Application of regulated oxygen-enriched negative pressure-assisted wound therapy in combating anaerobic infections
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- Topaz, M., Bisker, O., Litmanovitch, M. et al. Eur J Plast Surg (2011) 34: 351. doi:10.1007/s00238-010-0514-5
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Regulated negative pressure-assisted wound therapy (RNPT) applies non-pharmaceutical technology for enhanced healing of hard to cure, acute and chronic wounds. Although used for over two decades, wound pO2 levels, an essential physical parameter inherent in RNPT that follows Henry’s Law of gases, have not been reported. Necrotizing fasciitis (NF), a severe soft tissue infection, necessitates prompt intensive therapeutic response using pharmaceutical, surgical, and hyperbaric oxygen treatment when anaerobes are involved. We report a linear decrease in pO2 values in direct correlation with sub-atmospheric pressure and concurrent changes in wound atmospheric pO2 by supplemented oxygen in an in vitro phantom leg simulated wound model treated with RNPT. We designed a system using regulated oxygen-enriched negative pressure therapy (RO-NPT) for controlling wound atmospheric pO2. A representative patient case report treated for NF with RO-NPT is presented. RNPT follows Henry’s law of gases and leads to a decrease in wound atmospheric pO2. The application of RNPT in anaerobic wound infections should be contraindicated. Wound pO2 is enhanced by simultaneous application of oxygen by RO-NPT. We have demonstrated the rationale for the possible use of RO-NPT for prevention of anaerobic wound infections and as a supplemental mode of treatment of NF.
KeywordsRNPTRO-NPTHenry’s lawWound infectionAnaerobic infectionNecrotizing fasciitisHBO
Regulated negative pressure-assisted wound therapy (RNPT) is an established method of wound treatment and one of the most important therapies developed and practiced to enhance wound healing [1, 2]. When properly applied, it is a relatively simple, safe tool that can be utilized in a wide range of conditions without the need for sophisticated technology or elaborate surgery.
RNPT has frequently been applied in our institution, mainly in trauma and for treatment of diabetic and peripheral vascular disease-affected limbs, with good clinical outcome. Although RNPT has been applied in clinical practice for over two decades and extensive data have been published in recent years, only few attempted to clarify the scientific basis of its mechanisms of action and physiologic effects. An important and fundamental physical aspect associated with the very basics of relevant gas laws seem to have been ignored.
In this expression, Henry’s law equilibrium constant for a specific gas, K(gas), is an empirical constant with the dimensions of pressure divided by concentration, that has a value depending on the units chosen for the solubility of the gas, C(gas), and the gas partial pressure, p(gas).
Following assessment of Henry’s law, we claim that lower air pressures, as in the atmosphere of wounds treated by RNPT, have lower pO2 levels relative to pO2 at atmospheric pressure, and the wound’s interface under RNPT contains less dissolved oxygen than untreated wounds. This phenomenon, inherent in any RNPT application, may place a substantial limitation on its clinical use. Severe anaerobic infections such as necrotizing fasciitis (NF) require intensive care with prompt therapeutic response using pharmaceutical, surgical, and oxygen enrichment by hyperbaric oxygen (HBO). We hypothesize that this inherent limitation of RNPT may be reduced or totally eliminated by the simultaneous administration of oxygen into the sub-atmospheric-sealed wound cavity.
In order to confirm our hypothesis and better understand the physiology of wounds treated with RNPT, an in vitro experimental model was structured to determine the spectrum levels of pO2 in the environment of wounds treated with RNPT and oxygen-enriched RNPT. A representative case of our clinical experience with the application of regulated oxygen-enriched negative pressure-assisted wound therapy (RO-NPT) will be presented.
Materials and methods
The pO2 was measured at various oxygen flows and negative pressure ranges as indicated in clinical applications for wound treatment. The pO2 values were recorded in relative percent units following calibration of the sensor. Negative pressure was regulated to the desired pressure levels that could be applied in clinical cases (in the range of 50–200 torr below atmospheric pressure, in 25–50 torr increments). Oxygen flow was controlled and delivered to the simulated wound by an oxygen regulator (Silbermann Technologies Ltd., Petah Tikva, Israel) in predetermined 1 L/min flow increments in a range of 1 to 10 L/min. One hundred percent oxygen was administered to the opposite edge of the vacuum source so that oxygen flow was homogeneously distributed through the sponge cavities in contact with the wound over the entire wound surface area. Pressure was readjusted following administration of oxygen to the system. The pO2 in the wound atmosphere was continuously monitored by a pO2 detector (Teledyne Electronic Devices, City of Industry, CA, USA). Calibration of the sensor was performed at sea level and room atmosphere and in 100% pO2 atmosphere prior to initiation of each experimental set. The probe was positioned at the outflow suction port, and measurements were recorded after equilibration was reached (within 10–30 s).
