Impact Of Hyperoxia On Wound Healing

The discovery that oxygen is an essential nutritional ingredient of healing has stressed the importance of adequate oxygen supply to the repair tissue. Reports from several laboratories have indicated that in many types of wounds hyperoxia enhances healing and conversely, hypoxia inhibits

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repair.

Measurements of local tissue oxygen tensions in a rabbit ear chamber model by means of polarographic ultramicro electrodes have shown that cell replication is hardly seen in portions of wounds where PO2 falls below 20 mmHg. Fibroblasts and endothelial cells replicate best in the range of 30-80 mmHg.2'5'16

Collagen synthesis is crucially dependent on the availability of molecular oxygen. Oxygen is incorporated into the peptide chain to form hydroxyprolyl and hydroxylysyl residues. Hutton17 found a close correlation between the rate proline hydroxylation and oxygen concentration over the range of 0.51-14.9 volumes per cent of oxygen by using a partially purified chick embryo hydroxylase. The Km value for oxygen was 2.6 volumes per cent, equaling a PO2 of about 20 mmHg. These results were supported by Myllyla18 and more recently challenged by De Jong and Kemp19 who suggested a value closer to 100 mmHg. The former estimates suggest that collagen deposition will become maximal at about 200 mmHg, and the later estimate predicts that it will not become maximal until a PO2 of 1000 mmHg is reached. Because hydroxylation of proline is one of the essential steps of collagen synthesis, its rate seems to limit the rate of collagen deposition. This means that the deposition of collagen is limited by the oxygen tension under normal circumstances, and even more so under hypoxic circumstances. By the rules of enzyme kinetics, this data gives a target PO2 for hyperbaric oxygen given in pursuit of wound healing.

The rate of collagen accumulation in healing wounds is also a function of arterial PO2 and of wound PO2 over the entire physiologic range.2'5 This agrees with observations made with ultramicro oxygen electrodes in rabbit ear chambers in which the minimal PO2 in the area of newly formed collagen fibers is of the order of 20-30 mmHg.2,4,16 The ear chamber studies have also shown, that oxygen tensions in healing tissues are heterogeneous. Areas of extremely low oxygen tensions, that are not optimal for healing, are found in wounds even in normal physiologic circumstances.

In addition to its effect upon collagen deposition, PO2 also influences collagen crosslinking, an important factor in the development of wound strength. Chvapil20 reported that the crosslinking of collagen in chick embryo skin slices increased almost linearly when oxygen concentration in the incubating gas was elevated from 20 to 95 volumes per cent. Lysyl oxygenase, which catalyses an important extracellular step in formation of covalent bonds that crosslink collagen peptides also uses molecular oxygen as a substrate. The critical or limiting range of oxygen tensions is in the same range as in the case of prolyl hydroxylase. Thus, crosslinking of collagen, which determines its mechanical strength, is also a function of oxygen tension.1

Niinikoski9 reported that the tensile strength of incisional skin wounds in rats increases as ambient oxygen concentration increases from 18 to 70 volumes per cent. When 70% oxygen was administered, the tensile strength was 35% above the control level in 10-day wounds. Systemic hypoxia suppressed the rate of gain of tensile strength and optimal conditions were passed when the oxygen treatment was extended to 100% oxygen at 1 ATA ambient pressure. At this level, however, evidence of lung oxygen toxicity was found, because the animals were exposed for a considerable period of time to this high oxygen concentration. Parallel observations in subcutaneous cellulose sponge implants demonstrated that the favourable effect of oxygen resulted from enhanced accumulation of collagen, augmented crosslinking of collagen and increased synthetic activity of wound cells as indicated by the rise in their RNA/DNA ratio. Studies of the synthesis of RNA and DNA as well as collagen by means of incorporation of specific radioactive isotopes into granulation tissue in vitro supported and further confirmed the beneficial action of oxygen.

Vihersaari8 investigated enzyme activities in the limiting steps of glycolysis, citric acid cycle and pentose phosphate cycle in subcutaneously implanted hollow cylindrical cellulose sponge implants of rats chronically breathing 12% O2, air or 55% O2 in ambient pressure. Respiratory gas tensions and concentrations of puryvate and lactate were measured in wound fluid aspirated from the central dead space of the implants. Significant portions of repair tissue existed in conditions of extremely low oxygen tension. Probably because all added oxygen was readily consumed, the wound fluid PO2 increased only slightly in hyperoxic environment. The wound PCO2 increased in parallel with the inspired PO2, probably due to enhanced production of carbon dioxide. Hyperoxia shifted the wound metabolism from anaerobic towards aerobic glycolysis. This occurred concurrently with activation of citric acid cycle. The activity of succinic dehydrogenase, a linking enzyme between citric acid cycle and electron transfer chain, also increased with increasing oxygen tension. It has to be stressed, however, that even in animals breathing 55% O2 wound fluid lactate levels remained as high as at the level of 6 mmol/l as opposed to 1 mmol/l or less in blood.

In addition to oxygen, the supply of other substrates is of vital importance for healing. If the Pasteur effect pertains, it indicates that in hyperoxic environment lactic acid is oxidized to carbon dioxide and water and the energy yield per molecule of glucose is considerably increased. Hence, the energy needs of the cell can be met by consumption of considerably less glucose. In spite of this the granulation tissue consumes increased amounts of glucose if added oxygen is available.8,21 It is possible that the cells in the vicinity of the capillary consume glucose so extensively that the supply to the most peripheral cells is limited. This imbalance could probably be corrected by increasing the mean capillary blood PO2 which would decrease the glucose utilization of cells adjacent to capillaries. Relatively more glucose would then be available for the most peripheral cells at hypoxic areas. This could explain the elevated glucose consumption in the wound at increased oxygen supply. This hypothesis was later supported by the finding of Silver who reported with a use glucose electrodes that intercapillary glucose gradients in the rabbit ear chamber tissue partially level off under hyperoxic conditions (personal communication).

During wound healing new blood vessels grow rapidly from areas of high oxygen tension and low lactate concentration to areas of low oxygen tension and high lactate. Recent data indicate that several angiogenic factors, e.g. VEGF and IL-8 are preferentially expressed in areas of low oxygen tension. More recent measurements indicate that not only low oxygen tension, but also high lactate causes the same effect, and that secretion of these factors is even greater when both are present.1,6 Interestingly, the angiogenic response from the intact venules in which blood is flowing freely at the wound edge is greatly enhanced by hyperoxia. No mechanism for this is known, but the strongest clinical evidence of it comes from reports on HBO of chronic wounds.22

Epithelization of open wounds is also clearly dependent on the oxygen tension. Medawar23 noted that epithelial cells grow in culture at a rate that is proportional to their oxygen tension, and others have refined the observation.24,25 Simple acceleration of epithelization is rarely an indication for HBO, but because epithelization does require vascularized base, there is a role for hyperbaric oxygen in preparing this base in certain ischemic, chronic wounds.22,26

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