WHO Needs High FIO2?

World Health Organization and the United States Center for Disease Control have recently recommended the use of 0.8 FIO2 in all adult surgical patients undergoing general anaesthesia, to prevent surgical site infections. This recommendation has arisen several discussions: As a matter of fact, there are numerous studies with different results about the effect of FIO2 on surgical site infection. Moreover, the clinical effects of FIO2 are not limited to infection control. We asked some prominent authors about their comments regarding the recent recommendations.

Stimulating Fracture Healing in Ischemic Environments: Does Oxygen Direct Stem Cell Fate during Fracture Healing?

Bone fractures represent an enormous societal and economic burden as one of the most prevalent causes of disability worldwide. Each year, nearly 15 million people are affected by fractures in the United States alone. Data indicate that the blood supply is critical for fracture healing; as data indicate that concomitant bone and vascular injury are major risk factors for non-union. However, the various role(s) that the vasculature plays remains speculative. Fracture stabilization dictates stem cell fate choices during repair. In stabilized fractures stem cells differentiate directly into osteoblasts and heal the injury by intramembranous ossification. In contrast, in non-stable fractures stem cells differentiate into chondrocytes and the bone heals through endochondral ossification, where a cartilage template transforms into bone as the chondrocytes transform into osteoblasts. One suggested role of the vasculature has been to participate in the stem cell fate decisions due to delivery of oxygen. In stable fractures, the blood vessels are thought to remain intact and promote osteogenesis, while in non-stable fractures, continual disruption of the vasculature creates hypoxia that favors formation of cartilage, which is avascular. However, recent data suggests that non-stable fractures are more vascularized than stable fractures, that oxygen does not appear associated with differentiation of stem cells into chondrocytes and osteoblasts, that cartilage is not hypoxic, and that oxygen, not sustained hypoxia, is required for angiogenesis. These unexpected results, which contrast other published studies, are indicative of the need to better understand the complex, spatio-temporal regulation of vascularization and oxygenation in fracture healing. This work has also revealed that oxygen, along with the promotion of angiogenesis, may be novel adjuvants that can stimulate healing in select patient populations.

The role of oxygen during fracture healing.

Oxygen affects the activity of multiple skeletogenic cells and is involved in many processes that are important for fracture healing. However, the role of oxygen in fracture healing has not been fully studied. Here we systematically examine the effects of oxygen tension on fracture healing and test the ability of hyperoxia to rescue healing defects in a mouse model of ischemic fracture healing. Mice with tibia fracture were housed in custom-built gas chambers and groups breathed a constant atmosphere of 13% oxygen (hypoxia), 21% oxygen (normoxia), or 50% oxygen (hyperoxia). The influx of inflammatory cells to the fracture site, stem cell differentiation, tissue vascularization, and fracture healing were analyzed. In addition, the efficacy of hyperoxia (50% oxygen) as a treatment regimen for fracture nonunion was tested. Hypoxic animals had decreased tissue vascularity, decreased bone formation, and delayed callus remodeling. Hyperoxia increased tissue vascularization, altered fracture healing in un-complicated fractures, and improved bone repair in ischemia-induced delayed fracture union. However, neither hypoxia nor hyperoxia significantly altered chondrogenesis or osteogenesis during early stages of fracture healing, and infiltration of macrophages and neutrophils was not affected by environmental oxygen after bone injury. In conclusion, our results indicate that environmental oxygen levels affect tissue vascularization and fracture healing, and that providing oxygen when fractures are accompanied by ischemia may be beneficial.

Collagen Organization Critical Role in Wound Contraction.

