Hyperbaric oxygen delays healing in an ischemic wound model

The true etiology of non-healing, chronic wounds remains elusive. One common denominator, whether the wounds are due to pressure, diabetes, or arterial insufficiency is localized tissue ischemia in the wound bed. Angiogenesis is an important process in tissue growth and development and therefore plays an integral role in wound healing. This study was designed to test the hypothesis that hyperbaric oxygen (HBO) will promote angiogenesis and therefore improve healing in a rat model of tissue ischemia. A rat model of tissue ischemia was used in which excisional wounds were centered on a 2.0 cm dorsal bipedicle skin flap with an intervening silicone sheet. In previous studies we have shown that the tissue remains ischemic up to 14 days, wound healing and tensile strength are impaired and lactate is elevated. After flap creation, 60 rats were divided into treatment and control groups, with the treatment group receiving daily HBO at 2.4 atm for 90 minutes. Wound surface area tracing and excision of the wound beds was performed on days 1, 3, 7, 10 and 14, with n = 6 per data point. The concentration of VEGF in wound extracts was quantified by ELISA and standardized for total protein. Data were expressed as mean ± SEM. The wound healing curves diverge at day 5, with HBO-treated wounds consistently larger and delayed in healing compared to control (day 7: HBO - 0.27 ± 0.02 cm² vs control - 0.17 ± 0.07 cm², day 14: HBO - 0.06 ± 0.04 vs cm² vs control - 0.03 ± 0.02 cm²). VEGF is greater at all time points in the control wounds with a peak at day 10 (HBO: 159 ± 55 pg/mg protein vs control: 244 ± 61 pg/mg protein). Non-ischemic wounds expressed low levels of VEGF, which was unchanged by hyperbaric treatment throughout the course of healing. HBO suppresses the expression of VEGF and delays healing in a model of tissue ischemia. Ongoing studies will analyze the effect of HBO on matrix metalloproteinase expression and the relationship between the angiogenic factors, oxidative stress and neovascularization.     

Influence of platelet-derived growth factor on microcirculation during normal and impaired wound healing

The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.     

Hyaluronan, heterogeneity, and healing: the effects of ultrapure hyaluronan of defined molecular size on the repair of full-thickness pig skin wounds.

The extracellular matrix macromolecule, hyaluronan, is thought to modulate wound healing. However, the molecular size of hyaluronan and contaminating associated proteins may be important determinants of these effects. We have examined the results of seven daily topical treatments of full-thickness skin wounds in pigs with ultrapure hyaluronan of defined molecular size. High molecular weight hyaluronan (>1000 kd) enhanced, whereas low molecular weight hyaluronan decreased, the rate of early wound contraction as compared with intermediate hyaluronan (molecular weight = 100 kd) and saline solution controls. Fracture strength at 21 days was reduced by high and intermediate molecular weight hyaluronan but not by low molecular weight hyaluronan. Wound perfusion, measured by means of a scanning laser-Doppler technique as a noninvasive indicator of angiogenesis, showed depression by high and intermediate molecular weight hyaluronan on day 3, but all forms of hyaluronan caused elevated blood flow on day 7. The architecture of granulation tissue in this wet healing model was highly organized, but no gross histologic differences were seen because of treatment. Different molecular species of hyaluronan have differential effects on contraction, angiogenesis, and the evolution of wound strength. Where hyaluronan is used as a treatment or vehicle for wounds, its precise composition should be specified.     

