Re-establishing Macro Vascular Flow and Wound Healing: Beyond the Vascular Intervention

Introduction

Since approximately 70% of wounds treated at outpatient wound centers in the United States are lower extremity venous ulcerations, we will use a leg ulcer as case study for the purposes of this article. A 65-year-old Caucasian female presents to the wound center with a 10 x 8 cm wound just above the medial malleoli. The wound has been present for 2 years and is slowly increasing in size. The skin surrounding the wound is hyperpigmented and the soft tissue is firm on palpation. The ankle circumference is 16 cm, while the mid-calf circumference is 35 cm giving the leg an “inverted champagne bottle” appearance. The wound bed is 80% yellow with a thick adherent slough noted. The edges of the wound are rolled over and there is no evidence of epithelial migration. The patient complains of pain that is worse at night and she develops calf pain within a 2 block ambulation distance that is relieved by standing for 5 minutes. The patient in this case suffers from a “mixed” venous/arterial ulcer, further complicating her care. A wound care work-up consists of a comprehensive history, physical exam, and a series of laboratory tests in order to arrive at a provisional diagnosis.² As part of this work-up, the wound bed must be assessed, measured, photographed, and oftentimes, biopsied.

Much has been written about the wound micro-environment and the impact it has on healing.³ Important issues that will be discussed in this paper include the role of bacteria, inflammation, cellular proliferation, and tissue perfusion on the microenvironment.

Wound Bio-Burden

It is now accepted that all chronic wounds are colonized with bacteria making routine cultures unnecessary. There is a natural balance between the quantity of bacteria present (bio-burden) and the host’s immune status. When equilibrium is reached, there is no clinical infection. A sterile wound is not necessarily a goal of treatment, as the presence of granulation tissue is stimulated by the presence of some bacteria, which stimulate inflammation and healing.⁴ If the inoculums of bacteria is increased (> 10⁵ organism/gram tissue) or the host suffers a decrease in immunity, clinical infection occurs.⁵ Many describe the failure of skin grafts, delayed closures, and overall wound healing problems when the bacterial bioburden exceeds 10.^5,6 This value is accepted by many as a quantitative definition of infection, except in the presence of beta-hemolytic streptococcus, where the value is somewhat lower (10³).⁷ The presence of necrotic debris, foreign body, or the desiccation of a wound bed enhance bacterial growth. The concept of colonization (the mere presence of organisms) and infection (the invasion of organisms into the tissue) can usually be determined by the physical exam.⁸ The cardinal signs of inflammation; erythema, pain, swelling, and increased temperature may be clues to impending infection. Many patients are clinically unable to mount an inflammatory response, and in those patients, the use of quantitative culturing along with clinical intuition are necessary. There are numerous ways to obtain cultures of a wound, but the quantitative biopsy remains the gold standard.⁹ The use of techniques such as PCR will enable an even more sensitive approach to identifying the wound bio-burden, but the therapeutic dilemma of whether to treat or not will remain.10 Wound bio-burden and infection must be thought of as a continuum and not a point in time when a specific number of organisms are present. In between the state of an infection and colonization, there is a condition in which the bacteria compete with the wound for nutrients and oxygen resulting in a non-healing wound. This microenvironmental condition has been referred to as “critical colonization” of the wound.¹¹ A wound can initially proceed along a healing trajectory and then suddenly plateau, a condition known as the stunned wound.¹² Interestingly, systemic antibiotics penetrate very poorly into granulation tissue and scar limiting their usefulness in non-infected, critically colonized wounds.¹³ Several papers have reported on a potential negative outcome with the use of antibiotics without infection.^14,15 Topical applications of antiseptics and wound modalities such as ultraviolet light, pulsed lavage and low frequency non-contact ultrasound may be of benefit in controlling the bioburden without subjecting the patient to unnecessary risks of bacterial resistance.¹⁶

The impact of bacteria and infection induce a systemic effect on host healing as well. Changes in cicatrix formation in a wound have been attributed to the impact of infection in very early work by Alexis Carrel.¹⁷ There are contrasting reports on the correlation of healing impact and tensile strength with the presence of microorganisms.^18–20 Laato et al delivered further support of a yin-yang impact of bacteria on healing.²¹ Marks et al demonstrated that wound location and depth are also significant factors in determining the clinical importance of a specific bacterial isolate.²² This reference discusses the actual biological impact of bacteria on healing, however, most references focus on the impact of bacteria on flap and skin graft survival.