A 49-year-old male presented with multiple trauma injuries including pneumothorax and multiple fractures. On admission to our hospital, he was conscious with flail chest, afebrile, tachycardic, and hypertensive, with normal hemoglobin level (14.9 gr%), and mildly elevated total white blood cell counts (14.6 × 109/L). Total body CT revealed multiple rib fractures with lung contusion, multiple pelvic fractures, and closed fractures of both legs. He was intubated, broad spectrum antibiotic therapy was commenced, fractures of lower limbs were put under traction, and he was transferred to the ICU.
On day 2 following admission, his clinical condition deteriorated. The patient experienced high fever (39.6°C), his white blood cell count dropped (3.5 × 109/L), as did his hemoglobin (8.2 gr%). Skin discoloration was observed and crepitus was palpated under the skin. NF involving gas-forming bacteria infecting the IV site in the left cubital region was diagnosed, spreading along the fascial plane to the forearm, arm, and left chest wall. Blood and wound cultures eventually yielded anaerobic bacterial growth of Acinetobacter baumannii. Immediate exploration of infected tissue with wide fasciotomy and aggressive debridement were performed (Fig. 4a), and broad spectrum antibiotics were continued. Due to the chest wall trauma and the need for mechanical ventilation, transfer to an HBO chamber was not possible. As an alternative, continuous RO-NPT treatment (Fig. 4b) applying 10 L/min of oxygen in combination with sub-atmospheric pressure of 150 torr generating a wound pO2 atmosphere in the range of 50–60% was commenced. Treatment yielded successful results; the anaerobic infection was rapidly extinct. Repeated anaerobic cultures taken after the administration of oxygen to the wound revealed no residual anaerobic growth.
The patient was moved to the orthopedic ward 12 days after admission to our hospital. Following continuous RO-NPT treatment and staged excisions of necrotic tissue (Fig. 4c), wide skin grafting was applied for closure of all wounds. The patient was discharged from hospital after 7 weeks and has remained well (Fig. 4d).
Anaerobes proliferate in an oxygen-depleted environment and can be a major setback to the healing process of various wounds, becoming a potentially life-threatening risk in severe, complicated cases such as NF .
Anaerobes cannot grow in the presence of oxygen because they lack enzymes and antioxidants that can effectively scavenge intracellular and membranal toxic oxidizing agents. Enzymes such as superoxide dismutase, catalase, and peroxidase were demonstrated as crucial necessary factors in the defense mechanism of aerobic bacteria in detoxifying the pathway of oxygen radicals. Anaerobic bacteria contain few or none of these enzymes. As a consequence, anaerobes prefer the oxygen-free habitat, and the proliferation of anaerobic pathogens in body tissues depends on the level of tissue pO2.
Numerical data concerning the sensitivity of anaerobic bacteria to oxygen have suggested that the size of growth inhibition zones is directly proportional to the logarithm of the partial pressure of oxygen used . Anaerobes differ in their sensitivity to oxygen. Inhibition of bacterial agar growth of strict anaerobes was demonstrated at pO2 levels greater than 0.5%. Growth of moderate anaerobes was inhibited at pO2 levels greater than 2–8% .
Anaerobes are the predominant fraction of normal human skin and mucous membrane bacterial flora, and are consequently a common cause of endogenous infection. Because of their fastidious nature, they are difficult to isolate and are often undetected . In situations such as trauma, vascular occlusion, or surgical manifestations when oxygen concentration is reduced in tissue, the anaerobic indigenous flora can multiply quickly and turn into ruthless pathogens that induce local infection and sepsis.
In a recent study, biofilms appeared to be more abundant in chronic wounds, and molecular analysis of the specimens revealed diverse polymicrobial communities, including strictly anaerobic bacteria, not revealed by cultures. In a study on the pathogenesis and current treatment of chronic wounds, anaerobic bacteria were perceived in most wounds when sensitive culturing techniques were applied .