Effect of hyperoxia on vascular endothelial growth factor levels in a wound model

HYPOTHESIS: Hyperbaric oxygen (HBO) therapy increases vascular endothelial growth factor (VEGF) levels in wounds. DESIGN: Wounds were monitored for oxygen delivery during HBO treatment, and wound fluids were analyzed for VEGF and lactate on days 2, 5, and 10 following wounding. SETTING: Experimental animal model. INTERVENTIONS: Rats were randomized to HBO therapy and control groups. The HBO therapy was administered for 90 minutes, twice daily with 100% oxygen at 2.1 atmospheres absolute. Treatment was administered for 7 days following wounding. MAIN OUTCOME MEASURES: Vascular endothelial growth factor, PO(2), and lactate levels in wound fluid were measured on days 2, 5, and 10. RESULTS: Wound oxygen rises with HBO from nearly 0 mm Hg to as high as 600 mm Hg. The peak level occurs at the end of the 90-minute treatment, and hyperoxia of lessening degree persists for approximately 1 hour. The VEGF levels significantly increase with HBO by approximately 40% 5 days following wounding and decrease to control levels 3 days after exposures are stopped. Wound lactate levels remain unchanged with HBO treatment (range, 2.0-10.5 mmol/L). CONCLUSIONS: Increased VEGF production seems to explain in part the angiogenic action of HBO. This supports other data that hypoxia is not necessarily a requirement for wound VEGF production.     

Noninvasive device readouts validated by immunohistochemical analysis enable objective quantitative assessment of acute wound healing in human skin

Objective evaluation of cutaneous wounds through use of noninvasive devices has important implications for diagnosis, monitoring treatment efficacy, progression and may lead to development of improved theranostic treatment strategies. However, there is a lack of validation in the use of certain devices in wound repair, where objective measurements taken by noninvasive devices have been corroborated by immunohistochemical analysis. Thus, data from three acute wound‐healing studies in healthy volunteers using three noninvasive objective devices were further evaluated by immunohistochemistry. One hundred ten participants had 5‐mm diameter skin biopsies to their arms. Spectrophotometric intracutaneous analysis (SIAscopy), full‐field laser perfusion imaging, and three‐dimensional imaging provided quantitative measurements of melanin, hemoglobin, collagen, blood flow, and wound size; all of which were validated by immunohistochemistry. Full‐field laser perfusion imaging showed blood flow increased to D7 and decreased by 40% to D14. SIAscopy showed that hemoglobin increased to D7 and reduced to D14. CD31 analysis corroborated this by showing a 76% increase in blood vessel density to D7 and a reduction by 14% to D14. Three‐dimensional imaging showed that wound surface area reduced by 50% from day 7 to day 14. Alpha‐smooth muscle Actin (Alpha‐SMA) staining supported these trends by showing increased levels by 72% from D0 to D14 (corresponding to wound contraction). Collagen, measured by SIAscopy, decreased to D7 and increased to D14, which was validated by collagen III analysis. Additionally, collagen I increased by 14% from D0 to D14. SIAscopy measurements for melanin showed an increase at D7 and a slight reduction to D14, while melanogenesis increased by 46.7% from D0 to D14. These findings show the utility of noninvasive objective devices in the quantitative evaluation of wound‐healing parameters in human skin as corroborated by immunohistochemistry. This may contribute to the development of prognostic parameters for assessment of response to wound therapy.     

Bacterial load in relation to vacuum-assisted closure wound therapy: a prospective randomized trial.

Vacuum-assisted closure has become a new technique in the challenging management of contaminated, acute, and chronic wounds. Although promising clinical results have been described, scientific proof to substantiate the mechanism of action of this therapy is scarce. In the present study, we examined whether the positive effect on wound healing found in vacuum-assisted closure-treated wounds could be explained by an effect on the bacterial load. Fifty-four patients who needed open wound management before surgical closure were included in this study. Wounds were randomized to either vacuum-assisted closure therapy (n= 29) or treatment by conventional moist gauze therapy (n= 25). Healing was characterized by development of a clean granulating wound bed ("ready for surgical therapy") and reduction of wound surface area. To quantify bacterial load, biopsies were collected. No significant difference was found in time needed to reach "ready for surgical therapy" comparing both therapies. Wound surface area reduction was significantly larger in vacuum-assisted closure-treated wounds: 3.8 +/- 0.5 percent/day (mean +/- SEM) compared to conventional-treated wounds (1.7 +/- 0.6 percent/day; p < 0.05). The total quantitative bacterial load was generally stable in both therapies. However, nonfermentative gram negative bacilli showed a significant decrease in vacuum-assisted closure-treated wounds (p < 0.05), whereas Staphylococcus aureus showed a significant increase in vacuum-assisted closure-treated wounds (p < 0.05). In conclusion, this study shows a positive effect of vacuum-assisted closure therapy on wound healing, expressed as a significant reduction of wound surface area. However, this could not be explained by a significant quantitative reduction of the bacterial load.     