It has been established that the bacterial type, wound location, depth of injury, bacterial quantity, and host response are all parameters that require analysis when determining the impact of bacteria on wound healing and granulation tissue formation. It is a commonly held, general, surgical assumption that the presence of granulation tissue denotes a healthy wound bed. Harding described subclinical characteristics of granulation tissue that should alert the observer to the possibility of a granulation tissue infection.²³ Harding further alerts the clinician to the importance of the presence of abnormal granulation tissue in an indolent wound that was previously progressing. The presence of bacteria in an open wound therefore creates a clinical dilemma. The characteristics of both pathogen and host must be considered when determining the clinical relevance of bacterial presence and the subsequent treatment recommendations. The presence of bacteria can also lead to a pro-inflammatory condition in the wound bed that is not conducive to wound contraction or epithelial migration. Pro-inflammatory cytokine levels have been shown to be markedly elevated in chronic wound fluid when compared to acute wound fluid.^24,25 Matrix metalloproteases are necessary in small quantities for digestion of the basement membrane and to allow for cellular migration but in large quantities they will impede healing in part, by digesting endogenous growth factors. Recently, wound dressings have been designed that specifically bind MMPs in an attempt to improve the micro-environment and re-establish a healing trajectory.²⁶ Either the excess quantity or survival of activated polymorphonuclear cells leads to the release of matrix metalloproteinases (MMPs), which can tip the balance towards matrix breakdown.

Wound Micro-environment

Another important issue in the wound micro-environment is the status of the extracellular matrix and the proliferative capacity of the resident cells. The extra cellular matrix (ECM) can either provide a structural scaffolding or act as an impediment for cellular migration.²⁷ Wound healing requires a balancing but not complete elimination of these MMPs.²⁸ Without, for example, collagenase-1, the migration and directionality of keratinocytes would be lost, leading to impaired resurfacing of the wound.²⁹ The hostile environment of the chronic wound as described above might also result in the loss of proliferative capacity for the cells involved in healing.³⁰ Growth rates and migrating capacity of fibroblasts has been shown to be reduced in the periwound tissue of patients with venous ulcers.^31,32 Fibroblasts are dysfunctional in diabetic patients.³³ Much of the current work on cellular senescence has focused on the impact of telomere shortening the activity of telomerase.³⁴

