Oxygen in Acute and Chronic Wound Healing

S. Schreml; R.M. Szeimies; L. Prantl; S. Karrer; M. Landthaler; P. Babilas

Disclosures

The British Journal of Dermatology. 2010;163(2):257-268. 

In This Article

Physiological Wound Healing

Physiological (syn. acute) wound healing is a dynamic stepwise process consisting of partially overlapping phases that are determined by interacting events on a molecular, cellular and extracellular matrix (ECM) level. This process, which is not yet fully understood, starts with a disturbance of tissue integrity and ends with a restitutio ad integrum or a scar formation within an appropriate length of time. Nearly every step of wound healing requires oxygen.[5,7,10,15,16] For didactic reasons, the course of physiological wound healing is schematically divided into three overlapping phases: the inflammatory phase, the proliferative phase (neoangiogenesis, tissue formation, re-epithelialization) and the tissue remodelling phase (Fig. 1).[1,2,17]

Figure 1.

Wound healing phases. The inflammatory phase starts after tissue injury. At this stage, cytokines, chemokines and reactive oxygen species are released and cells are recruited to the wound site. In the subsequent proliferative phase (neoangiogenesis, tissue formation, re-epithelialization) new tissue is formed by endothelial cells, fibroblasts and keratinocytes. After these initial steps, tissue remodelling starts. EGF, epidermal growth factor; FGF, fibroblast growth factor; IL, interleukin; KGF, keratinocyte growth factor; MMPs, matrix metalloproteinases; PDGF, platelet-derived growth factor; ROS, reactive oxygen species; TGF, transforming growth factor; TNF, tumour necrosis factor; VEGF, vascular endothelial growth factor.

Inflammatory Phase

Physiologically, the inflammatory phase lasts between 4 and 6 days and starts immediately after wounding. Blood vessels constrict after traumatization, and platelets aggregate along the activated endothelium. Vascular disruption and vasoconstriction cause a hypoxic microenvironment that is intensified by increased oxygen consumption due to metabolically active cells contributing to wound healing. Hypoxia actuates the initial steps of wound healing by boosting ROS activity, by activating platelets and endothelium, and by inducing cytokines released from platelets, monocytes and parenchymal cells [e.g. vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, tumour necrosis factor (TNF)].[9,18] However, even if acute hypoxia initiates wound healing, the recovery of wound tissue oxygenation is of major importance for physiological healing as chronic hypoxia impairs all processes necessary for healing. The aggregated platelets initiate the coagulation cascade leading to a blood clot, which prevents the leakage of blood and forms a provisional ECM. The provisional ECM, which is composed of fibronectin, fibrinogen, fibrin, thrombospondin and vitronectin, fills the tissue defect and enables migration of the different cytokines and cells required for the healing process.[19] Besides these structural contributions, the activated platelets direct the healing process through the secretion of several mediators of wound healing such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), TGF-β1 and TGF-β2.[2] ROS stimulate cytokine and chemokine release as well as their functions. The primary effect of these mediators is the recruitment and activation of neutrophils and macrophages to the wound site and the activation of fibroblasts. However, these platelet-derived processes are not the only ones that initiate healing. The injury activates epithelial and nonepithelial cells in the wound area. Consecutively, cytokines and chemokines are secreted, which initiate stress pathways and activate the complement cascade, both oxygen-dependent processes. In consequence, a set of secreted factors [TGF-α, TGF-β1, keratinocyte growth factor (KGF), EGF, PDFG and insulin-like growth factor (IGF)] is released, which attracts and stimulates relevant players of wound healing such as inflammatory leucocytes and fibroblasts. Just recently, it has been shown that hydrogen peroxide (H2O2) is an important mediator in wound–leucocyte interaction.[20] A tail-fin model of a zebrafish with a genetically encoded H2O2 sensor showed that H2O2 at the wound margins peaks as early as a few minutes after injury, and that leucocytes are recruited to the wound site by a tissue-scale H2O2 gradient. Whether H2O2 is produced by damaged cells or by their neighbours, and how neutrophils sense H2O2 gradients yet remains to be answered.[21]