BACKGROUND: Open wound closure by wound contraction produces a healed defect made up mostly of dermis. Generating thicker collagen fibers condenses granulation tissue, which pulls surrounding skin into the defect. THE PROBLEM: What is the mechanism for open wound contraction? Is it through the generation of contractile force using sustained myosin ATPase, thus causing cell contraction or by rapid myosin ATPase that condenses collagen fibrils into fibers? BASIC/CLINICAL SCIENCE ADDRESSED: The mechanism for wound contraction is not often debated after the discovery of the myofibroblast. Myofibroblasts are the major cell phenotype in maturing granulation tissue. It is concluded, not quite accurately, that myofibroblasts are responsible for wound contraction. As wound contraction progresses, polarized light microscopy reveals birefringence patterns associated with ever-increasing thickening of collagen fibers. Collagen fibers thicken by eliminating water between fibrils. Wound contraction requires collagen synthesis and granulation tissue compaction. Both myofibroblasts and fibroblasts synthesize collagen, but fibroblasts, not myofibroblasts, compact collagen. Free-floating fibroblast-populated collagen lattices (FPCL) contract by rapid myosin ATPase, thus resulting in thicker collagen fibers by elongated fibroblasts. The release of an attached FPCL, using sustained myosin ATPase, produces rapid lattice contraction, now populated with contracted myofibroblasts in the absence of thick collagen fibers. DISCUSSION: In vivo and in vitro studies show that rapid myosin ATPase is the motor for wound contraction. Myofibroblasts maintain steady mechano-tension through sustained myosin ATPase, which generates cell contraction forces that fail to produce thicker collagen fibers. The hypothesis is that cytoplasmic microfilaments pull collagen fibrils over the fibroblast's plasma membrane surface, bringing collagen fibrils in closer contact with one another. The self-assembly nature of collagen fixes collagen fibrils in regular arrays generating thicker collagen fibers. CONCLUSION: Wound contraction progresses through fibroblasts generating thicker collagen fibers, using tractional forces; rather than by myofibroblasts utilizing cell contraction forces.

Optimization of clinical breast examination.

BACKGROUND: Breast examination is necessary for evaluation of the 8% to 17% of cancers missed by mammograms, but it is being done less often and less effectively. To improve the use of breast examination, we tested whether a technique to focus attention could improve the call rate (the percent of examinations leading to further evaluation), a measure of quality, without retraining in examination technique. METHODS: Clinicians were randomized to complete 1 of 2 dedicated, de-identified forms after routine breast examination: a long form intended to focus attention by requesting general breast descriptors along with clinical information and breast examination findings (10 clinicians recorded 964 examinations) or a short form recording only clinical information and examination findings (11 clinicians recorded 558 examinations). There was no technique retraining. Study call rates were compared with historical controls (298 breast examinations by 16 clinicians). RESULTS: The call rates by the study groups of clinicians were similar, but the call rate using either form (8.3%) was significantly higher than the call rate in the preceding year when no dedicated form was used (4.7%; P=.031), suggesting a Hawthorne effect in which altering conditions of data collection (using the dedicated forms) functioned as an independent variable. Surveillance, Epidemiology, and End Results data predicted 3.4 cancers in all 1822 patients; 4 cancers were found. CONCLUSION: Breast examination call rate doubled when attention was focused on examination results using a dedicated form, and we found the anticipated cancers. Breast examination quality can be improved by focusing clinician attention without retraining in technique.

Human skin wounds: a major and snowballing threat to public health and the economy.

ABSTRACT In the United States, chronic wounds affect 6.5 million patients. An estimated excess of US$25 billion is spent annually on treatment of chronic wounds and the burden is rapidly growing due to increasing health care costs, an aging population and a sharp rise in the incidence of diabetes and obesity worldwide. The annual wound care products market is projected to reach $15.3 billion by 2010. Chronic wounds are rarely seen in individuals who are otherwise healthy. In fact, chronic wound patients frequently suffer from "highly branded" diseases such as diabetes and obesity. This seems to have overshadowed the significance of wounds per se as a major health problem. For example, NIH's Research Portfolio Online Reporting Tool (RePORT; http://report.nih.gov/), directed at providing access to estimates of funding for various disease conditions does list several rare diseases but does not list wounds. Forty million inpatient surgical procedures were performed in the United States in 2000, followed closely by 31.5 million outpatient surgeries. The need for post-surgical wound care is sharply on the rise. Emergency wound care in an acute setting has major significance not only in a war setting but also in homeland preparedness against natural disasters as well as against terrorism attacks. An additional burden of wound healing is the problem of skin scarring, a $12 billion annual market. The immense economic and social impact of wounds in our society calls for allocation of a higher level of attention and resources to understand biological mechanisms underlying cutaneous wound complications.

