Oxygen and Wound Healing Phases
Nearly every step in the wound healing process requires oxygen. Even though acute hypoxia stimulates wound healing, oxygen recovery (tissue oxygenation) is required, because chronic hypoxia will impair the healing.
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. 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. 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. 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. 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, 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.
Once the skin and vasculature is disrupted, there is an increased amount of oxygen consumption which in turn creates a hypoxic event. 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). Neutrophils and monocytes produce ROS as described in this respiratory burst, consequently inducing neutrophil chemotaxis. Certain antibiotics, such as aminoglycosides, have been shown to work synergistically with oxygen. Oxygen is known to have a preventive effect against anaerobic wound infections.
A prospective study of 300 patients with a colorectal resection was randomized into 2 groups. 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.
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.
Human keratinocytes in patients more than 60 years of age have been shown to have slower motility than people half their age. 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.
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. Siddiqui et al 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. 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. Oxygen is needed for lysine and proline hydroxylation, which is the step required for collagen to be released from cells. 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.
Hypoxia stimulates angiogenesis but cannot sustain the process. The most influential growth factor for angiogenesis is VEGF. In vitro studies have proven the expression of VEGF increases in both states of hypoxia and hyperoxia. Angiogenesis will proceed and can only be maintained when there is sufficient oxygen and VEGF will be released at higher oxygen tensions.
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. 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. In turn, cells develop a process in which structures are developed for adhesion to the extracellular matrix and developing actin filaments for cell migration. 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." 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. 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.
Schematic review of the phases of wound healing over time with oxygen availability. Reproduced with permission.65
Wounds. 2016;28(8):264-270. © 2016 HMP Communications, LLC