The Presence of Oxygen in Wound Healing

Oxygen must be tightly governed in all phases of wound healing to produce viable granulation tissue. This idea of tight regulation has yet to be disputed; however, the role of oxygen at the cellular and molecular levels still is not fully understood as it pertains to its place in healing wounds. In an attempt to better understand the dynamics of oxygen on living tissue and its potential role as a therapy in wound healing, a substantial literature review of the role of oxygen in wound healing was performed and the following key points were extrapolated: 1) During energy metabolism, oxygen is needed for mitochondrial cytochrome oxidase as it produces high-energy phosphates that are needed for many cellular functions, 2) oxygen is also involved in the hydroxylation of proline and lysine into procollagen, which leads to collagen maturation, 3) in angiogenesis, hypoxia is required to start the process of wound healing, but it has been shown that if oxygen is administered it can accelerate and sustain vessel growth, 4) the antimicrobial action of oxygen occurs when nicotinamide adenine dinucleotide phosphate (NADPH)-linked oxygenase acts as a catalyst for the production of reactive oxygen species (ROS), a superoxide ion which kills bacteria, and 5) the level of evidence is moderate for the use of hyperbaric oxygen therapy (HBOT) for diabetic foot ulcers, crush injuries, and soft-tissue infections. The authors hypothesized that HBOT would be beneficial to arterial insufficiency wounds and other ailments, but at this time further study is needed before HBOT would be indicated. 

Oxygen is a significant factor in wound healing. In general, living tissue needs oxygen and nutrients to thrive, and with wounds, it is needed to regenerate healthy tissue. In normal wound healing, the wound either requires conditions of hypoxia or normal levels of oxygen (ie, normoxia). These different conditions occur in all phases of wound healing. A wound is dependent on both the supply of oxygen to the wound tissue, which is determined by the pulmonary gas exchange, and the blood hemoglobin level. The cardiac output of the patient, the perfusion rate, and the amount of capillaries around the wound along with the consumption rate of parenchymal and stromal cells determine these levels.¹ This paper will discuss the role of oxygen in healthy wound healing. The discussion will examine how oxygen is produced, consumed, and used in the various stages of wound healing at both a molecular level and a cellular level. Finally, there will a brief discussion on the use of oxygen as therapy.

In the aerobic metabolism of glucose, cells use oxygen as the final electron acceptor to generate adenosine triphosphate (ATP), which fuels the majority of cellular processes during wound healing.² Healing tissue requires an increased energy demand.³ This additional energy is generated from the oxidative metabolism which in turn increases the oxygen demand of the healing tissue.⁴ Thus, the ATP that is generated from this process helps supply the power for tissue repair. During the inflammatory phase of wound healing, platelets and disintegrating cells can contribute ATP.⁵ This extracellular ATP can act as a signalling mechanism for many aspects of wound healing such as the immune response, inflammation, epithelial cells, and angiogenesis.⁶ When ATP is released during an injury to the skin, it acts as an early signal in an epidermal-like growth factor which, downstream, signals epidermal growth.⁷ Another signalling function of ATP is that it is released from the cells in the injured tissue, thereby activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is required to produce the redox signals in wound healing.⁸ The first discussion of the killing of bacteria by an oxidase occurred in 1978.⁹ When the phagocytosis of bacteria occurs, the immune system increases oxygen consumption through NADPH oxidase that in turn generates metabolites.¹⁰ These metabolites catalyse the production of a reactive oxygen species (ROS) by cells that then stimulate a high demand for oxygen or “respiratory burst.”¹¹ The majority of the oxygen consumed by neutrophils occurs during this respiratory burst.¹² Nicotinamide adenine dinucleotide phosphate oxidase is vital in the survival of macrophages, and it also enables phagocytosis of dead cells.¹³

Initially, free radicals were thought to be destructive to normal tissue, and it also was thought that these free radicals should be bound to antioxidants to stop their destructive nature.¹⁴ Low-level free radicals were then later recognized as possibly serving as signalling messengers.¹⁵ Inflammation after an injury occurs as a site for significant production of ROS due to the amount of phagocytosis occurring. As wound healing progresses, things like cell proliferation and migration are present due to redox signalling of ROS.¹⁶ Production of hydrogen peroxide also occurs during wound healing.⁸ When hydrogen peroxide is decomposed, it generates oxygen as an end product.¹⁷ Redox signals are generated, and decreased tissue oxygen and tissue hypoxia will limit the signalling of redox; thus disabling the function of several growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and also limit some molecular mechanisms such as leukocyte recruitment.¹⁵ 

Nearly every step in the wound healing process requires oxygen.¹⁸ Even though acute hypoxia stimulates wound healing, oxygen recovery (tissue oxygenation) is required, because chronic hypoxia will impair the healing.¹

During the inflammatory phase, the most significant cellular processes occur when oxygen is involved in the oxidative phosphorylation in the mitochondria which results in the production of ATP.¹⁹ The ROS have more roles than just the oxidative killing of bacteria; after hemostasis, hypoxia occurs and activates the initial steps of wound healing by boosting ROS activity. Hypoxia also activates platelets and endothelium by inducing cytokines released from platelets, monocytes, and growth factors.²⁰ This usually occurs at low concentrations. Hypoxia-induced factor (HIF) results in a transcription HIF, which binds to hypoxia response elements in gene promoter regions. 