Hyperbaric oxygen stimulates vasculogenic stem cell growth and differentiation in vivo.

We hypothesized that oxidative stress from hyperbaric oxygen (HBO(2), 2.8 ATA for 90 min daily) exerts a trophic effect on vasculogenic stem cells. In a mouse model, circulating stem/progenitor cell (SPC) recruitment and differentiation in subcutaneous Matrigel were stimulated by HBO(2) and by a physiological oxidative stressor, lactate. In combination, HBO(2) and lactate had additive effects. Vascular channels lined by CD34(+) SPCs were identified. HBO(2) and lactate accelerated channel development, cell differentiation based on surface marker expression, and cell cycle entry. CD34(+) SPCs exhibited increases in thioredoxin-1 (Trx1), Trx reductase, hypoxia-inducible factors (HIF)-1, -2, and -3, phosphorylated mitogen-activated protein kinases, vascular endothelial growth factor, and stromal cell-derived factor-1. Cell recruitment to Matrigel and protein synthesis responses were abrogated by N-acetyl cysteine, dithioerythritol, oxamate, apocynin, U-0126, neutralizing anti-vascular endothelial growth factor, or anti-stromal cell-derived factor-1 antibodies, and small inhibitory RNA to Trx reductase, lactate dehydrogenase, gp91(phox), HIF-1 or -2, and in mice conditionally null for HIF-1 in myeloid cells. By causing an oxidative stress, HBO(2) activates a physiological redox-active autocrine loop in SPCs that stimulates vasculogenesis. Thioredoxin system activation leads to elevations in HIF-1 and -2, followed by synthesis of HIF-dependent growth factors. HIF-3 has a negative impact on SPCs.

Lactate stimulates vasculogenic stem cells via the thioredoxin system and engages an autocrine activation loop involving hypoxia-inducible factor 1.

The recruitment and differentiation of circulating stem/progenitor cells (SPCs) in subcutaneous Matrigel in mice was assessed. There were over one million CD34(+) SPCs per Matrigel plug 18 h after Matrigel implantation, and including a polymer to elevate the lactate concentration increased the number of SPCs by 3.6-fold. Intricate CD34(+) cell-lined channels were linked to the systemic circulation, and lactate accelerated cell differentiation as evaluated based on surface marker expression and cell cycle entry. CD34(+) SPCs from lactate-supplemented Matrigel exhibited significantly higher concentrations of thioredoxin 1 (Trx1) and hypoxia-inducible factor 1 (HIF-1) than cells from unsupplemented Matrigel, whereas Trx1 and HIF-1 in CD45(+) leukocytes were not elevated by lactate. Results obtained using small inhibitory RNA (siRNA) specific to HIF-1 and mice with conditionally HIF-1 null myeloid cells indicated that SPC recruitment and lactate-mediated effects were dependent on HIF-1. Cells from lactate-supplemented Matrigel had higher concentrations of phosphorylated extracellular signal-regulated kinases 1 and 2, Trx1, Trx reductase (TrxR), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) than cells from unsupplemented Matrigel. SPC recruitment and protein changes were inhibited by siRNA specific to lactate dehydrogenase, TrxR, or HIF-1 and by oxamate, apocynin, U0126, N-acetylcysteine, dithioerythritol, and antibodies to VEGF or SDF-1. Oxidative stress from lactate metabolism by SPCs accelerated further SPC recruitment and differentiation through Trx1-mediated elevations in HIF-1 levels and the subsequent synthesis of HIF-1-dependent growth factors.