The presence of aerobic or facultative infections creates a habitat that supports growth of anaerobes by reducing the oxygen concentration in the infected tissue. This may even be of greater significance when applying occlusive dressings, creating an airtight sealed environment, as in RNPT.
NF is a life-threatening, rapidly progressive bacterial infection spreading through the deep fascial plane, with secondary necrosis of the fascia and subcutaneous tissues. This deep fascial infection causes vascular occlusion, ischemia, tissue discoloration, and necrosis. Superficial nerves are damaged, producing the characteristic localized loss of sensation .
Type 1 NF is often a mixed infection and can be caused by both aerobic and anaerobic bacteria, most commonly occurring in patients following surgical procedures and following trauma. Type 2 NF accounts for 10–15% of cases and refers to a mono-microbial infection caused mainly by group A Streptococcus pyogenes, the so-called “flesh-eating” bacterial infection. Two-thirds of Type 1 NF have both aerobic and anaerobic bacteria; consequently, the treatment of anaerobes is essential in Type 1 NF.
Once NF is suspected, treatment should be initiated without delay. Empirically, broad spectrum antibiotics must be administered immediately. Early, aggressive, and frequent surgical debridement and exploration of necrotic tissue is essential, combined with intensive medical care and HBO as an adjunctive therapy . The mortality rate of patients with NF is in the range of 20–30%. The strongest prognostic factor associated with NF is age; patients under the age of 35 years have a significantly lower (0%) mortality rate compared to mortality (65%) for patients over the age of 70 .
HBO is considered standard treatment in cases where anaerobic infection of soft tissue is documented or suspected .
HBO temporarily increases the oxygen partial pressure to extreme levels, diminishing anaerobic growth. HBO increases plasma solubility of oxygen, generates peaks of high tissue pO2, and is associated with improved survival and limb salvage in necrotizing soft tissue infections . Wound tissue pO2 levels are a major determinant of susceptibility to infections, as has been shown both in experimental models and in human subjects. HBO therapy may be beneficial in situations where oxygen supply to tissue is compromised by local injury and particularly if anaerobic infection is present .
In a study indicating the beneficial effect of HBO on experimental staphylococcal osteomyelitis in rabbits , the use of oxygen as a defensive line against infections was demonstrated when the phagocytic killing of Staphylococcus aureus was markedly decreased at a pO2 of 23 torr, significantly improved at 45 and 109 torr, and was found most effective at 150 torr.
HBO is not a suitable solution for every patient. HBO therapy has the self-evident limitation of accessibility to the chamber as well as issues of safety and expense. For example, the administration of this treatment may be problematic for patients suffering from lung trauma or in cases of ventilated patients who are hard or impossible to mobilize. Contraindications to HBO use include recent ear or sinus surgery, seizure disorder, and febrile disorders; in general, HBO may pose risks of oxygen toxicity . Moreover, treatment costs for HBO are significantly high, and may reach $20,000 per conventional wound healing protocol.
Following tissue injury, three phases of repair mechanisms are activated: inflammation, proliferation, and remodeling. All physiologically active processes involved in these phases are in need of an adequate supply of oxygen . Problems with delayed healing and infection of wounds in most cases are related to oxygen delivery to the wound tissue .
Wounds that are well oxygenated have been shown to be less likely to become infected. The effect of oxygen as a therapeutic agent essential for wound healing and its importance in the defense mechanism against infections has long been recognized. Destruction by oxidation, oxidative killing, is the most important defense against pathogens and depends on the oxygen partial pressure in the contaminated tissue . The ability of supplemental O2 to reduce infection is mediated by reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in wound neutrophils and macrophages .
For therapeutic applications, O2 can be given be systemically or locally delivered to the wound using a topical device. Systemically administered O2 relies on the vascular bed to deliver oxygen to the healing tissue. It is therefore reasonable to assume that ischemic areas of the wound will not significantly benefit from this form of oxygen administration. These wounds will be more susceptible to infections, as already mentioned .
The concept of topical oxygen therapy as an alternative to HBO has also been acknowledged. Not only is it potentially less toxic, it may also be much more convenient, less expensive, and have few, if any, complications. Topical delivery of oxygen can oxygenate the wound directly when the wound is severed from circulation or when the vascular bed is impaired .