These regions upregulate glucose metabolism, control vessel tone, and angiogenesis.²¹ Hypoxia-induced factor regulates oxygen hemostasis in the wound, and ROS stimulates cytokine and chemokine-receptor activation as well as other functions necessary for wound repair. The main effect of these mediators is the recruitment and activation of neutrophils and macrophages to the wound site and the activation of fibroblasts.²² Once the cytokines and chemokines are secreted, they activate the oxygen-dependent complement cascade. At this time, a set of growth factors are released that stimulate and attract the major components of wound healing such as wound leukocytes and fibroblasts. Hydrogen peroxide has been shown to be a mediator of these interactions. In an experiment by Niethammer and colleagues,²³ a Zebra fish larval tail fin had a mechanically created wound induced. This was done to prove hydrogen peroxide arrives first to a new wound site from the epithelial cells of the tail fin. Eventually the hydrogen peroxide recruits leukocytes and fibroblasts in this study of inflammatory and regenerative chemical response to wounds.²³

Figure 1 and Figure 2

Once the skin and vasculature is disrupted, there is an increased amount of oxygen consumption which in turn creates a hypoxic event.¹⁹ Reactive oxygen species activity is initiated by hypoxia, which causes platelets and monocytes to release transforming growth factor beta (TGF-β), VEGF, and tumor necrosis factor alpha (TNF-α).²⁴ Neutrophils and monocytes produce ROS as described in this respiratory burst, consequently inducing neutrophil chemotaxis.²⁴ Certain antibiotics, such as aminoglycosides, have been shown to work synergistically with oxygen.²⁵ Oxygen is known to have a preventive effect against anaerobic wound infections.²⁶ 

A prospective study of 300 patients with a colorectal resection was randomized into 2 groups.²⁷ The first group of 148 patients received 80% oxygen supplementation intraoperatively and 80% postoperatively for 6 hours, while the other group of 143 patients received 30% supplementation intraoperatively and 30% oxygenation postsurgically for 6 hours. The latter group (30% oxygen) had a greater rate of infection in contrast to the group receiving 80% oxygen. In conclusion, it was demonstrated that patients receiving higher concentrations of oxygen resulted in lower rates of postcolon or postrectal surgery infections.²⁷

In the proliferative phase, hypoxia has been shown to increase keratinocyte motility. This was shown in vitro producing proteins that are involved in cell motility.²⁸

Human keratinocytes in patients more than 60 years of age have been shown to have slower motility than people half their age.²⁹ It has been hypothesized that matrix metalloproteinases (MMPs) 1 and 9 are required in keratinocyte migration on type I and type IV collagen, respectively. These MMPs in young keratinocytes are induced by hypoxia yet not induced in older keratinocytes.³⁰

Transforming growth factor beta one (TGF-β1) is the growth factor responsible for the transcription of the procollagen gene, which has been proven to increase the migration of young cultured human fibroblasts.³¹ Siddiqui et al³² have also demonstrated that acute hypoxia increases fibroblast proliferation, collagen synthesis, and expression of TGF-β1 messenger RNA (mRNA). Oxygen is needed in the later steps of collagen synthesis for proline and lysine hydroxylation and cross-linking.³³ For fibroblasts to lay collagen down properly, oxygen tensions are needed to be between 30-40 mm Hg because the production of collagen is proportional to the oxygen tension.³⁴ Oxygen is needed for lysine and proline hydroxylation, which is the step required for collagen to be released from cells.³⁵ In order for collagen to form a triple helix, oxygen must be present. Without oxygen, the pro-alpha peptide chains fail to form the triple helix.³⁶

Hypoxia stimulates angiogenesis but cannot sustain the process.³⁷ The most influential growth factor for angiogenesis is VEGF.³⁸ In vitro studies have proven the expression of VEGF increases in both states of hypoxia and hyperoxia.³⁹ Angiogenesis will proceed and can only be maintained when there is sufficient oxygen and VEGF will be released at higher oxygen tensions.⁴⁰

Epidermal keratinocytes differentiate, proliferate, and migrate on the wound surface to start the reepithelization of a wound. Wound injury causes stress pathways to be activated which then cause the oxygen-dependent release of certain cytokines and chemokines, such as keratinocyte growth factor (KGF), epidermal growth factor (EGF), PDGF, insulin-like growth factor (IGF), and tumor necrosis factor (TNF) superfamily.⁴¹ The TNF is the main cytokine that seems to stimulate epidermal cells at the wound edges and hair follicles in an autocrine manner, which is an oxygen-dependent process.⁴² In turn, cells develop a process in which structures are developed for adhesion to the extracellular matrix and developing actin filaments for cell migration.⁴³ There has to be a significant cell migration accompanied with oxygen-dependent cell proliferation for large wounds to close. Cytokines and chemokines that are most likely released from keratinocyte stem cells stimulate the proliferation of keratinocytes in a process called a “proliferative burst.”⁴⁴ This process has a high amount of metabolic activity since there are different steps that require oxygen and ROS. 