Lactate modulates gene expression in human mesenchymal stem cells.

PURPOSE: Surgical wounds are characterised by elevated tissue lactate concentrations. This accumulated lactate is capable of stimulating collagen synthesis and new vessel growth as well. Recently, it has been shown in vivo that lactate is also able to favour homing of stem cells. The aim of this investigation was to test the hypothesis that lactate has an impact on gene expression of mesenchymal stem cells (MSC). MATERIALS AND METHODS: MSC were isolated from human bone marrow using the density gradient technique and incubated with alpha-methoxyethoxymethyl containing 10% fetal calf serum at 37 degrees C under 95% air and 5% CO(2). Cultured MSC were characterised by in vitro differentiation assays and fluorescence-activated cell sorting (FACS) analysis. Characterised MSC were treated with 15 mM lactate for different time periods (1, 6 and 24 h and 3 and 7 days). Gene expression analysis was performed using a custom-designed oligonucleotide microarray. A significant alteration of gene expression was defined as a two-fold stimulation or inhibition. The phenotype of MSC was investigated by FACS analysis of specific surface epitope patterns. RESULTS: Gene expression analysis shows 63 up- and 51 down-regulated genes after 1 h of treatment, 45 up- and 47 down-regulated genes after 6 h of treatment, 57 up- and 72 down-regulated genes after 24 h of treatment, 103 up- and 28 down-regulated genes after 3 days of treatment and 50 up- and 101 down-regulated genes after 7 days of treatment with lactate. The majority of the modulated genes are related to the expression of cytokines, transcription factors and cell-cycle- or cellular-matrix-associated proteins. In particular, lactate up-regulates the expression of interleukin-6 (3 days, 4.11-fold), of heat shock protein 70 (3 days, 2.36-fold) and of hypoxia-inducible factor-1alpha (3 days, 2.09-fold). A down-regulating effect of lactate is observed for superoxide dismutase 2 (1 h, 0.5-fold; 24 h, 0.4-fold; 7 days, 0.32-fold) and BCL2-associated X protein (24 h, 0.42-fold; 7 days, 0.4-fold). Expression of cell surface antigens clusters of differentiation 29, 44, 59, 73, 90, 105, 106 and 146 does not change over the time period of lactate treatment. CONCLUSIONS: Lactate modulates expression of genes involved in wound healing. However, lactate does not profoundly change the phenotype of MSC. In addition to providing new insights into the wound healing physiology, these data could also be the rationale for new treatment strategies for chronic non-healing wounds.

Lactate, with oxygen, incites angiogenesis.

Lactate has been reconsidered! As we now know, most is produced aerobically We report that lactate accumulation commonly occurs in the presence of oxygen and is sufficient to instigate signals for angiogenesis and connective tissue deposition. These include vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF beta), interleukin-1 (IL-1), and hypoxia-inducible factor (hif-1alpha). This paper, a mini-review, is occasioned by new data showing increased presence of VEGF and angiogenesis in an oxygenated site by adding a slow-release source of lactate into Matrigel and implanting the Matrigel subcutaneously in mice.

Aerobically derived lactate stimulates revascularization and tissue repair via redox mechanisms.

Hypoxia serves as a physiologic cue to drive an angiogenic response via HIF-dependent mechanisms. Interestingly, minor elevation of lactate levels in the tissue produces the same effect under aerobic conditions. Aerobic glycolysis contributes to lactate accumulation in the presence of oxygen, especially under inflammatory conditions. We previously postulated that aerobic lactate accumulation, already known to stimulate collagen deposition, will also stimulate angiogenesis. If substantiated, this concept would advance understanding of wound healing and aerobic angiogenesis because lactate accumulation has many aerobic sources. In this study, Matrigel plugs containing a powdered, hydrolyzable lactate polymer were implanted into the subcutaneous space of mice. Lactate monomer concentrations in the implant were consistent with wound levels for more than 11 days. They induced little inflammation but considerable VEGF production and were highly angiogenic, as opposed to controls. Arterial hypoxia abrogated angiogenesis. Furthermore, inhibition of lactate dehydrogenase by using oxamate also prevented the angiogenic effects of lactate. Lactate monomer, at concentrations found in cutaneous wounds, stabilized HIF-1alpha and increased VEGF levels in aerobically cultured human endothelial cells. Accumulated lactate, therefore, appears to convey the impression of "metabolic need" for vascularization, even in well-oxygenated and pH-neutral conditions. Lactate and oxygen together stimulate angiogenesis and matrix deposition.