The effect of differing ambient oxygen tensions on wound infection.

Wound infections were studied in rabbits using two standard inocula (approximately equal to 10-4 and approximately equal to 10-6) of Pseudomonas aeruginosa injected into subcutaneous wound dead space made by implantation of standard wire mesh cylinders. The inoculation was done on the fourth day after implantation of the cylinders in animals kept from the day of implantation in atmospheres of 12%, 21%, or 45% oxygen content. Samples of wound fluid (0.2 ml) were removed for quantitative culture just before inoculation and 3, 7, 14, and 21 days later. No positive cultures resulted from samples taken before inoculation. One uninoculated wound served as a control in each animal. None of these control wounds became infected. Culture counts were significantly highest in the anoxic group and lowest in the hyperoxic group. Established infections were significantly lowest in the hyperoxics and highest in the hypoxics. The percent of wounds showing a significant culture count showed a similar trend. The mechanisms of this effect is not known, but a possible mechanism lies in the relative inability of leucocytes to kill this bacterium under hypoxic conditions.     

Hyperbaric oxygen therapy in adriamycin extravasation: an experimental animal study

Adriamycin extravasation is a serious problem that can lead to severe local necrosis and damage to underlying nerves, tendon, and muscle. There are some experimental studies in which conflicting results were reported about the beneficial effects of hyperbaric oxygen (HBO) in adriamycin-induced skin lesions. In this study the effect of HBO on the healing of adriamycin-induced wounds was investigated in the Wistar-Albino rat model. Ninety-five rats underwent injection of a 0.7-ml adriamycin solution (2 mg per milliliter) subcutaneously on the right upper hind legs. The HBO group (n = 43) received HBO treatment (at 2.5 ATA for 80 minutes) twice a day for 28 days. The control group (n = 42) did not receive any therapy. Lesion size was measured for 4 weeks. There was no significant difference in lesion size between the groups on days 7 (p = 0.1364) and 14 (p = 0.1123), but the lesion size was significantly smaller in the HBO group than in the control group on days 21 (p = 0.0251) and 28 (p = 0.0001). Of 36 animals with lesions in the HBO group, complete wound healing occurred in 16 animals by day 40. However, there was no complete wound healing in any of the 36 animals with lesions in the control group. The beneficial effect of HBO in adriamycin-induced skin lesions was observed in this experimental study.     

TIME伤口床准备联合封闭式负压引流用于慢性伤口护理

目的探讨基于TIME原则的伤口床准备联合封闭式负压引流治疗慢性伤口的效果。方法将80例患者随机分为观察组和对照组各40例。观察组采用TIME原则进行伤口床准备,并应用封闭式负压引流,对照组在TIME原则指导下采用传统清创联合无菌敷料换药方法进行伤口护理。比较两组干预前和干预后7d、14d、21d的伤口面积缩小率、深度缩小率、局部症状体征以及观察期内伤口愈合率及所有愈合伤口的愈合时间、伤口换药时数、伤口换药费用。结果观察组伤口面积、深度缩小率和局部症状体征改善显著优于对照组(均P0.01);观察组90d内伤口愈合率显著高于对照组,伤口愈合时间、护理时数和换药费用显著低于对照组(均P0.01)。结论基于TIME原则的伤口床准备联合封闭式负压引流能够显著促进慢性伤口愈合,减少护理工作量及患者经济负担。     

The effect of hyperbaric oxygen on reperfusion of ischemic axial skin flaps: a laser Doppler analysis.