A study of dermal wound healing following treatment with topically applied pure oxygen in a pig model showed that exposure of open dermal wounds to topical oxygen treatment increased tissue pO2 of superficial wound tissue . In another rabbit model, the importance of topical administration of oxygen to an ischemic epithelial wound was confirmed. The study supported the theories about the role of oxygen as a signaling molecule in therapeutic processes .
The inflow of oxygen in the in vitro experimental model was in the range of 1–10 L/min distributed in a relatively small volume of compressed porous mold covering the wound (total space volume in a range of 50–100 cc). Because of the small space volume enclosed underneath the drape, oxygen concentration reached equilibrium relatively fast, within 10–20 s. When reviewing Fick’s law of diffusion, it is trivial to predict that this Phantom leg model would closely correlate to in vivo pO2 levels as the only difference between the in vitro and the in vivo setup would be a relatively negligible volume of oxygen that would diffuse into the open wound in the in vivo setup in a flow that is expected to be of few orders of magnitude less than the inflow of oxygen into the limb covering mold.
While topical O2 is not likely to diffuse into deeper tissues in significant volumes, it can potentially oxygenate superficial areas of the wound not supplied by proper vasculature or even diffuse into biofilms in chronic wounds not affected by antibiotics. By correcting pO2 levels of cells at the wound core, topical oxygen can support NADPH oxidase function in the superficial wound’s cells, contributing to processes such as cell motility, angiogenesis, extracellular matrix formation, and reactive oxygen species formation, operating against infections. The optimal pO2 levels for wound healing still need to be determined.
The concept of using topical RNPT to create a suction force for enhancing wound healing is well established, and the beneficial effect of sub-atmospheric pressure application has been demonstrated in many studies . The occlusive RNPT dressing provides a moist environment that prevents cellular dehydration, and stimulates collagen synthesis and granulation tissue for accelerated wound healing. The drainage may also prevent the penetration of bacteria to deeper tissues. The VAC® device (Kinetic Concepts, Inc., San Antonio, TX) for applying RNPT was designed for particular chronic wounds, but it has rapidly evolved into an accepted treatment for a wide range of wound types. The technique is simple, broadly applicable, cost-effective, and beneficial . Two basic mechanisms are believed to account for its physiologic effects: (1) the removal of fluids, thereby decreasing edema, relieving interstitial pressure, and increasing blood flow, which in turn reduces tissue bacterial flora, and (2) the mechanical deformation or stress within the tissue accelerating protein and matrix synthesis. Another important factor should be considered part of the physical evacuation of fluids from the wound bed that may flush out excessive proteolitic, zinc-dependent enzymes: various matrix metalloproteins (MMPs; elastases, collagenases, and gelatinases), enzymes involved in both the turnover and degradation of the extracellular matrix, compromising or inhibiting wound healing. MMPs are generated both by various cells in the wound bed and by bacteria colonizing the open wound .
Although the above data and other studies have been published in recent years attempting to shed light on and clarify the scientific basis of the physiologic effects of RNPT, the fundamental feature associated with the very basics of gas laws and other physical effects went unnoticed. The few case reports that relate to the development of infection associated with RNPT recommend the discontinuation of RNPT in anaerobic wound infections . In order to minimize this complication, it was recommended to use VAC only on clean healthy wounds, thus limiting the scope of indications for RNPT. None of the reports, however, refer to the basic physical laws of gases to substantiate this recommendation.
A combined staged treatment using the VAC® system and HBO was recently studied in a rabbit ear model, evaluating the significance of consecutive use of sub-atmospheric pressure by the VAC® system and HBO in an ischemic full-thickness wound. The conclusion of this study was that HBO did not significantly improve the rate of healing, whereas the sub-atmospheric pressure significantly increased the wound healing process . This model, however, did not address the simultaneous combination of these modalities or the treatment of infected wounds.
