly Khona; Chandan K. Sen    Indiana Center for Regenerative Medicine and Engineering, Department of Surgery, Indiana University Health Comprehensive Wound Center, Indiana University School of Medicine, Indianapolis, IN, United States

Abstract

Diabetes is an ongoing global epidemic associated with pathologies such as foot ulcers, which are a leading cause of hospitalization. Ulceration, infection, gangrene, and amputation are outcomes of underlying vascular, neurological, and immunological dysfunction, leading to an estimated burden of billions of dollars every year in health care costs. Improving the treatment of ulcers together with prevention are key facets in foot ulcer control and they require integrated, multidisciplinary approaches.

Keywords

Diabetes; Foot ulcers; Vascular; Neurological and immunological dysfunctions; Multidisciplinary approaches

1 Introduction

Diabetic foot ulcers (DFU) are a common reason for hospitalization of diabetic patients and frequently result in amputation of lower limbs. Of the one million people who undergo nontraumatic leg amputations annually worldwide, 75% are performed on people who have type 2 diabetes mellitus (T2DM) [1,2]. The risk of death at 10 years for a diabetic with DFU is twice as high as the risk for a patient without a DFU [3]. The amputation rate in patients with DFU is 38.4% [4]. Infection is a common (> 50%) complication of DFU [1,5–13]. Emerging evidence underscores the importance of biofilm infection in the progression of nonhealing DFU [12,14]. Eighty-five percent of amputations in diabetic patients is attributed to DFUs made chronic by infections [15]. DFUs occur due to a combination of mechanical changes in the foot, peripheral neuropathy and peripheral arterial disease [16,17]. In the United States, DFU management alone is estimated to cost somewhere between $9 and $13 billion [18]. DFU management includes the use of therapeutic footwear to offload the wound [19,20] together with maintenance of a moist wound environment [21]. Debridement together with aggressive antibiotic therapy is necessitated for infected wounds [22,23]. Additionally, the maintenance of optimal blood glucose and treatment of vascular insufficiencies are key elements of wound management.

This chapter provides a brief overview of the pathology of the diabetic foot. Details of the component elements are presented in other chapters within this book.

2 Clinical classification

2.1 Definition

A diabetic foot is characterized by ulceration that is driven by neuropathy and/or peripheral artery disease of the lower limb and is a complex, frequent, and expensive complication in diabetic patients.

2.2 Risk factors

Several direct and indirect risk factors are associated with the development of a DFU. These include (a) lifestyle factors such as smoking, uncontrolled diabetes, poor nutrition, immobility, age, etc.; (b) physiological factors such as neuropathy (loss of sensation), vasculopathy (insufficient oxygen availability), shear stress and trauma, and bone deformities (c) genetic and ethnic factors have been implicated in the development of diabetes and diabetic complications [24,25]. DFU is of particular concern among Latinos African Americans, and in Native Americans, who have the highest risk for diabetes worldwide [26].

2.3 Etiology

DFUs develop due to a combination of neuropathy [27], arterial disease [28], pressure [19], and plantar deformity [6]. Diabetic neuropathy is found in 80% of diabetic persons with foot ulcers and is a key factor in development of DFUs.

Biomechanical factors, such as tissue stiffness and thickness, may contribute to DFUs. In a study involving 39 subjects, the heel pad of the foot without an ulcer was found to be stiffer than that with an ulcer [29]. In patients with neuropathy, the plantar soft tissue is thicker and less stiff in specific parts of the foot that are more prone to developing into ulcers. Therefore, mechanical properties of plantar soft tissue could have predictive value for DFU prognosis [30].

2.4 Epidemiology

According to the Centers for Disease Control, 30 million Americans are estimated to have diabetes [31]. DFUs, in particular, are associated with increased hospitalizations [18]. Fifteen percent of diabetic patients develop a foot ulcer, and 38.4% of these require amputation. Indeed, in the United States, most nontraumatic lower-extremity amputations are associated with diabetes [32].

Diabetes and complications such DFUs have been reported globally and are indicators of the growing and profound impact of this pervasive disease [8,33–41].