This study evaluates the microvascular reperfusion of ischemic skin flaps with and without acute hyperbaric oxygen (HBO) treatment. Thirty-two axial pattern epigastric skin flaps (3 x 6 cm) in male Wistar rats were subjected to 8 hours of global ischemia by pedicle clamp occlusion. The rats were divided into the following control and two experimental groups: Control (n = 12) with ischemia, no HBO; Group 1 (n = 11) with HBO treatment (three 1.75-hour dives, 2.5 absolute atm, 100% O2) during ischemia; and Group 2 (n = 9) with HBO treatment (two 1.75-hour dives) immediately after ischemia. Laser Doppler flows were recorded in two distal standardized flap locations at 0.5, 2, 4, and 18 hours after reperfusion in control rats and Group 1 rats and at 18 hours only in Group 2 rats, using a Med-Pacific 6000 laser Doppler unit. Mean distal flap laser Doppler flows (mV) were Control: 0.5 hours = 23.2 +/- 11.9, 2 hours = 52.8 +/- 27.3, 4 hours = 53.6 +/- 32.1, 18 hours = 40.2 +/- 36.2; Group 1: 0.5 hours = 71.8 +/- 30.9 (p less than 0.05 vs. control), 2 hours = 74.3 +/- 27.3, 4 hours = 67.4 +/- 20.6, 18 hours = 79.1 +/- 40.3 (p less than 0.05 vs. control); and Group 2: 18 hours = 90.3 +/- 47.9 (p less than 0.05 vs. control). It is concluded that acute HBO treatment of ischemic rat skin flaps improves distal microvascular perfusion as measured by laser Doppler flowmetry. This effect is observed for HBO treatment given either during or immediately after prolonged global ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laser Doppler flowmetry assessment of peristernal perfusion after cardiac surgery: beneficial effect of negative pressure therapy

Negative pressure therapy has been successfully applied to clean, closed incisions in patients at high-risk for wound complications. Using laser Doppler flowmetry, we evaluated peristernal perfusion after cardiac surgery via median sternotomy, assessing the influence of mammary artery harvesting and the impact of negative pressure therapy. Twenty adult patients underwent median sternotomy for cardiac surgery followed by routine closure. Negative pressure was applied at 125 mm Hg for 4 days postoperatively in patients with increased risk for wound complications (n = 10, negative pressure group); standard dressings were applied to control incisions postoperatively (n = 10). Presternal perfusion was determined at baseline and daily for 4 days postoperatively using laser Doppler flowmetry. Results within and between groups were compared with analysis of variance. No wound complications were encountered in either group. Perfusion increased among the patients who underwent negative pressure therapy and decreased among the controls (P = 0.004). Mammary artery harvesting reduced peristernal perfusion by 25.7% in the controls, but negative pressure increased perfusion by 100% after mammary harvesting (P = 0.04). Negative pressure therapy increased perfusion relative to controls and compensated for reduced perfusion rendered by mammary artery harvesting, providing additional support for 'well wound therapy' in high-risk patients.     

CCN1 secreted by human adipose-derived stem cells enhances wound healing and promotes angiogenesis through activating the AKT signalling pathway

The study aimed to explore the role of cellular communication network factor 1 (CCN1) an extracellular matrix protein in hADSC-treated wound healing. Immunofluorescence and enzyme-linked immunosorbent assays (ELISA) were used to demonstrate the secretion of CCN1 by hADSCs, isolated from human fat tissue. We investigated the role of CCN1 in wound healing by knockdown of CCN1 expression in hADSCs using CCN1 siRNA. Conditioned medium of hADSCs or hADSCs with CCN1 knocked down (hADSC-CM^CCN1↓) was collected. After treatment with plain DMEM/F12, hADSC-CM, hADSC-CM^CCN1↓, or recombinant human CCN1 (rhCCN1), the wound healing abilities of human umbilical vascular endothelial cells (HUVECs) were assayed, and the AKT, also known as protein kinase B (PKB), signalling pathway was detected using western blotting. Next, we created full-thickness skin wounds on the backs of the mice and different treatments were applied to the wound surface. Wound size was measured using a digital camera on days 0–10, and evaluated. H&E and immunohistochemical staining were performed, and laser Doppler perfusion imaging was used to evaluate blood perfusion. The wound model and wound-healing assay showed that the hADSCs-CM and rhCCN1 groups had enhanced wound healing compared to the hADSCs-CM^CCN1↓ group. Further, CCN1 and hADSCs-CM promoted the proliferation and migration of HUVECs through the AKT signalling pathway. We concluded that CCN1 secreted by hADSCs enhances wound healing and promotes angiogenesis by activating the AKT signalling pathway. CCN1 plays a vital role in the regulation of hADSCs-CM during wound healing.     