Although the rationale of simultaneous oxygen supplementation to RNPT systems by HBO may be compelling, it is expected to be technically complex. The use of the VAC® system was defined as contraindicated in an HBO chamber by the manufacturer due to combustion hazard. Still, the optional advantages of simultaneously joining the two strategies of supplemental oxygen together with sub-atmospheric pressure for the combined prevention and treatment of anaerobic wound infections and the acceleration of wound healing seem to have significant merit. Our laboratory measurements, jointly with our positive wide clinical experience, point to the benefits of enriching the wound atmosphere with oxygen, reaching levels of pO2 greater than 60%, at a working sub-atmospheric pressure range of 50–75 torr, with oxygen flow of 4 L/min (Fig. 3). It was possible to achieve an increase in baseline pO2 levels by a factor of 4.2, with greater supplemental oxygen flow, while maintaining a range of clinically effective sub-atmospheric pressure in the wound cavity.
According to Henry’s law, stated over two centuries ago, the solubility of oxygen in fluid solutions is roughly proportional to the partial pressure of oxygen in the surrounding atmosphere. In the sub-atmospheric pressure range of wounds treated with RNPT (applicable to all currently available devices), pO2 was found, as expected, to be less than 21%, meaning lower solubility of oxygen in the wound’s fluids, creating an enhanced microenvironment for anaerobic bacterial growth. Our measurements of pO2 in a simulated wound atmosphere without supplemental oxygen (Fig. 2) showed a decline of pO2 from 160 torr at atmospheric pressure to pO2 of 106 torr at 200 torr sub-atmospheric pressure. In a more accurate extrapolation to clinical pO2 values, wounds may be even more vulnerable to infections as pO2 may be even further reduced due to oxygen consumption by bacteria and catabolic processes under the sealed wounds.
In light of our findings, those sporadic reports suggesting treatment of NF with VAC [24, 25] should be reassessed. Type 1 NF is an example of an infection involving anaerobic bacteria in which RNPT is contraindicated, where RO-NPT may be of imperative clinical significance, making it an alternative or even at times preferable treatment to intermittent tissue oxygenation with HBO. Oxygen levels in the wound, treated with RO-NPT, can be monitored, calculated, and adjusted by altering oxygen inflow and/or sub-atmospheric pressure to attain the desired pO2 level, as seen in Fig. 3. These levels of pO2 may be sufficient for prevention or inhibition of anaerobic infection, or treatment of apparent anaerobic infections.
In the last 2 years, we have successfully applied RO-NPT in three NF cases, and routinely apply RO-NPT as prophylaxis in major trauma injury and in diabetic and PVD wounds. All anaerobic infections were rapidly terminated with no need for HBO. No anaerobic infection developed in any of the prophylaxis treated cases of RO-NPT. Even in severe cases, most of the treated wounds could be healed by simple surgical procedures such as skin grafts.
The same rationale of hyperbaric oxygen treatment goes with the synergistic effect of continuous oxygen flow, increasing oxygen partial pressure in the isolated ground atmosphere of wounds for the treatment of most soft tissue infections. The physical–mechanical non-pharmaceutical platform technology combined in RO-NPT has the potential to be utilized on a wide scale in an intensive care setup in a hospital or in a home care treatment, for critically ill patients and chronic wounds, for large populations under disaster conditions, field settings, and in underprivileged societies where HBO and advanced surgical and therapeutic conditions are not available or feasible. Our clinical experience should be regarded as first evidence for the benefit of RO-NPT for the treatment of NF. A larger number of clinical cases need to corroborate our findings. Further laboratory research and prospective randomized clinical trials are mandatory to better understand the role of oxygen in treatment and prophylaxis of anaerobic infection and wound healing applying RO-NPT.
The application of RNPT follows Henry’s law of gases and leads to decreased pO2 in wound atmosphere. In this paper, we present our laboratory measurements of the spectrum levels of pO2 that were detected in a simulated in vitro model of wound atmosphere treated with RNPT and RO-NPT. The lowered level of wound-dissolved oxygen in the closed, moist microenvironment of sub-atmospheric pressure-treated wounds risks development of anaerobic infections. The use of RNPT in anaerobic wound infections should be contraindicated. Wound pO2 is enhanced by simultaneous application of oxygen to RNPT. We have demonstrated the rationale for the possible use of RO-NPT for prevention of anaerobic wound infections and as a supplemental mode of treatment in NF.
We thank Naomi Meyerstein (Experimental Hematology Laboratory, Department of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel) and Dan Meyerstein (Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel) for their obliging assistance and enlightening comments, and Emily Galam for her editorial support.