Skin Burns: Review of Molecular Mechanisms and Therapeutic Approaches

Burn wounds are one of the main causes of skin damage. Based on World Health Organization statistics, almost 300 000 people worldwide die of burns each year. In severe burns, the cells and blood vessels are often injured and the blood supply to the wound is disturbed. Many factors such as oxygenation, infection, aging, hormones, and nutrition potentially can influence burn progression and disrupt repair with unbalanced release of various growth factors and cytokines. Different treatment approaches such as dressings and skin substitutes have been applied to aid wound healing. A thorough understanding of the effective factors on burns can improve wound healing outcomes. This review evaluates articles published on the Scopus, EMBASE, and PubMed databases that attempt to explain the pathophysiology, molecular components, and therapeutic approaches involved in the burn wound healing process.

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Skin, composed of the epidermis, dermis, and hypodermis layers, is the largest organ of the body. Its functions are absolutely necessary for self-preservation. The skin is prone to damage by microorganisms and thermal, mechanical, and chemical factors. An important cause of skin damage is burn wounds.^1,2 According to World Health Organization reports, about 300 000 individuals worldwide die of burns annually.^3,4 In the United States, 500 000 patients undergo burn wound treatment annually, of which more than 40 000 are hospitalized and 3400 die.⁴ 

Burn wounds lead to various local and systemic pathophysiological processes in the body. They, like all wound types, initiate a continuous inflammatory process associated with the release of different cytokines.⁵ The study of the molecular and physiological bases of cutaneous wound healing could lead to more therapeutic possibilities. For example, studies have proposed the expression of inflammatory mediators (cytokines, chemokines, and growth factors) within a damaged area is essential for the wound healing process.^6,7 Therefore, this review discusses local and systemic pathophysiological responses, molecular components, and some of the therapeutic approaches involved in the burn wound healing process.

There are various models for burn wound evaluation. One of most commonly used is Jackson’s model in which 3 concentric areas can be detected based on the severity of tissue damage and changes in blood flow of a burn wound (Figure).^2,7,8 Briefly, the first zone is the zone of coagulation; this is the point of maximum damage with irreversible tissue loss due to coagulation of the proteins and tissue necrosis. Surrounding the coagulation zone is the zone of stasis, which is characterized by decreased perfusion. This ischemic zone may progress to full necrosis unless the ischemia is reversed. Therefore, the main aim of burn resuscitation is to increase tissue perfusion here and prevent any further damage. The outermost layer is the zone of hyperemia. Tissue perfusion is increased and the tissue here invariably is recovered, unless there is severe sepsis or prolonged hypoperfusion.

In burn injuries, released chemical factors from damaged cells are activated in a 2-phase proinflammatory and anti-inflammatory response. In the first phase, a transcriptional activator (protein), nuclear factor κB, is activated immediately after a severe burn injury to regulate the induction of several proinflammatory mediators, including tumor necrosis factor alpha (TNF-α) and intercellular adhesion molecule-1 (ICAM-1).⁸ These mediators activate neutrophils and monocytes and trigger antimicrobial activity. In addition, TNF-α is responsible for the secretion of other proinflammatory mediators, including interleukins 1 and 6 (IL-1, IL-6), and inducing apoptosis of various cells in the wound area. Thermal injuries increase hypermetabolism, which leads to increased production of anti-inflammatory cytokines, reactive oxygen species (ROS), and reactive nitrogen species (RNS).⁹ The anti-inflammatory phase of a burn injury are related to T helper (T_H) 2 lymphocytes and the secretion of 3 cytokines: IL-4, IL-10, and TNF-α.¹⁰  

Wound healing starts with the expression of various chemokines that initiate a topical response to constrict the impaired area. This topical response is associated with inflammation and local infection, which starts with sympathetic stimulation and may lead to hypovolemia, renal failure, and myocardial dysfunction. Progressive organ dysfunction is accompanied by a high mortality rate.^11,12 Patient-dependent factors such as nutrition, resuscitation, aging, oxygen therapy, and stress can play a crucial role in the systemic response. When the topical pathways become insufficient for a therapeutic response, a systemic response is induced. 