The use of hyperbaric oxygen therapy to treat chronic wounds: A review

Chronic wounds, defined as those wounds which fail to proceed through an orderly process to produce anatomic and functional integrity, are a significant socioeconomic problem. A wound may fail to heal for a variety of reasons including the use of corticosteroids, formation of squamous cell carcinoma, persistent infection, unrelieved pressure, and underlying hypoxia within the wound bed. Hypoxia appears to inhibit the wound healing process by blocking fibroblast proliferation, collagen production, and capillary angiogenesis and to increase the risk of infection. Hyperbaric oxygen therapy (HBOT) has been shown to aid the healing of ulcerated wounds and demonstrated to reduce the risk of amputation in diabetic patients. However, the causal reasons for the response of the underlying biological processes of wound repair to HBOT, such as the up‐regulation of angiogenesis and collagen synthesis are unclear and, consequently, current protocols remain empirical. Here we review chronic wound healing and the use of hyperbaric oxygen as an adjunctive treatment for nonhealing wounds. Databases including PubMed, ScienceDirect, Blackwell Synergy, and The Cochrane Library were searched for relevant phrases including HBOT, HBO/HBOT, wound healing, and chronic/nonhealing wounds/ulcers.     

Regional disturbances in blood flow and metabolism in equine limb wound healing with formation of exuberant granulation tissue

As in other fibroproliferative disorders, hypoxia has been suggested to play a key role in the pathogenesis of exuberant granulation tissue (EGT). The purpose of this study was to investigate metabolism and blood flow locally in full‐thickness wounds healing with (limb wounds) and without (body wounds) formation of EGT. Microdialysis was used to recover endogenous metabolites from the wounds, and laser Doppler flowmetry was used to measure blood flow. Measurements were performed before wounding and 1–28 days after wounding. Blood flow was consistently lower in limb wounds than in body wounds throughout the study period with no change over time. After wounding and throughout the study period, the glucose concentration was significantly lower in limb wounds than in body wounds, whereas the lactate level showed a significantly higher concentration in limb wounds. The lactate/glucose ratio displayed a significant difference between body and limb wounds. In conclusion, the metabolic disturbances may suggest an inadequate oxygen supply during the wound healing process in equine limb wounds healing with EGT. This may be related to the inherently decreased perfusion in the wound bed of limb wounds.     

In vivo effect of hyperbaric oxygen on wound angiogenesis and epithelialization

Hyperbaric oxygen (HBO) therapy is increasingly being used in different areas of medical practice. While demonstrated to be effective in several settings, its mechanism of action is not well understood. In the present study, we determined the effects of HBO on wound epithelialization and neovascularization in an in vivo hairless mouse ear "impaired" wound model. To impair wound healing, macrophages were depleted by pretreatment with iota-carrageenan. Wound epithelialization and neovascularization were measured using intravital microscopy and computerized planimetry. Metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and tumor necrosis factor-α (TNF-α) were measured on days 2 and 7 using immunohistochemistry. In nonimpaired healing wounds, the rate of epithelialization and neovascularization was significantly accelerated in the groups treated with HBO. Time to wound closure was significantly delayed in impaired compared with nonimpaired healing wounds and HBO treatment completely reversed this delay. Neither HBO treatment nor macrophage depletion caused significant alterations in MMP-2 expression in wounds. In contrast, TNF-α, MMP-9, and TIMP-1 were significantly up-regulated in the impaired healing group receiving HBO treatment. These results show that HBO therapy effectively reversed the negative effect exerted by macrophage reduction on wound epithelialization and neovascularization. This beneficial effect could be due to stimulation of TNF-α production and, to a lesser degree due to release of metalloproteinases.     