3 The complicated diabetic foot

The diabetic foot develops as a consequence of several independent and interdependent complications discussed briefly below.

3.1 Vascular

Diabetes is associated with microvascular and macrovascular etiologies, including cerebrovascular, cardiovascular, and peripheral arterial disease. DFU patients have higher premature mortality due to cardiovascular complications [42,43]. Diabetic adults are 2–4 times more at risk for heart disease and stroke than their nondiabetic counterparts. In fact, 65% of deaths in diabetics are associated with vascular complications [31]. In addition to the association of ischemic heart disease and mortality, chronic ulceration could induce chronic inflammation, which could promote the development of atherosclerosis [44]. Furthermore, the ischemia caused by poor blood flow promotes nonhealing ulceration of the foot, leading to amputation. The key impact from this complication is the lack of oxygen supply to the foot and wound and therefore, aggressive treatment of limb ischemia is vital to managing the wound and preventing amputation [45,46].

3.2 Neural

It is believed that 45%–60% of all DFUs are purely neuropathic, while up to 45% have neuropathy combined with ischemia [47,48]. Peripheral sensory neuropathy (PSN) is a major factor leading to DFUs [27,47,49,50]. PSN, even with adequate arterial perfusion, will often promote infection progression primarily due to a lack of sensation in the foot [51,52]. Unnoticed excessive temperature exposures, pressure from ill-fitting shoes, or trauma may cause blistering and ulceration. Motor neuropathy results in muscle atrophy causing foot deformities such as hammertoe, foot drop, etc. [53,54]. Neuropathy induced muscular atrophy together with vascular deficiencies are significant risk factors for limb loss in diabetic patients. Autonomic neuropathy leads to skin breakdown creating a portal for entry of microbes that cause infection [55,56]. In diabetic people with neuropathy [57], the wound recurrence rate is 66% with an associated increase in amputation rate by 12%. Cardiovascular autonomic neuropathy (CAN) is another complication of diabetes that is associated with increased mortality and silent myocardial ischemia [58]. Somatic neuropathy has been implicated in the calcification of the arterial wall leading to cardiovascular mortality [59].

Sensory and motor neuropathy in the foot together with bone and joint deformities and associated vascular changes cause a progressive condition called Charcot foot (neuropathic osteoarthropathy), which physically manifests as a convex foot with a rocker-bottom appearance [60–62]. It is thought to affect about 2% of diabetic persons. Excessive inflammation and vascular calcification common in diabetic patients are implicated in the etiology of this condition [61,63–66]. Neuropathic feet with low intrinsic muscle volume and plantar aponeurosis dysfunction may contribute to the development of claw toes, manifesting as extension and flexion of the metatarsophalangeal and interphalangeal joints, respectively [67].

3.3 Skin and soft tissue

The diabetic skin is mechanically less competent due to glycation of fibrillar collagen and may have defects in the epidermal barrier [68]. The defective vascular supply and neuropathy affects the skin resulting in poor skin nutrition and regenerative capacity and increased vulnerability to cutaneous injuries [1,13,69,70].

3.4 Bone

Bone disease is a complication in diabetes resulting in impaired bone quality that contributes to increased vulnerability to fracture [71]. Underlying mechanisms include modification of bone cells by advanced glycation end products, changes in the incretin hormone response, and microvascular deficiencies. The diabetic Charcot foot shows increased bone resorption and inflammation stimulating osteoclastic hyperactivity leading to gross deformities [72].

3.5 Infection

DFUs are prone to infections that could be more severe than those found in nondiabetic patients, affecting not only the skin and soft tissue but underlying bone [73]. The incidence of DFU infection (DFI) ranges from 25% in all persons with the diagnosis, to 4% yearly in patients undergoing treatment. The combined effect of hyperglycemia, poor vascularization (poor oxygenation), neurological problems, and immunological disturbances contribute to sustaining DFIs [74–77]. DFIs are polymicrobial in nature and complicated by the biofilm mode of growth [5,9,11,52,78–84]. Uncontrolled diabetes impairs the ability of host immune defenses to control microbial pathogens. Ischemia promotes infection by decreasing oxygenation of tissue and impairs the delivery of immune cells and systemic antibiotics to the site of infection.