Local factors and responses in burn injuries

Cytokines. Cytokines are a broad category of proteins, which are secreted by certain cells of the immune system and interact with cell signaling. They include ILs, lymphokines, and signaling molecules (eg, interferons and TNF-α). Chemokines or chemotactic cytokines are small cytokines that can induce direct migration of leukocytes to the wound area (chemotaxis). For example, IL-1α, IL-1β, IL-6, and TNF-α may control various processes in the wound area, including keratinocyte and fibroblast proliferation and differentiation, synthesis and destruction of extracellular matrix (ECM) proteins, and immune response regulation. In the inflammatory phase of wound healing, macrophages and neutrophils are the major sources of these cytokines.^6,13,14 

Growth factors. Growth factors are several classes of endogenous signaling molecules that regulate cellular responses to wound healing processes. They consist of platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), transforming growth factor beta (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and amnion-derived cellular cytokine solution (ACCS). Growth factors play a role in many biological processes, such as proliferation, migration, chemotaxis of fibroblasts and inflammatory cells, stimulation of endothelial cells, and angiogenesis. They interact with the production of the ECM, which inhibits apoptosis, and mediate synthesis of other cytokines and growth factors (Table 1^6,15). Growth factors and cytokines have therapeutic importance in burn wounds to accelerate wound healing; however, they may not be effective in healing extensive and/or deeper burn wounds. While not well understood, it seems the use of these factors applied with skin grafts may be helpful in severe burn wounds.^6,13,15

Oxidative stresses. A severe burn is associated with the secretion of free radicals (ROS and RNS) that cause local and systemic pathophysiological responses in burns. Research has shown released ROS and RNS could protect the wound area against infection.^16,17 On the other hand, increased ROS secretion is associated with immunosuppression, systemic inflammatory response syndrome (SIRS), infection, sepsis, tissue damage, and multiple organ failure.¹⁷ Thus, burn wound improvement depends on the balance between secretion and excretion of free radicals. Particularly, lipid peroxidation of cell membrane polyunsaturated fatty acids with free radicals activates the accumulation of cytotoxic products, such as lipid-derived aldehydes (LDAs). Hydroxynonenal, malondialdehyde, and acrolein are toxic LDAs that interact with normal cell functions such as cell signal transduction, DNA synthesis, and enzyme activities. They cause severe damage to DNA, resulting in genotoxic and mutagenic alternation. Therefore, oxidative stress is an important mechanism in the development of burns. Some studies^18-20 also suggest proinflammatory cytokines (IL-6 and TNF-α) release chemokines, (IL-8), leading to a continuous cascade of ROS and producing severe cellular injuries.

Inflammation. Within the burn area, inflammatory mediators increase the vascular hydrostatic pressure, leading to vessel dilation and systemic edema. As a response to inflammation, the endothelial cell junctions are disjointed and the cell membrane barrier functions are disrupted. It has been shown that thermal injury alters reorganization and contraction of endothelial cell actin and induces general vascular hyperpermeability. It also causes the formation of stress fibers in endothelial cells as well as disruption of integrity of the endothelial barrier.²¹ Furthermore, kinins, specifically bradykinin released from mast cells, cause vasodilation, smooth muscle contraction, and increased microvascular permeability.²² Not surprisingly, in the first week following a burn injury, bacteria can grow within the wound area and infections of the skin and soft tissue commonly occur early in hospitalization.²³ This can cause a prolonged inflammatory phase associated with increased levels of matrix metalloproteases (MMPs) and the breakdown of collagen and ECM. If the inflammatory phase takes longer, the wound may enter a chronic state that fails to heal (Figure).²⁴ 

Infection. The skin acts as a barrier from the external environment to maintain homeostasis and body temperature. Any type of damage to this barrier can disrupt the immune system and increase its susceptibility to bacterial infection. Infection leads to delayed wound treatment, prolonged hospitalization, and increased costs and mortality rate.²⁵ In vitro research has demonstrated bacterial infection in burn wounds can colonize to other tissues after 5 to 7 days and exacerbate to sepsis and death.²⁶ Burn wound infections have been widely investigated in the literature. The rate of systemic infections and mortalities have decreased since the development of topical antibiotics (eg, silver sulfadiazine).²⁷ Moreover, bacterial culture also has helped identify appropriate antibiotics for the invasive bacterial species.²⁸ However, fungi are a culprit for infections with high mortality rates in severe burn wounds.²⁹