The last step or phase of wound healing is remodeling which can last up to 2 years. Gradually, the provisional collagen, which is mostly type III, is replaced with type I collagen produced strictly in oxygen-dependent fibroblasts. The wound then gains tensile strength, and the collagen fibers contract so the wound shrinks.⁴⁵ The most prominent mediators of this collagen process are MMPs and tissue inhibitors of metalloproteinases (TIMPs), which are released by macrophages, keratinocytes, endothelial cells, and fibroblasts, which are all dependent on oxygen.⁴⁶

Throughout the phases of wound healing there is a control of oxygen maintained in a narrow range. This point of normoxia is important because it is used to prevent abnormal periods of hypoxia or hyperoxia which can create damage to cell membranes.⁵ This point of normoxia is the state of oxygenation where the cell or tissue does not report hypoxia nor does it report hyperoxia which would be oxygen toxicity.⁴⁷ If there were a change, the cells or tissue would react by switching on either a hypoxic or hyperoxic response. Depending on the organ of the body, the normoxic set point would be different due to the amount of oxygen required.⁴⁸ Hypoxia sensing and response is implicated in ischemic disease conditions, but is required for development where there is a changing state of oxygenation sending a signal to continue the wound-healing process. This sensing is either considered HIF-dependent or HIF-independent.²¹

Intermittent hypoxia, a periodic exposure to hypoxia, is interrupted by a return to normoxia where less hypoxic periods occur in many circumstances.⁴⁹ This intermittent hypoxia is mostly found in obstructive sleep apnea, but in a study by Khayat et al,⁵⁰ the authors have shown patients with this condition commonly have nonhealing wounds. Even though hyperoxia may induce some positive effects, if this occurs for a period of time exceeding the normoxic set point it can be a risk factor.⁵¹ In areas where the wound has pockets of hypoxia, the goal is to reestablish normoxia in the areas of hypoxia without exposing the wound to high levels of oxygen which might cause oxygen toxicity.⁵² Wound healing might be delayed in extreme hyperoxia which can cause growth arrest and cell death by mitochondria apoptosis.⁵³ The normoxic set point can be tuned when the cells are exposed to modest changes of oxygen and there is a physiological change that can possibly be an adaptive process.⁵⁴

This review would be incomplete without a brief discussion of the use of oxygen in the treatment of wounds. Hyperbaric oxygen therapy (HBOT) is usually administered in a single patient or multipatient chamber that delivers 100% oxygen at 2 atmospheres of pressure. Hyperbaric oxygen therapy has proven to raise tissue oxygen 10 to 20 fold above room air.⁵⁵ One theory as to why HBOT might work is the synergy with PDGF since PDGF requires oxygen-derived hydrogen peroxide for functioning.⁵⁶ Another oxygen therapy is topical oxygen. This therapy utilizes either a chamber or a plastic bag to create a closed environment to deliver 100% oxygen converted from room air.⁵⁷ It is hypothesized that 100% oxygen applied locally to a wound increases VEGF expression, which may induce angiogenesis.⁵⁸ The evidence for clinical use of HBOT is moderate at best. In a review from the Cochrane Library Database, Kranke et al⁵⁹ presented 12 randomized trials that included participants with foot ulcers/wounds and diabetes. Short-term (up to 6 weeks) HBOT was found to be effective in improving healing but there were no significant findings that the wounds were completely healed after 1 year. For chronic wounds in patients with decreased blood supply or pressure ulcers, no evidence could confirm or deny any effects of HBOT.⁵⁹ In another study, Fedorko et al⁶⁰ published a randomized, placebo-controlled study for patients with both types I and II diabetes, diabetic wounds, or lower extremity injuries. Hyperbaric oxygen therapy did not offer any additional advantages in wound care nor did the therapy support a reduction in lower limb amputations or wound size in patients with diabetic foot ulcers over a 12-week period.⁶⁰

Throughout all phases of wound healing, oxygen plays a substantial role. Its effects vary depending on whether the wound is in a hypoxic, normoxic, or in a hyperoxic state. The following are the key points. First during energy metabolism, oxygen is needed for mitochondrial cytochrome oxidase.⁶¹ This in turn produces high-energy phosphates which then are needed for many cellular functions.⁴⁰ Second, in collagen synthesis oxygen is involved in the hydroxylation of proline and lysine into procollagen which leads to collagen maturation.⁶² Third, in angiogenesis, hypoxia is required to start the process, but it has been shown that if oxygen is administered it can accelerate and sustain vessel growth.⁶³ Finally, the antimicrobial action of oxygen occurs when converted by leukocytic NADPH oxidase to a superoxide ion which kills bacteria.⁶⁴

Affiliations: Case Western Reserve University School of Medicine, Cleveland, OH; and Louis Stokes VA Medical Center, Cleveland, OH

Correspondence:
Howard M. Kimmel, DPM, MBA
Case Western Reserve University School of Medicine
Cleveland, OH
buckeyefootcare@sbcglobal.net

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