Biofilms are estimated to account for 60% of chronic wound infections [85]. Recent studies demonstrated a role for biofilm infection in compromising the functionality of repaired skin. Biofilm infection promoted the down-regulation of epithelial junctional proteins, resulting in wounds that appeared to close visually, but lacked barrier function. There are no reliable point-of-care diagnostic methods for direct biofilm detection in wounds, but, a promising indirect alternative could be the measurement of trans-epidermal water loss (TEWL) using a commercial handheld device [86–89].

4 Care and management

4.1 Treatment

The treatment of DFUs involves at minimum surgical interventions (debridement, revascularization etc.), wound coverage (dressings and maintenance of moisture) [21], antibiotic therapy (infection treatment) [22,23], the use of appropriate therapeutic footwear [19,20], optimal control of blood glucose, and evaluation and correction of vascular insufficiency [90].

Surgical interventions include debridement of infected tissue and sometimes bone, removal of excess callus, skin grafting, and revascularization. Debridement may not eliminate microbial biofilms from the wounds and could possibly drive biofilm debris deeper into the wound, aiding in recurrent infection [89]. The use of cultured human cells [91, 92], recombinant growth factors [93–95], placenta or grafts for coverage, and hyperbaric oxygen treatments may promote wound healing, if vascular supply is adequate. Hyperbaric oxygen therapy (HBOT) increases the supply of oxygen to wounds [96,97] and has proven to be beneficial in the treatment of ischemic wounds as seen in DFUs [98]. Multiple hyperbaric oxygen treatments (85 min daily, 5 d/wk for 8 wk) were shown to result in the complete healing of 52% of patients with DFU in the treatment group compared to 29% in the placebo group [99]. The dosing of oxygen needs to cater to the specific needs of the wound to be most effective [100–102].

4.1.1 Other factors

The management of systemic and local factors [103–106] such as sugar control, limited activity (offloading and bed rest) and physiological parameters such as obesity, hypertension, hyperlipidemia, heart disease, and smoking cessation are key elements in preventing and treating DFU [107, 108].

Ongoing and completed clinical trials (https://clinicaltrials.gov/) on DFU treatments include over 500 studies testing therapies, such as maggot debridement, oxygen, platelet gel, human amniotic membrane, laser, shockwave, vacuum-assisted closure (VAC) combined with various dressings, erythropoietin hydrogels, pulsed radiofrequency energy, phage therapy (infection related), surfactants, mesenchymal stromal cells and derivatives, fish skin grafts, phototherapy among others.

4.2 Prevention

Routine preventive podiatric care, appropriate shoes, and patient education are key factors in preventing ulcer formation and amputation [75]. Eighty-five percent of DFUs are estimated to be preventable with appropriate medical evaluation, supportive care, including the use of well-fitting shoes and prompt treatment of wounds. Additionally, physical activity and exercise decreased ulcer occurrence significantly better than no physical activity (0.02 vs 0.12, respectively). DFU patients who had more activity also had improved peripheral neurological parameters [109].

5 Conclusion

DFUs are a complicated manifestation of several interconnecting pathologies that may not be managed by any single intervention or therapeutic. A productive solution would involve interdisciplinary approaches that seamlessly integrate medical interventions, community outreach, nutritional support, and consideration of socioeconomic and cultural factors.

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Chapter 2

Role of oxidants and antioxidants in diabetic wound healing

Manuela Martins-Green; Shayan Saeed    Molecular, Cell and Systems Biology Department, University of California, Riverside, CA, United States