Systemic factors in burn injuries

Systemic inflammatory response syndrome. This syndrome is a common inflammatory condition associated with various injuries, such as infection, trauma, and burns. The probability of SIRS increases 3-fold in patients with burns with a total body surface area (TBSA) of more than 30% and is characterized by elevated levels of IL-6, IL-2, and IL-8. The released cytokines induce the synthesis of prostaglandin E2 (PGE2), IL-6, and platelet-activating factor by endothelial cells and macrophages in cutaneous burns. Expression of PGE2 suppresses the lymphocyte reactivity and provokes T_H cells to differentiation to T suppressor. Therefore, this alternation in lymphocyte population inhibits pro-inflammatory cytokines such as IL-2 and IL-1β.^30,31

Patient-dependent factors in burn injuries

Oxygen supply. Oxygen is essential for cell metabolism, adenosine triphosphate production, and wound healing. In the early phases (hemostasis and inflammatory phase), the oxygen supply to burned tissue is important for neutrophilic function because it produces ROS required to inhibit bacterial colonization and wound infection. Furthermore, ROS, such as superoxide and hydrogen peroxide, act as free radicals that help wound healing via PDGF signal transduction, angiogenesis, and cell migration. Because of vascular impairment and high oxygen demand, the microenvironment of the burn wound is susceptible to hypoxia. Some cytokine and growth factors (eg, VEGF, TNF-α, PDGF, TGF-β, and endothelin-1) are generated through hypoxia from macrophages, fibroblasts, and keratinocytes, which are important in the development of chemotaxis and proliferation of cells in burn wound healing.^11,32,33 

Nutrition. Severe burns cause hypermetabolism, which is also a consequence of several hormonal changes. Burn trauma stimulates major increases in catabolic hormones (eg, epinephrine, cortisol, and glucagon) and accelerates gluconeogenesis, glycogenolysis, and muscle proteolysis. Catabolic hormones neutralize the effect of insulin and raise blood sugar levels; protein synthesis and lipogenesis are inhibited. Similarly, growth hormone is antagonized and less effective.³⁴ Therefore, in burn wound healing, prevention of hypermetabolic conditions and good nutritional support are determinant factors.³⁵ 

Nutritional support in burn injuries is complicated and includes several nutritional possibilities. For example, excessive utilization of carbohydrates may cause hyperglycemia, which causes systemic inflammation and muscle damage. Also, excessive intake of fatty acids results in immunosuppression that can impose serious risks of infection and sepsis. It has been shown vitamins and insulin administration reduce protein catabolism, increase protein synthesis, and consequently, improve healing time.^36,37 Insulin also can be replaced with other recombinant growth factors (EGF and TGF) in wound healing, however, the use of such factors is still controversial and expensive.^38,39

Resuscitation. Severe thermal wounds (> 20% TBSA) require fluid resuscitation. The purpose of fluid resuscitation is to maintain the perfusion of all organs with a minimal volume of liquid. Traditional resuscitation fluid includes modified Brock and Park formulations, crystalloid solutions (lactate ranger containing sodium, chloride, calcium, potassium, and lactate), and colloid solutions (fresh frozen plasma, albumin).⁴⁰

Aging. Aging is recognized as a common cause of a temporary interruption in wound healing, but it does not seriously endanger wound healing. Aging leads to a change in the inflammatory reaction, such as lower macrophage phagocytic capacity, T-cell infiltration into the wound site, and chemokine production. In addition, reduction of collagen synthesis and reassembly, wound angiogenesis, and re-epithelialization were observed in aged mice.^41,42 

Stress. Stress impacts human health in different ways as seen in many diseases and impaired wound healing. Stress reduces the level of pro-inflammatory cytokines and decreases the expression of chemoattractant factors such as IL-1α and IL-8 at the wound site, which are important for the initiation of the inflammatory phase of wound healing.⁴³

First aid

First aid is an essential intervention before receiving medical treatment. Among the current recommendations for the initial treatment of burns is to keep burn wounds cool at about 15°C for 20 minutes. Research has found the use of 2°C to 15°C water is beneficial for wound healing.⁴⁴ This first aid technique would increase the rate of reepithelialization, reduce pain, and improve the appearance and thickness of the resultant scar.⁴⁵ Nevertheless, using water at 2°C, especially for children or hypothermic patients, is dangerous. 