Lactate stimulates endothelial cell migration.

The significance of the high lactate levels that characterize healing wounds is not fully understood. Lactate has been shown to enhance collagen synthesis by fibroblasts and vascular endothelial growth factor (VEGF) production by macrophages and endothelial cells. VEGF has been shown to induce endothelial cell migration. However, it has not been shown whether accumulated lactate correlates with the biological activity of VEGF. Therefore, we investigated the effect of lactate on migration of endothelial cells. Human umbilical vein endothelial cells and human microvascular endothelial cells were cultured to subconfluent monolayers in standard six-well tissue culture plates. Following a 24-hour serum starvation, cells were treated with the indicated concentrations of l-lactate. Cell migration was assessed using a modified Boyden chamber. VEGF protein in the cell culture supernatant was measured by enzyme-linked immunoassay. Lactate-enhanced VEGF protein synthesis in a time- and dose-dependent manner. Lactate added into the bottom well did not stimulate cellular migration from the upper well. However, lactate when added together with endothelial cells to the bottom well of the Boyden chamber increased cellular migration in a dose-dependent manner. This effect was blocked by anti-VEGF and by cycloheximide. Lactate enhances VEGF production in endothelial cells, although lactate, itself, is not a chemoattractant. We conclude that the lactate-mediated increase in cellular migration is regulated by VEGF.

Using physiology to improve surgical wound outcomes.

Despite major advances in surgical management and approaches, including aseptic techniques, prophylactic antibiotics, and laparoscopic surgery, surgical wound infection and wound failure remain common complications of surgery. In a review of the literature, the authors found that a growing body of literature supports the concept that patient factors are a major determinant of wound outcome after surgery. In particular, wounds are exquisitely sensitive to hypoxia, which is both common and preventable. Perioperative management can be adapted to promote postoperative wound healing and resistance to infection. The most important factors are fluid management, temperature management, pain control, increased arterial oxygen tension, and, as has been long recognized, appropriate sterile techniques and administration of prophylactic antibiotics. This article reviews how knowledge of and attention to physiology can improve quality of care in both acute and chronic wounds.

Perflubron emulsion increases subcutaneous tissue oxygen tension in rats.

Oxygen plays a central role in wound healing. The hypothesis of this study was that the administration of a perfluorocarbon emulsion, which dissolves oxygen will increase subcutaneous tissue oxygen tension (PsqO2) in normovolemic and hemorrhaged animals. In the first set of experiments, PsqO2 was measured with a polarographic oxygen electrode along the dorsum of normovolemic-anesthetized rats (n=20) breathing supplemental oxygen. After baseline equilibration of tissue oxygen tension, perflubron emulsion (n=12) or saline control (n=8) was administered. Perflubron administration increased PsqO2 by 32.0 +/- 7.2 mmHg, whereas saline administration had no effect (-1.0 +/- 6.6 mmHg). In a second set of experiments, PsqO2 (n=12) was measured in rats breathing 100 percent oxygen after being bled 20 percent of their blood volume. Creating hypovolemic rats allowed for the study of the effect of perflubron emulsion administration on subcutaneous oxygen tension in tissues with compromised blood flow. Perflubron emulsion was only effective at increasing oxygen tension in tissues with oxygen extractions less then 2.0 vol%. These findings agree with those results analytically predicted based on the oxygen solubility of the perflubron emulsion.