Tissue perfusion: Assessment of microcirculation. An additional aspect of the wound microenvironment is the status of local tissue perfusion. The microcirculation is connected in series to the macrocirculation, however, the presence of an adequate macrocirculation does not guarantee adequate microcirculatory flow.³⁵ The microcirculation refers to the web of capillaries and arterioles that represent the primary resistance bed for the circulation. There are several tests that can evaluate the status of the micro-circulatory system. Intravital capillaroscopy is a non-invasive technique that utilizes a microscope to identify nutrient capillaries.³⁶ Transcutaneous pulse oximetry (TcPO₂) is a technique widely used to detect the skin oxygen tension. A dime-sized Clarke type solid state polarographic electrode containing a platinum cathode with a reference electrode of silver chloride is housed in a probe tip along with a heater and thermistor. The tip of the probe is covered with a permeable membrane. An electrolyte solution fills the reservoir inside the probe. The reduction of oxygen at the cathode generates a current, which is then fed into the pO₂ channel of a monitor and converted into a voltage and digitized. The electrode is attached via a fixation device to the immediate periwound skin and heated to 43–45 degrees Celsius, which induces hyperemia and the dissolution of keratin lipids, thereby increasing gas permeability. The examination takes 20 minutes and should be repeated at the same tissue site at each examination. The final TcPO₂ result may be influenced by capillary temperature, blood flow, and metabolic oxygen consumption. Although there is a short distance from the probe tip to the capillary (0.3 mm), the oxygen has to pass through metabolically active tissue and is, therefore, partially consumed. The TcPO₂ value, therefore, does not directly correlate to the arterial blood gas, often causing confusion among healthcare providers working with this equipment. The technique generates a value that is more a reflection of the difference between oxygen delivery and utilization than an approximation of arterial PO₂. Various researchers have attempted to utilize the technique to help in the clinical selection of appropriate amputation levels.^37–39 Clyne et al studied the TcPO₂ values in the gaiter distribution (medial lower leg) of patients with chronic venous insufficiency and found very low levels.40 This technique can and should be used in patients with diabetes because the Doppler-derived ankle pressures are frequently falsely elevated secondary to calcification of blood vessels. This problem can be overcome with the adjunctive use of a TcPO₂ monitor because transcutaneous oximetry is not influenced by this process. There are numerous clinical uses for TcPO₂ monitoring in the wound care industry. Colin et al described the use of TcPO₂ monitoring in the evaluation of support surfaces and the prevention of pressure ulcers.⁴¹ Chomard et al evaluated the effects of a synthetic prostacyclin on limb salvage by monitoring TcPO2 levels.42 More recently, similar studies have employed TcPO2 for monitoring Iloprost therapy in limb ischemia.43 Laser Doppler perfusion imaging is a recently developed technique which utilizes a low intensity laser (Helium-Neon) light.44 The device measures the backscattering created by moving red blood cells over a specific rectangular area, analyzing up to 4,096 individual points. The wavelength of this monochromatic light is 670 nanometers (nm) with a maximum accessible power of 1mW. The penetration of the laser beam reaches 500 µm when applied to intact skin, however, penetration can reach 2.5 times greater in other non-skin tissues like granulation tissue.⁴⁵ Other tests for microcirculation include positron emission tomography, nuclear magnetic resonance spectroscopy, xenon washout, and near-infrared spectroscopy.⁴⁶

Decisions about limb salvage need to include the results of testing from both the macro and the microcirculation. The clinical usefulness of monitoring the microcirculation was demonstrated in a paper by Ubbink et al, in which 111 patients with non-reconstructable vascular disease were categorized into 3 groups based on the results of transcutaneous oximetry, laser Doppler, and capillaroscopy.⁴⁷ Patients in the poor outcomes group were likely to end up with amputation, while those in the intermediate and good categories had significantly higher limb salvage rates. This type of analysis might lead to a more appropriate use of resources in the future for limb salvage cases. Arora et al have recently published a paper that evaluated the impact of successful bypass surgery on the microcirculation.⁴⁸ The results showed that although impaired vasodilation was improved, it was not completely reversed with successful bypass, thereby explaining how a patient can fail to heal a wound despite the adequate restoration of macrovascular flow.

Treatment Plan

Patients can also have good macro arterial perfusion and poor micro flow. The patient described at the beginning of this article had an ankle brachial index of 0.70. The periwound transcutaneous oxygen value was 25, a value of greater than 40 is considered normal. The patient, therefore, has a mixed venous/arterial ulcer and suffers from inadequate macro and microcirculation. Her symptoms from the macrovascular standpoint include 2-block claudication and the presence of a non-healing wound. The microcirculation is also suboptimal and this is likely due to the lipodermatosclerosis and scar tissue in the peri-wound soft tissue.

It is important at this point for communication and collaboration between the vascular and the wound care team. There are actually a number of therapeutic pathways that could be followed depending on the patient’s symptoms, anesthesia risk, patient desires, and status of the wound pain, and local medical expertise. Since the micro-circulatory studies were abnormal, a trial of treatments aimed at enhancing the microcirculation (i.e., electrical stimulation, growth factors, bio-engineered tissue, and therapeutic ultrasound) could be used, along with aggressive wound care for a short course. Systemic therapy along with life style modifications should also be employed. If the wound heals and the patient continues to have quality of life claudication issues, then an interventional procedure might be indicated. If after 2–4 weeks of local therapy, however, there was little or no improvement in wound healing, then the patient would likely require a macrovascular interventional procedure. If compression therapy was considered an integral component of the treatment plan, and the patient could not tolerate this secondary to the underlying arterial disease, then a macrovascular intervention could be considered as a primary therapy. Oftentimes, a patient with a “mixed” venous/arterial ulcer requires a bypass procedure in order to allow for adequate compression therapy, which is a critical component of the treatment for venous insufficiency and ulceration.