Research⁴⁶ also has shown cold water inhibits the release of histamine from damaged tissues and significantly inhibits the activation of kallikrein in human plasma. Kallikrein is one of the kinins (bradykinin and kallidin) that cause vasodilatation and hypotension, and it increases fluid leakage from the vessels and causes local edema. Therefore, kinins inhibition causes a reduction in vasodilatation, vascular permeability, and edema. There is no evidence showing the use of ice in burn wounds is more harmful, but direct contact with ice may increase vascular contractions and damage progression or cause frostbite and protein denaturation in tissue.^44,45

Traditional and drug treatments 

Cooling burn wounds with water eliminates heat, preventing further burn development. This is effective if carried out within 20 minutes by immersion in water at 15°C. The current recommendations for first aid treatment of burn injuries should be to use cold running tap water (between 2°C–15°C) on the burn; ice should not be used.⁴⁷ Chemical burns should be washed with plenty of water.⁴⁸ 

Several studies^49,50 have found that the treatment of inflammation is difficult in severe burns.Traditional anti-inflammatory treatments such as nonsteroidal anti-inflammatory drugs or glucocorticoids downregulate prostaglandin synthesis and negatively impact wound healing. However, steroid therapy decreases inflammation, pain, and hospitalization time of burn patients.⁵¹ It also has been reported that non-anti-inflammatory drugs, such as opioids, postpone the early inflammatory phase, accelerate the proliferative phase, and stimulate keratinocyte migration in vitro.^52,53 However, large-scale clinical trials have not yet been conducted to evaluate the efficacy of opioids in wound healing. 

Burn dressings 

Since infection prevention is critical, wound coverage should be applied as soon as possible. However, coverage is one of the main challenges in the treatment of severe burns. Wound dressings can be divided into 4 groups: conventional, biological, biosynthetic, or antimicrobial.

Conventional coverage (eg, petroleum gauze or silicone sheets) are used to cover the wounds temporarily; these do not contain antibiotics or medications and tend to stick to the surface of the wound. The frequent conventional dressing changes required also can affect reepithelialization and delay wound healing.

Biological coverages include cadaver allograft skin, xenograft skin, and human amnion and are used to cover burn wounds temporarily to help reepithelization. For immunological reasons, biologics cannot be used as a permanent substitute.

Biosynthetic coverages are composed of epidermal, dermal, or epidermal-dermal combination substitutes that simulate skin function. Several skin substitutes have been used successfully for burn wound treatment such as Karoskin (human cadaver skin with dermal and epidermal cells; Karocell Tissue Engineering AB, Karolinska, Sweden), Glyaderm (glycerol preserved acellular dermal collagen-elastin matrix; Euro Skin Bank, Beverwijk, The Netherlands), and OASIS Wound Matrix (porcine acellular lyophilized small intestinal collagen matrix; Smith+Nephew, Fort Worth, TX).

Antimicrobial dressings are widely used to protect against infections. These products may contain either silver, nanocrystalline silver, cadexomer iodine, or honey as an antimicrobial agent.⁵⁴ Antimicrobial dressings, such as silver dressings, can have a preventive effect against infection within the first 48 hours of a burn injury. Generally, a silver dressing should be soaked in sterile water and applied to the wound bed, not in normal saline solution, because chlorine ions can attach to silver ions and reduce the amount of silver delivered to the ulcer. After 48 hours, the silver dressing is removed and the wound is assessed for further treatment. Although silver is poisonous to bacteria, there is evidence that silver prevents keratinocytes and fibroblasts from proliferation and can potentially slow the healing process. It also is recommended to use a moist dressing after the initial antimicrobial dressing.⁵⁵ Standard wound management with dressings can significantly rehabilitate the stasis zone and prevent further coagulation. The general purpose of each wound dressing, regardless of burn size and depth, is to prevent infection, promote wound healing, reduce pain, and enhance motion capacity and performance. Burn injury is a dynamic process of changes, especially in the first 48 hours after injury. Therefore, a suitable burn dressing should be firmly used for 48 hours to prevent infection. 