Abstract

It has been extremely difficult to study the mechanisms involved in the development of chronic wounds because they do not contain a single defect. One of the critical parameters for proper healing is the redox state of the wound. The redox state is maintained by a balance between oxidant and antioxidant molecules. Oxidative stress (OS) is present in tissues and cells when there is an imbalance between the levels of reactive oxygen species (ROS) and the ability of antioxidants in the tissues and cells to remove these species and repair the damage they cause. Low levels of ROS are important for initiation and progression of proper healing, whereas high levels of ROS derail the cell and molecular mechanisms involved in healing leading to cell death and paralyzes of the healing process. Antioxidants inactivate reactive species by donating their electrons to these species and preventing them from capturing electrons from other important molecules such as DNA, proteins, and lipids. When the levels of antioxidant molecules are not sufficient to detoxify the cells and tissues from excessive OS, damage occurs. Examples of reactive radical forms of O2 are superoxide (O2•−), hydroxyl (•OH), and peroxyl (LOO•), whereas examples of nonradical reactive forms are hydrogen peroxide (H2O2), hypochlorous acid (HClO), and peroxynitrite (ONOO). Antioxidants can work through enzymatic or nonenzymatic reactions that can occur intracellularly in the cytosol and/or in organelles such as the mitochondria or in the extracellular environment. Chronic wounds in humans have high levels of OS. This is particularly true for chronic wounds of diabetic patients. A better understanding of how wounds become chronic is critical for future success in the treatment of such wounds after debridement. Because high levels of OS are present in chronic wounds and are critical for chronic wound initiation and development, we speculate that treatment of human chronic wounds with antioxidants both systemically and locally after debridement, coupled with other treatments such as antibiotics, may be effective in resolving chronicity.

Keywords

Oxidative stress; ROS; H2O2; Catalase; GPx; NOX; Nrf2; AGE

1 Introduction

Healing of cutaneous wounds involves a series of complex processes that occur sequentially with each phase overlapping the previous one, leading to a partial regeneration of the dermal tissue and reestablishment of the epithelial barrier. Upon wounding, hemostasis occurs with formation of a clot that stops the bleeding and releases factors that stimulate the inflammatory phase, which involves the chemoattraction of various types of leukocytes to the site of wounding. The first type of leukocytes to appear are the neutrophils that kill bacteria by producing reactive oxygen species (ROS), and in this manner prevent wound infection. Following the influx of neutrophils, monocytes arrive at the wound site and become proinflammatory macrophages. These cells are involved in cleaning the damage caused by neutrophils when phagocytosing the bacteria. At the same time antiinflammatory macrophages secrete growth factors and cytokines that promote healing. Furthermore, keratinocytes, fibroblasts, and endothelial cells also migrate to the wound site to close the wound. While keratinocytes form the epidermis, fibroblasts produce the extracellular matrix molecules (ECM) that confer structural and biochemical properties to the wound tissue and endothelial cells that contribute to the development of new blood vessels. These processes form the so-called granulation tissue named so because of its granular appearance. In the final phase of wound healing, remodeling, the surplus cells undergo apoptosis and the excess ECM produced is removed by phagocytes that remodel the wound tissue, ultimately resulting in scar tissue.

Acute wound healing follows this path of events. However, when the processes involved in acute healing are either stalled or fail to occur in the proper order, impaired healing occurs, the wounds do not close properly, skin barrier is not established, and the granulation tissue does not evolve into a scar tissue. Furthermore, when impaired wound healing occurs and is accompanied by chronic inflammation and infection with biofilm-forming bacteria, the wounds become chronic. Therefore, chronic wounds develop as a result of defective regulation of the complex cellular and molecular processes involved in proper healing (e.g., [1–4]). They impact ~ 6.5 M people and cost ~$25B/year in the United States alone (e.g., [5]). Although a number of studies have been performed to address processes involved in wound chronicity, until now the scientific community in the wound healing field has been unable to crack the complex and multidimensional processes involved in initiation and development of chronic wounds, in particular of diabetic chronic wounds. This is primarily because it is virtually impossible to study initiation and development of wound chronicity in humans because by the time these wounds present in the clinic, the initial stages of development are long gone. In addition, we cannot experiment in humans with chronic wounds. The critical need for a cure of diabetic chronic wounds is underlined by the continuous increase in type II diabetes (which accounts for 90%–95% of all diabetes). It has been reported that diabetes affects 387 M people globally and 28 M in the United States and prediabetes affects 316 M more globally and 86 M more in the United States [6]. These statistics are daunting, considering that a significant number of these patients will go on to develop foot ulcers and that about 12% of them will require amputation (e.g., [5]); the 5-year survival after amputation is ~ 50%.