Approaching the wound patient in this manner will avoid unnecessary high-risk procedures, and the limited treatment time would minimize potential harmful outcomes of a persistent non-healing wound.

After a vascular intervention, the wound team needs to monitor the healing trajectory and measure the microcirculatory status. The presence of chronic ischemia in the lower extremity results in an adaptive peripheral arteriolar vasodilation. The microcirculation, however, is both structurally and physiologically altered in the ischemic state.⁴⁹ Re-establishing macro flow results in a large volume of blood entering a dysfunctional microcirculation with resulting “revascularization edema”, which further compromises tissue perfusion. Caselli demonstrated that trascutaneous oxygen levels increase over a 4-week period after successful revascularization.⁵⁰

The soft tissue and skin are subjected to the effects of reperfusion injury as described in the cardiac literature. The production of oxygen free radicals and intra-cellular calcium overloading are two mechanisms of action for ischemic-reperfusion (IR) injury. Single episodes of IR can result in myocardial stunning.⁵¹ Stunning refers to the reversible mismatch of perfusion-contraction that occurs despite adequate macro-flow. Extended periods of ischemia can lead to a more permanent condition known as tissue hibernation, or ultimately necrosis.⁵² Another post revascularization process reported in the cardiac literature is the concept of “no-reflow”. The endothelium can become dysfunctional in the revascularization period, leading to cell swelling, leukocyte induced inflammatory responses, in-situ thrombosis, decreased nitric oxide production, vasoconstriction, and possibly distal embolization.⁵³ The reversibility of no-reflow has been dependent on the patient’s risk factors and the device used in the revascularization procedure.⁵⁴ In lower extremity revascularization procedures with non-healing wounds and, in particular, with open guillotine amputations, our team has observed progressive tissue necrosis and a heterogeneous pattern of granulation tissue formation that might be a result of the no-reflow process. In the cardiac literature, distal protective devices, Glycoprotein IIb/IIIa inhibitors, receptor antagonists, calcium channel blockers, and potassium channel activators have all been studied as preventive and therapeutic options for no-reflow states.⁵⁵ Diagnosis of this condition has been improved in the heart with the advent of myocardial contrast echocardiography.⁵⁶ Recently, an index of microcirculatory resistance using a pressure/thermistor wire has resulted in the earlier diagnosis of no-reflow and has opened the possibility for earlier treatment.⁵⁷

These concepts have been adapted to our wound healing/limb salvage model. Our patients have analogous clinical situations. The limb is ischemic (pump) and end organ damage has already occurred (ulcer/gangrene). The distal resistance vessels have compensated through vasodilation and the microcirculation is dysfunctional at baseline. Revascularization results in a large volume of blood entering the microcirculation with resulting reperfusion injury. The increased use of catheter-based approaches to re-establishing blood flow to the extremity likely results in an increase in micro-thrombolic events. Endothelial cell swelling, capillary plugging, and vasoconstriction are noted clinically by advancing tissue necrosis despite debridement, heterogeneous development of granulation tissue, and an absence of wound contraction and epithelialization. Although difficult to monitor through intact skin, the open guillotine amputation patient provides a window to the micro-environment.