Laser Doppler imaging in the investigation of lower limb wounds

Good blood supply is a primary determinant for the healing of acute and chronic wounds alike. Chronic wounds commonly occur in the lower extremity in man. Scientists are forever looking for objective techniques with which tissue perfusion may be measured with accuracy. Laser Doppler imaging is a very useful technique for measuring microvascular perfusion in wounds because it involves no contact and produces a color image representing flow distribution over an area of tissue. In this article, the authors discuss its application to the assessment of lower limb wounds, and they review published studies in which it has been used to investigate a range of wound types. This review examines the study of healing mechanisms, treatment effects, risk prediction, healing potential, and underlying pathology. It also considers areas in which laser Doppler imaging is still to be exploited fully.     

The evaluation of subatmospheric pressure and hyperbaric oxygen in ischemic full-thickness wound healing

We evaluated the efficacy of subatmospheric pressure and hyperbaric oxygen (HBO) as adjuncts in the treatment of hypoxic full-thickness wounds in a rabbit model. We hypothesized that subatmospheric pressure and HBO independently are effective in improving wound healing in the ischemic wound model and that when they are used in combination there is an increased positive effect on wound healing. Using a standard ischemic wound model four full-thickness wounds were created on each ear of 41 male New Zealand white rabbits (N = 82 ears). On each rabbit one ear was dressed with the vacuum-assisted closure (VAC) device and connected to suction; the other was dressed identically without the suction and suction tubing. Twenty rabbits were treated with HBO daily for 10 days at 2.0 atmospheres absolute for 90 minutes plus descent and ascent times. Necropsy on all rabbits was performed on postoperative day 10. Four ischemic wound treatment groups were evaluated: Group 1 (N = 21) VAC dressing alone; Group 2 (N = 20) VAC dressing plus HBO; Group 3 (N = 21) VAC dressing to suction alone; and Group 4 (N = 20) VAC dressing to suction and HBO. Using light microscopy a veterinary pathologist blinded to treatment groups quantified peak granulation tissue, granulation tissue gap, and epithelialization tissue gap. Data were analyzed by analysis of variance with significance indicated by P < 0.05. Statistical significance was found in a comparison of VAC dressing to suction and VAC dressing alone for peak granulation tissue and granulation tissue gap both with and without use of HBO. VAC device use appears to increase the rate of healing in a rabbit ischemic wound model. HBO therapy did not significantly affect the rate of healing in this model.     

Negative pressure therapy is effective to manage a variety of wounds in infants and children

Negative pressure therapy (NPT) has been accepted as a valuable adjunct for wound closure in adults; however, reports on its effectiveness in young children and infants, including neonates, are limited. A retrospective chart review was conducted on children treated with NPT at a single institution between January 2003 and December 2005. Wound volumetric measurements were calculated at the start and end of therapy. Sixty-eight patients with 82 wounds were identified. The mean age was 8.5 years (range 7 days-18 years). Twenty patients (29%) were 2 years of age or younger, including eight neonates. Wound types included: pressure ulcers (n=13), extremity wounds (n=18), dehisced surgical wounds (n=19), open sternal wounds (n=10), wounds with fistulas (n=3), and complex abdominal wall defects (n=6). Low suction pressures (<100 mmHg) were generally used in children younger than 4 years of age. Following NPT, 93% of wounds decreased in volume. The average wound volume decrease was 80% (p<0.01, n=56). NPT can be effectively used to manage a variety of wounds in children and neonates. No major complications were identified in our retrospective review. Prospective studies are required to better refine the use of this technology in children.