Animal models for study of the genesis and development of nonhealing wounds are critical to elucidate the processes involved in wound chronicity. Although a number of such animal models have been developed previously to study chronic wounds, these models only mimic some of the critical elements of wound chronicity in humans [7–26]. Recently, the TallyHo Polygenic Mouse Model of Diabetes was developed as a model with potential to study diabetic chronic wounds. However, only males were hyperglycemic and the authors needed to introduce biofilm-forming bacteria for the wounds to become infected [27,28]. A more recent model used Wister rats fed with high-fat diet and treated with multiple injections of streptozotocin (STZ) to develop type II diabetes. This treatment caused noticeable insulin resistance, persistent hyperglycemia, moderate degree of insulinemia, as well as high serum cholesterol and high triglyceride levels. Furthermore, the rat wounds showed excessive wound inflammatory response, excessive and prolonged ROS production, excessive production of MMPs, and delayed healing [29]. However, these wounds did not become chronic nor did they develop biofilm much like chronic wounds in diabetic patients.

We have recently developed a novel chronic wound model using db/db−/−, a type II diabetic mice, which has all the characteristics present in human chronic wounds. What makes it a powerful model is the fact that this model develops biofilm naturally and without having the need to introduce extraneous bacteria. To develop our model, we took advantage of the fact that chronic wounds in humans contain toxic levels of oxidative stress (OS) and biofilm-producing bacteria [20–23]. High levels of OS lead to deregulation of gene expression, damage to DNA, proteins, and lipids, a hostile proteolytic environment, and cell death [30–32]. OS also leads to impaired keratinocyte migration in vitro, potentially inhibiting re-epithelialization and leading to poorly developed granulation tissue [11,29]. To create chronic wounds in our db/db−/− mouse model, we increase the OS to high levels immediately after wounding. The wounds go on to become chronic 100% of the time [20–23]. All of the defects found in human chronic wounds are present in our model including the presence of naturally formed biofilm. Therefore, we now have a mouse model that can be used to understand the basic cell and molecular mechanisms of initiation and development of wound chronicity under diabetic conditions.

2 Oxidative stress and wound healing

Oxygen is essential for just about all cellular functions (e.g., cell proliferation, migration, differentiation, death), and it is a prerequisite for successful wound healing. Sufficient oxygenation of the wound tissue is needed for adequate energy levels, which are essential for proper cellular function during the healing process. Under normoxic conditions, oxygenation of the wound is sufficient for proper healing. Under hypoxic conditions oxygenation of the wound tissue is insufficient for healing and under hyperoxic conditions, oxygenation is excessive, which can lead to increased ROS adversely affecting healing.

Oxidative stress is present in tissues/cells when there is an imbalance between the levels of ROS and the ability of antioxidants in the tissues/cells to remove these species and repair the damage they cause. Balanced levels of ROS kill pathogens, stimulate immune cells, keratinocyte migration, granulation tissue formation, angiogenesis, and collagen synthesis. Imbalanced levels of ROS cause DNA damage, lipid peroxidation, protein nitration, and eventually cell death.

ROS are highly reactive forms of O2-derived radicals or atoms/molecules that contain one or more unpaired electrons [33]. Examples are superoxide (O2•−), hydroxyl (•OH) and peroxyl (LOO•) radicals. Nonradical reactive forms of O2 can also occur in tissues such hydrogen peroxide (H2O2), hypochlorous acid (HClO), and peroxynitrite (ONOO). O2•− are produced when NADPH oxidases (NOX) oxidize NADPH to NADP + and in the process give one electron to O2 making O2•−. O2•− is produced primarily in the mitochondrial electron transport chain as small amounts of O2 leak from this system during the oxidative-phosphorylation reactions and by phagocytic cells, such as neutrophils, in the process of killing bacteria [34].