Skin grafting 

Skin flap grafting is also part of the gold standard for treatment of full- and deep partial-thickness wounds, because early excision helps to reduce infection and scarring. Puri et al⁵⁶ found excision of wound debris within 24 to 48 hours can reduce the amount of fluid loss and infection, decrease the duration of hospitalization and mortality rate, and increase the possibility of transplantation.⁵⁶

Moist wound healing 

The wound environment is defined as the environment in direct contact with its surface. A dry dressing is defined when there is no obstruction to extracellular fluid and ECM in the wound. The dressing is described as moist when a moisture-containing, controlled hydration dressing is used to cover the wound surface. Finally, it is described as a wet or an occlusive dressing when covered with an impermeable membrane that adheres to the wound border.⁵⁷ A dressing that creates and maintains a humid environment seems to provide optimal conditions for wound healing. The moisture under the occlusive dressing not only increases the rate of epithelialization but also improves it by maintenance of moisture similar to an incubator as well as maintenance of wound exudate, which contains cytokines and vital proteins in response to injury. Low oxygen tension in these dressings also promotes the inflammatory phase. However, a dry dressing with gauze does not exhibit these properties; it can impair wound healing and damage tissues when removed. In dry wounds, keratinocytes migrate at a deeper level in order to most effectively proliferate, but in a moist wound environment, keratinocytes can more easily move toward the surface of the wound for closure.⁵⁸ A moist wound bed can reduce the risk of infection by creating a hypoxic environment. This hypoxic wound bed amplifies angiogenesis, reduces wound bed pH, and makes the wound uninhabitable by bacteria, ultimately protecting against infection. Moreover, the moist wound environment increases the production of collagen by fibroblasts and helps the ECM synthesis.⁵⁸ Wound drainage fluids are a source of soluble factors associated with wound healing. In particular, PDGF, FGF, and EGF; keratinocyte growth factor (KGF [IL-6 and MMP-8]); and VEGF (hepatocyte growth factor [HGF], IL-8, and MMP-1) have been found in the fluid under occlusive dressings.⁵⁹ Some wet dressing products (including hydrocolloids, hydrogels, foams, and alginates) are included in Table 2.^58,60 

Cell therapy in severe burn wound healing

Regeneration of skin after wound healing depends on several factors, including the availability of primary precursor cells, ECM components, and cytokines for angiogenesis, cell-to-cell and cell-to-matrix interactions. Until now, autologous skin grafts commonly have been used to treat severe burns; however, its efficacy in severe burns is not fully understood because of the limited availability of donated skins. Alternatively, cell therapy uses autologous or allogeneic cell components to repair or regenerate damaged tissue. Cell therapy for burn injury treatment began when Rhienwald and Green⁶¹ isolated and serially cultivated human keratinocytes from a skin biopsy. The following subsections are a small part of the described content to understand the types of cells and their potential role in burn wound healing.⁶² 

Keratinocyte stem cells (KSCs). Based on molecular biomarkers, regenerative capacity, and cell differentiation status, keratinocytes are categorized into KSCs, transient keratinocytes, and differentiated keratinocytes. The KSCs, which comprise about 4% to 8% of the total keratinocyte content in the skin,⁶³ are seen in the basal membrane of the epidermis and around the skin appendages. These cells asymmetrically are divided to 

keep undifferentiated stem cell numbers in the basal membrane. In addition, a group of protozoan keratinocyte cells recognized in the peripheral blood is capable of converting to keratinocytes and express cytokeratin, involucrin, and filaggrin proteins.^62,63

Skin fibroblasts. Fibroblasts are critical to skin production and improve postburn healing. They produce ECM proteins in the dermis and play a role in the biological functions of skin cells. These cells produce many growth factors and cytokines, including VEGF, PDGF-AA, KGF, basic fibroblast growth factor, HGF, TGF-β1, IL-6, and IL-8. Fibroblasts also can be differentiated into pluripotent stem cells with the capacity to regenerate various tissues.^62,63