Mechanotransduction

In order to preserve limb length, our team has created a treatment protocol that includes revascularization and open guillotine amputation (when digital gangrene exists and the remaining foot anatomy would result in an imbalanced bio-mechanical foot). Patients are treated in a dedicated, 25-bed, in-patient, sub-acute wound center. Negative pressure therapy is used in a large number of cases (VAC^™ KCI, Inc., San Antonio, Texas) Negative pressure has been shown to increase angiogenesis and improve partial foot amputation healing.58,59 The mechanism of action appears to be mechano-transduction.60 The intermittent mode of negative pressure therapy applies a stress/strain mechanism that induces growth factor release, cellular stimulation, and wound healing.⁶¹ Another therapeutic option used is non-contact, low-frequency ultrasound therapy. (MIST Celleration, Eden Prairie, Minnesota). Bertuglia noted an increase in perfusion postischemic reperfusion in a hamster cheek model, using low frequency ultrasound microbubbles.⁶² In another paper, Bertuglia was able to blunt the positive effects of low frequency ultrasound with LNAME (monomnethyl-L-arginine), which is an inhibitor of nitric oxide synthase.⁶³ These results were confirmed in a paper by Hightower et al.⁶⁴ Ennis et al demonstrated an increase in microcirculation in hard to heal wounds using non-contact low frequency ultrasound in leg ulcer patients.⁶⁵ Pulsatile blood flow results in shear forces at the endothelial surface and results in chemical signaling and transduction.⁶⁶ This intermittent pressure is vital to the health of the endothelium. Ischemic tissue does not have the benefit of pulsatile flow and after revascularization, the condition of no-reflow can prevent the transmission of pulsatile pressures to the tissue level. The use of pulsed ultrasound, intermittent negative pressure and other energy-based modalities have a final common pathway that results in forces at the cellular level initiating biochemical changes, including the production of growth factors, Nitric oxide production, and the reduction of oxidative stress.¹⁶

Conclusion

In summary, we are proposing that vascular teams and wound healing units should collaborate on complex limb salvage cases. The location of the wound must be considered when assessing the macrovascular status. For example, a patient with a wound on the lateral ankle may have a patent anterior tibial and posterior tibial vessel but a completely occluded peroneal with poor collateralization leading to poor healing. Simply referring the wound patient to vascular without collaboration is like sending the pathology specimen from an unusual case without discussing what you are looking for with the pathologist. Once the vascular anatomy, patient functional status, risk factors, and potential for healing have been reviewed, the best option for revascularization can be selected. Tissue salvage needs to continue throughout the revascularization period and for 4–8 weeks post procedure to monitor and support the microcirculation. Using the concepts from the cardiology literature, we have adapted our limb salvage program to maximize both the macro and microvascular environment. The clinician can treat the microcirculation through the use of various modalities that can increase angiogenesis and local blood flow to the wound bed.¹⁶ A concept know as the push-pull theory has been presented by the authors as a working theoretical construct.⁶⁷ The push is achieved by the macrovascular-based arterial reconstruction. Other forms of “push” include increasing cardiac output, volume resuscitation, and the use of medications in the treatment of shock. The pull component is achieved through the use of mechanical energy based modalities, which lead to vasodilation and subsequent angiogenesis.^16,64 These therapies “pull” the blood flow towards the microcirculation in a bi-modal pattern, which can be demonstrated through the use of scanning laser Doppler.⁶⁵ The “pull” is essentially created by decreasing peripheral resistance and increasing the quantity of available capillaries, a process known as capillary recruitment. After the initial increase in flow, mediated by nitric oxide release from the endothelium within the microcirculation, a second phase of increased microcirculatory flow is achieved through the process of angiogenesis. Local microcirculatory perfusion can also be influenced by both vasoconstriction and adequate volume status. Noxious stimuli such as hypothermia, stress, pain, and depression can all lead to increased sympathetic tone and subsequent decreased tissue perfusion.⁶⁸ Smoking, through the action of nicotine, can also result in decreased microcirculatory flow.⁶⁹ Several medications, including beta-blockers, have been thought in the past to negatively impact the microcirculation, but with improved imaging techniques, appear to be safe.^70,71 Other medications (pentoxifylline) can be used to augment microcirculation and tissue perfusion.⁷² By using mechanical forces and energy-based modalities in the post-operative period, we can stimulate microcirculatory blood flow and emulate the physical effects of pulsatile flow. We are currently collecting a series of open guillotine amputation patients that resulted in limb salvage for a case series publication this year that confirms these theoretical constructs.

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