Oxygen-derived radicals are not the only chemical species that damage tissues. Nitrogen and sulfur-derived radicals are also damaging. Nitric oxide (NO) is generated enzymatically by nitric oxide synthetases (NOS). In the presence of NO, O2•− will form ONOO−, which causes peroxidation of lipid molecules and oxidation of aromatic amino acids in proteins, therefore damaging their function [33]. O2•− can also be dismutated to H2O2, spontaneously or by superoxide dismutase (SOD), which in turn, in the presence of ferrous ions can produce both hydroxyl anions (OH−) and •OH, which are highly damaging of proteins and DNA. However, H2O2 can be broken down into H2O by glutathione peroxidase (GPx) and into H2O + O2 by catalase, two very effective antioxidant enzymes present in cells and tissues. If this system of antioxidants does not function correctly, excessive oxidative stress builds in the tissue.

A balanced redox state is critical for proper healing. During the hemostasis phase of healing, ROS contribute to blood vessel constriction to stop bleeding at the same time that thrombin activation leads to polymerization of fibrin fibers to which platelets and collagen adhere contributing to the formation of the clot [35]. As platelets degranulate, they release growth factors as well as proinflammatory cytokines and chemokines that are important in attracting neutrophils to initiate the inflammatory phase of healing. These leukocytes are very effective in killing bacteria via the respiratory/oxidative burst, a defense strategy that involves the production of high levels of O2•− and H2O2 via NADPH oxidase, into the bacteria-containing phagosome leading to the killing of these infectious agents [36]. Simultaneously, some of these ROS are released into the microenvironment causing tissue damage. At low concentrations (10 μM), H2O2 attracts leukocytes to the site of injury, whereas at concentrations 10 × higher, it induces levels of basic fibroblast growth factor (bFGF) that stimulate fibroblast proliferation and migration, increase vascular endothelial growth factor (VEGF), which stimulates angiogenesis. H2O2 also stimulates signaling by transforming growth factor beta 1 (TGFβ1), which leads to increased chemotaxis for keratinocytes and stimulation of matrix production [36–38]. H2O2 is the major signaling ROS during wound healing; it is easily synthesized and degraded, it moves readily through cell membranes and tissues because it is uncharged, it can have a long half-life and is selective in the molecules it reacts with.

3 Antioxidants and wound healing

The redox state of tissues and cells is maintained by a balance between oxidant and antioxidant molecules. The latter molecules remove the deleterious effects of the reactive species by donating their electrons to these species and preventing them from capturing electrons from other important molecules, such as DNA, proteins, and lipids. In the presence of excessive levels of ROS and/or RNS and when the levels of antioxidant molecules are not sufficient to detoxify the cells and tissues, damage occurs.

There are several types of antioxidants, those that perform enzymatic reactions and those that are nonenzymatic in their effects (Table 1). Some of these reactions occur in the extracellular microenvironment, others occur intracellularly in the cytosol and/or in organelles such as the mitochondria [39].

Table 1

3.1 Examples of enzymatic antioxidants

Examples of enzymatic antioxidants are SOD, glutathione S-transferases (GSTs), glutathione peroxidases (GPx), NADP(H), catalase, heme-oxygenase 1 (HO-1), peroxiredoxins (Prdx), thioredoxin-1 (Trx-1) and -2 (Trx-2).

3.1.1 SOD, GSTs, GPx, NADPH

The mechanisms of action of SOD, GSTs, GPx are well coordinated during normal wound healing, they are altered in chronic wounds. SOD + O2•− + 2H+ produces H2O2, which is then broken down by either catalase that takes 2H2O2 molecules to produce H2O + O2 or by GPx that takes one molecule of H2O2 with 2 glutathione (GSH) molecules to produce 2H2O plus one molecule of glutathione disulfide (GSSG). The latter in turn will regenerate GSH for the GPx reaction by reacting with the coenzyme NADPH + H+ to produce 2GSH + NADP+, a reaction that is catalyzed by glutathione reductase (GRX) (Fig. 1).

Fig. 1 Generation and breakdown of H 2 O 2 . Superoxide dismutase (SOD) catalytically converts the superoxide radical (O 2 • − ) in the presence of 2 protons to one molecule of hydrogen peroxide (H 2 O 2 ). H 2 O 2 is catalytically convert to water and oxygen by catalase, or combines with the antioxidant glutathione (GSH) and catalytically forms water and glutathione disulfide (GSSH) by the enzyme glutathione peroxidase (GPx). Upon the depletion, glutathione is regenerated by interacting GSSG with NADPH and hydrogen by the enzyme glutaredoxin (GRx).