Mesenchymal stem cells (MSCs). Mesenchymal stem cells are a specific group of multipotent cells able to self-renew with virtually unlimited differentiation capacity and can be derived from most autologous or allogeneic tissues. These cells can produce growth factors (VEGF, EGF, KGF, IGF, MMP-9, and stem cell factor 1) that are specific to angiogenesis, cell proliferation and differentiation, and establishment of the ECM during wound healing. The MSC also releases cytokines, including TNF-α, interferon lambda, IL-1α, IL-1β, and nitric oxide, to control host immune response.^62,63

Embryonic and induced pluripotent stem cells. Human embryonic stem cells (hESCs) are isolated from human embryos, which have an excellent capacity for tissue regeneration. Research has shown hESCs can be differentiated into functional keratinocytes.⁶⁴ Although hESCs can be used as a skin substitute in patients with burns, its application is problematic, owing to ethical issues. Takahashi et al⁶⁵ obtained pluripotent stem cells (iPSCs), which can be used as an alternative source for hESCs with potential for therapeutic programs. Embryonic stem cells or iPSCs could release growth factors VEGF, FGF-2, TGF-β, GM-CSF, and IL-6 and IL-8 that induce cellular proliferation, differentiation, and migration.^62,63

Honey has been used for thousands of years as a wound treatment, but more recently its efficiency has been the focus of scientific investigation. Molan and Rhodes⁶⁶ found honey is a biological therapy with numerous bioactive and physical properties that aid wound healing. The high osmolarity of honey absorbs fluid from the wound bed and creates a negative pressure for lymph flow from the lymphatic vessels into the wound bed. The acidity of honey releases oxygen from the hemoglobin, as a result, destructive proteases would not be able to function in the wound environment. In addition, honey has broad antibacterial properties mainly related to hydrogen peroxide, which is deactivated by the blood catalase enzyme, serum, and wound tissue. Used in wound care products, Manuka honey can resist dilution with significant amounts of wound exudate and maintain sufficient activity to inhibit bacterial growth. There is good evidence honey contains bioactivities that stimulate the immune response, suppress inflammation, and cause fast autolytic debridement, thus promoting the growth of tissues for wound repair.^66,67 

Hyperbaric oxygen treatment for burn wounds was first used in the 1960s⁶⁸ and further developed in the decades following. However, there are still problems regarding the risks and possible costs. A 2013 murine model⁶⁹ showed hyperbaric oxygen improved healing time and scar formation in burn injuries.

Mineral pitch (also known as mummy and shilajit) exists in mountainous areas and flows through the rocks as a brown to black sticky substance that is a physiologically active organic matter. It is used in folk medicine to treat bone fractures, gastric disorders, joint inflammation, impotence, neurological and cardiovascular diseases, and muscle and tendon strain. It also is said to be an effective treatment for gastric ulcers, ulcers, diabetes, and urinary tract infections. It has antioxidant, anti-inflammatory, and immune-enhancing properties. One study⁷⁰ showed fulvic acid, a key ingredient in mineral pitch, acts as an antioxidant and anti-inflammatory agent. Therefore, the use of mineral pitch may help reduce free radicals and cellular damage within injuries. Pant et al⁷¹ suggested mineral pitch may be a factor in a diet that may lower the risk of cancer and inhibit tumor growth.

There are different clinical challenges in the treatment of severe burns to balance healing, reducing risk of infection, and decreasing hospitalization time and treatment costs. The treatment options for burn wounds has significantly developed over the recent decades through preclinical and clinical research, including new grafts and dressings, inflammatory control, nutritional optimization, and unique drug interventions. It is critical that burn patients are treated as per their specific challenges and factors (eg, age, TBSA, and comorbidities). Research and increased knowledge on burn pathobiology, infection, stem cells, transplantation, and rehabilitation will cause improved individual care and new treatment options. 

Authors: Ahad Ferdowsi Khosroshahi, PhD¹; Jafar Soleimani Rad, PhD¹; Raziyeh Kheirjou, PhD student¹; Mohammad Reza Ranjkesh, MD²; and Leila Roshangar, PhD¹

Affiliations: ¹Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; ²Department of Anatomical Sciences,Faculty of Medicine, Tabriz University of Medical Sciences; and ³Department of Dermatology, Faculty of Medicine, Tabriz University of Medical Sciences

Correspondence: Leila Roshangar, PhD, Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran; A.Ferdosi.Kh@gmail.com 

Disclosure: The authors disclose no financial or other conflicts of interest.