Even very superficial traumas without the destruction of blood vessels and activation of platelets initiate wound healing.[1] Injured parenchymal cells secrete prostaglandins, histamine, bradykinin and serotonin, which induce vasodilatation and increase capillary permeability. Subsequently, diapedesis of cells is accelerated and oxygen supply to the wound site is increased. Clinical correlates of these processes are erythema and oedema, which become apparent at the wound edges during the inflammation phase. The infiltration of leucocytes, monocytes, and – 24–48 h later – macrophages is the key event in initial wound healing; their functions, such as degradation of cell detritus, counteraction of tissue infection and phagocytosis of microorganisms, are indispensable for wound healing. The amount and proportion of these inflammatory cells as well as the duration of the inflammatory phase depend on the wound extent, the degree of tissue infection, and the extent of debris that needs to be removed.[1] Metabolically, inflammatory cells are extremely active and therefore depend on high amounts of oxygen. The high consumption of oxygen may lead to areas of hypoxia, even in well-oxygenated wounds.[5] During the inflammatory phase, one central product of neutrophils, macrophages, monocytes, endothelial cells and fibroblasts is ROS. Thrombin, PDGF and TNF stimulate the release of ROS from endothelial cells, whereas interleukin (IL)-1 and platelet-activating factor stimulate ROS release from fibroblasts.[22] ROS are the main force against microorganisms and thus wound infection. As mentioned above, NADPH-linked oxygenase is responsible for the production of ROS, a highly oxygen-dependent process: the Km (half maximal velocity) for NADPH-linked oxygenase with oxygen as a substrate is a pO2 value of 40–80 mmHg.[8,23] Neutrophils were shown in vitro to lose their bacterial killing capacity at a pO2 level below 40 mmHg.[24,25] This loss may explain the significant bacterial colonization apparent in hypoxic chronic wounds. Besides their role in the oxidative killing of bacteria, ROS are able to augment neutrophil chemotaxis.[9,18] Activated inflammatory cells themselves produce cytokines and growth factors such as IGF, leucocyte growth factor, IL-1, IL-2, TNF, TGF-α, TGF-β, VEGF, PDGF and lactate.[26] VEGF and PDGF are both potent chemoattractants and mitogens for fibroblasts and angiogenic growth factors; their release initiates the formation of granulation tissue and thus the proliferative phase at day 4–5 after wounding.

Proliferative Phase

The proliferative phase lasts – depending on the extent of the wound – for a few weeks and comprises elementary processes such as neovascularization, formation of granulation tissue and ECM, and re-epithelialization. Endothelial cells and fibroblasts simultaneously invade the initially built haemostatic clot. Macrophages lead the way by degrading the clot and by releasing cytokines and chemokines that attract fibroblasts and stimulate angiogenesis.[27] Particularly macrophages and their metabolites play a pivotal role in granulation tissue formation as depletion of macrophages was shown to lead to impaired wound healing in an in vivo porcine model.[27] Fibroblasts and keratinocytes also secrete growth factors. Hereby, the cytokines of the TGF-β superfamily seem to play the most prominent role in granulation tissue formation.[28] Interestingly, ECM molecules such as fibrinogen, fibronectin, fibrin and vitronectin are interactive with cytokines and also regulate the proliferation, differentiation and migration of fibroblasts.[29] Important stimulators of angiogenesis are hypoxia and ROS. Both stimulate macrophages, fibroblasts, endothelial cells and keratinocytes to synthesize VEGF.[30–32] Again, acute hypoxia is the initiator of this process, whereas chronic hypoxia impairs neovascularization.[30,33] Hypoxia activates the transcription factor hypoxia-inducible factor (HIF)-1α. HIF-1α binds to the hypoxia response element in the gene promoter region of the VEGF gene, which in turn upregulates VEGF. VEGF, as the major angiogenic growth factor, stimulates endothelial cells to migrate, proliferate and form countless new capillaries.[32] Rossiter et al.[34] showed in a murine model system that keratinocyte-specific deletion of VEGF resulted in delayed wound healing due to impaired neoangiogenesis. Complementarily, Hong et al.[35] showed enhanced wound healing in transgenic mice with overexpression of VEGF in the skin. The new capillaries branch out and invade the provisional wound matrix, which is replaced piecemeal by a new ECM produced and deposited by fibroblasts. The emerging ECM, in which fibroblasts, myofibroblasts, leucocytes and macrophages are embedded, consists of immature collagen (type III), proteoglycans, glycosaminglycans, fibrin, fibronectin and hyaluronic acid.[36] In this context, the production and deposition of collagen represents a fundamental process as it reconstitutes skin alignment and integrity. The production and deposition of collagen is proportional to oxygen tension: fibroblasts need a pO2 of 30–40 mmHg for collagen synthesis.[37] A central oxygen-dependent step in the synthesis of collagen is the hydroxylation of proline and lysine residues. In addition, hydroxylase activity is critically dependent on cofactors such as iron and vitamin C. Lysyl hydroxylase and lysyl oxidase, both oxygen-dependent enzymes, catalyse collagen cross-linking, a step that aims at wound stability. Again, in hypoxia, acute hypoxic conditions must be distinguished from chronic hypoxia. Acute hypoxia may stimulate fibroblast proliferation, collagen synthesis and expression of TGF-β1, whereas chronic hypoxia decreases these processes as shown in vitro by Siddiqui et al.[38] in human dermal fibroblasts. Angiogenesis and ECM synthesis are interdependent processes as new blood vessels need new ECM as a three-dimensional scaffold for their ingrowth while the cell metabolism of, for example, fibroblasts needs new blood vessels that deliver oxygen and other nutrients. The fact that the same cytokines stimulate each process interconnects these steps of wound healing. Parallel to the formation of granulation tissue, re-epithelialization is initiated.