3.1.2 Heme-oxygenase 1 (HO-1)

HO-1 is primarily known for its ability to catalyze degradation of heme into CO and/or iron in the presence of O2 and NADPH giving rise to biliverdin that is converted into bilirubin, a very strong antioxidant, by biliverdin reductase in the presence of NADPH (Fig. 2A) [40,41]. CO is a vasodilator, antiinflammatory, antithrombotic, and antiapoptotic agent, whereas Fe++ through ferritin is an antioxidant and has cytoprotective properties. It was shown several decades ago that HO-1 is also highly induced by a variety of agents causing oxidative stress [42,43]. HO-1 is elevated during the first day after wounding, whereas HO-2 is not. HO-1 is expressed in keratinocytes of the wound epithelium and in inflammatory cells. Knockout mice for HO-1 showed delayed wound closure and decreased angiogenesis resulting in impaired wound healing.

Fig. 2 Antioxidant cycles for hemet oxygenase 1 (HO-1), thioredoxin (Trx), and peroxiredoxin (Prx). (A) The protein NRF2 stimulates expression of HO-1, which catalytically degrades the hemet complex, with the addition of oxygen and NADPH, to form biliverdin with the release of iron (Fe ² + ) and carbon monoxide (CO). Biliverdin is then reduced to bilirubin in the presence of NADPH by biliverdin reductase. (B) The oxidation of NADPH catalyzes the reduction of cysteines in Trx proteins. As Trx becomes oxidized, Prx is reduced and in the presence of H 2 O 2 , one oxygen is transferred to one of the SH groups in a cysteine of the Prx releasing water and regenerating reduced Prx.

3.1.3 Peroxiredoxins and thioredoxins

Peroxiredoxins and thioredoxins are antioxidant enzymes that function in various ways to reduce oxidative stress [44,45]. Prx(s) catalyze the reduction of H2O2 as well as a broad range of peroxides [46,47] (Fig. 2B). Prx 6 can also detoxify tissues and cells from peroxynitrite [48,49]. This enzyme is expressed at higher levels than other Prdx(s) in the wound tissue of mouse excisional wounds. In transgenic mice overexpressing Prdx(s) in keratinocytes, wound closure occurs more rapidly [49,50]. Trx(s) constitute a family of small redox proteins that function as antioxidant enzymes that facilitate the reduction of other proteins by cysteine thiol-disulfide exchange (Fig. 2B). These proteins contain 2 cysteines in a CXXC motif, which are critical for their ability to reduce other proteins such as insulin, the glucocorticoid receptor, and coagulation factors. Mice that are transgenic for Trx(s) are more resistant to inflammation [51].

3.2 Nonenzymatic antioxidants

Nonenzymatic antioxidants are low-molecular-weight molecules that are themselves used during the antioxidant process and are many times called sacrificial antioxidants [33,52]. Molecules such as vitamin C (ascorbic), vitamin E (α-tocopherol), vitamin D, glutathione, N-acetyl cysteine (NAC), alpha lipoic acid (αLA), carotenoids (e.g., lycopenes), bilirubin, and uric acid all belong to this class of antioxidant molecules. Several of these small molecules are found depleted after wounding indicating that they have been consumed to reduce oxidative stress in the wounded tissue and then recover to normal levels within 2 weeks of injury [53].

3.2.1 Vitamin C

Is a very important cofactor for the hydroxylation of prolines and lysines in fibrillar collagen. This hydroxylation is essential for the stabilization of the triplex helix in these collagens and providing integrity and strength to the connective tissue. It is, therefore, logical to think that Vit C is critical for scar formation where fibrillar collagens, in particular Col I, are important to confer strength to the tissue. However, two clinical trials that used Vit C as a supplement for the treatment of patients with significant wounds show contradictory results [54–56]. This outcome is most likely due to the lack of standardization on dose, time of application, and type of patients chosen. This is not an uncommon outcome of clinical trials in particular when the patients have different