Re-epithelialization aims at covering the wound surface by a layer of epithelium and is based on the differentiation, proliferation and migration of epidermal keratinocytes. The stress pathways activated by injury lead to the oxygen-dependent release of certain cytokines and chemokines (TNF, TGF-α, TGF-β1, KGF, EGF, PDFG and IGF) by parenchymal cells such as keratinocytes. These cytokines, foremost TNF, seem to stimulate epidermal cells at wound edges and hair follicles in an autocrine manner to restructure their cytoskeleton, a process that is oxygen dependent and starts within a few hours after injury.[39,40] The cells retract their intracellular tonofilaments, dissolve the desmosomal or hemidesmosomal connections, but establish adhesion structures for gripping to the ECM and develop cytoplasmic actin filaments for cell migration.[41–43] Stimulated by EGF, TGF-α, KGF, TGF-β1, hepatocyte growth factor (HGF) and IGF-1, cell migration toward the wound's central point, called shuffling, takes place.[44] Hereby, TGF-β1 is a key cytokine as it controls the expression of integrins in keratinocytes. Integrins are cell surface receptors that interact with ECM, particularly with fibrin and fibronectin.[45] Migration through the wound matrix, which is composed of necrotic material, bacteria, a haemostatic clot of platelets and fibrin, and later on of granulation tissue, is further supported by the activation of plasmin. This process is caused by a plasminogen activator produced by both epidermal cells[46] and matrix metalloproteinases (MMPs). MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-13) are released mainly by macrophages, keratinocytes, endothelial cells and fibroblasts[47] and degrade certain constituents of provisional wound tissue such as collagen I, III, IV and VII. It is of outstanding importance that MMP inhibitors such as α1-antiproteinase, secretory leucocyte protease inhibitor (SLPI), α2-macroglobulin and tissue inhibitors of MMPs (TIMPs) are sufficiently present, once provisional wound tissue has been removed. To achieve complete closure of larger wound areas, cell migration has to be accompanied by oxygen-dependent cell proliferation. For this, cytokines and chemokines (EGF, TGF-α, KGF, HGF, nerve growth factor, IGF-1, IL-1 and IL-6), most possibly released from keratinocyte stem cells, stimulate the proliferation of keratinocytes in a process called 'proliferative burst'.[48] As processes with a high metabolic activity the different steps of epithelialization are oxygen and ROS dependent. Taking all these considerations into account, the topical administration of pure oxygen on wounds could increase the rate of epithelialization.[49] However, O'Toole et al.[50] demonstrated in an in vitro study that hypoxic keratinocytes showed a decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility, but an increased expression and redistribution of the lamellipodia-associated proteins, cytoskeletal proteins which are involved in cell migration. However, the in vitro experiments discount the countless interactions of keratinocytes with, for example, inflammatory cells, bacterial colonization, granulation tissue etc. The same research group showed in another study that a very low concentration of H2O2 inhibits keratinocyte migration and proliferation.[51] The exact processes are not yet fully understood, but tools like chemiluminescent nanoparticle sensors for H2O2 enable us to study H2O2 functions in vivo in more detail.[52]

Tissue Remodelling Phase

The tissue remodelling phase starts as early as a few days after injury and lasts up to 2 years thereafter. In the beginning, wound contraction contributes to wound closure. This process is enabled due to the differentiation of a subgroup of fibroblasts to contractile myofibroblasts triggered by oxygen[53] and mediated by TGF-β1, TGF-β2 and PDGF.[54–56] After the main steps of the proliferative phase are fulfilled, unknown stop signals induce a redifferentiation of fibroblasts, keratinocytes and endothelial cells so that the accelerated proliferation and migration normalizes. Gradually, the provisional collagen (type III) is replaced by the more stable collagen type I that is produced strictly oxygen dependently by fibroblasts and is deposited in a physiological alignment. Thus, the healing wound gains increased wound tensile strength. The collagen fibres contract so that the wound tissue shrinks.[55,56] Prominent mediators of collagen anabolism and catabolism are MMPs, which are released oxygen dependently by macrophages, keratinocytes, endothelial cells and fibroblasts. Of great importance are the TIMPs, which contribute to a concerted maturation process that leads to a restitutio ad integrum or a scar formation depending on the MMP/TIMP ratio and activity.

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