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Wound Biomarkers: Measuring What We Manage
Acute wounds heal by a progression through a complex, but orderly, series of physiologic and molecular processes. In contrast, chronic wounds, those that fail to heal within 30 days, are characterized as having stalled in this healing progression due to a variety of systemic and local factors. There is an increasing number of nonhealing wounds worldwide leading to significant morbidity and mortality with abounding economic ramifications. It is estimated that even with specialized wound care in hospital-based outpatient wound centers, over one-third of patients may never have resolution of their wounds.1
Wound care providers and researchers have invested heavily in understanding specific biologic mechanisms contributing to delayed wound healing. It is widely accepted that wound healing is composed of an intricate process of sequential and overlapping phases of hemostasis, inflammation, proliferation, and remodeling.1 Intercellular, intracellular, and extracellular communications simultaneously occur within the wounded tissues to signal and support this multifaceted process.2 In certain instances, local, physiologic conditions and systemic factors exist that cause delays in tissue healing and contribute to wound chronicity.
As the population increases in age, so does the prevalence to develop chronic diseases such as diabetes, vascular impairment, inflammatory conditions, kidney disease, and malignancy.3 These conditions often result in impaired collagen deposition, lack of angiogenesis, and delayed infiltration of inflammatory cells.4 The resultant paucity of functioning macrophages and lymphocytes impacts host resistance to infection.4
Additionally, many of these same patients are on multiple concurrent medications. Non-steroidal anti-inflammatory drugs (NSAIDs), chemotherapy, immunosuppressive drugs, and corticosteroids are just a few agents known to disrupt the healing cascade.5 A subsequent failure to regenerate tissue because of comorbidities or concomitant medications results in ineffective restoration of the structure and functional integrity of the skin. Abnormalities in tissue regeneration leads to the formation of non-healing wounds including diabetic foot ulcers (DFUs), venous leg ulcers (VLUs) and pressure injuries (PIs).
The Basics of the Wound Healing Process
Wound healing is a complex process requiring multiple cellular and biochemical processes taking place at the same time. The wound healing cascade has been intently studied in the past few decades leading to a clearer understanding of what factors support tissue repair and what factors inhibit wound healing. Elevated protease levels have demonstrated deleterious effects on tissue healing by leading to chronic inflammation and inhibitions in cellular replication and migration.6 Further sustaining this inflammatory state, the polymicrobial microbiota of wounded tissue can tip from contaminated to critically colonized causing an inundation of proinflammatory cells leading to a decrease in the body’s response to infection and prolonging healing.6 Tissue regeneration is hampered when abnormalities in growth factors elicit diminished neovascularization and angiogenesis. As a result, the tissues will exhibit inadequate oxygenation and downregulation of necessary nutrients.6 Additionally, the pH of the wound can directly and indirectly impact many biochemical reactions that support wound healing.6 A change in wound tissue temperature has been shown to be a prognostic indicator of wound healing or infection.6 Tracking any one of these processes can potentially help to direct diagnosis and monitor healing progression in the patients we treat.
So, as wound management clinicians, we need to ask the question: how can we better measure what we manage?
Historically, wound assessments, biological indicators, and biomarkers have been used to identify and stratify subsets of healing and nonhealing wounds of varying etiologies. A biomarker is an objectively quantifiable substance or metric that can indicate the presence of a normal or pathophysiologic process.7 Predictive biomarkers can be useful in forecasting patient outcomes and therefore direct the most appropriate treatment pathway. Diagnostic biomarkers can help determine what physiologic parameters are influencing wound etiology and chronicity. While indicative, biomarkers are useful in monitoring disease progression or response to therapy. Determining which biomarkers are most clinically applicable in wound management involves investigating and validating factors that were thought to have effects on wound healing or inhibition. Examination of tissue samples revealed the first molecular markers correlating with healing interruptions. More recently point-of-care imaging technology has entered the wound management space allowing additional biomarker data to be collected at bedside providing health care providers a more complete clinical picture of each individual wound.
How Matrix Metalloproteinases Impact Wound Healing
Matrix metalloproteinases (MMPs) are critically important enzymes found in every phase of wound healing.7 During the healing cascade fibroblasts, keratinocytes, endothelial cells, and inflammatory cells synthesize and release MMPs.7 Tissue inhibitors of metalloproteinases (TIMPs) exist to balance MMP activity.7 Ideally MMPs function to remove damaged structural proteins like collagen to clear the way for fibroblasts to lay down the new scaffold supporting regeneration of the extracellular matrix.8 As fibroblast activity increases, the MMP level should taper off.7 This process is moderated by TIMPs. In chronic wounds, MMP levels remain high and TIMP levels are low, thus resulting in fibroblast inactivity and disorganization of the extracellular matrix (ECM).8 Increased levels of protease activity in nonhealing wounds have been shown to correlate with impaired wound healing.8 Previous studies have indicated that elevated protease activity (EPA) may be present in up to 28% of all chronic, nonhealing wounds.9
Therefore, MMP levels can be used as a predictive biomarker to aid in wound management. The key to MMP utility in wound healing is having the right amount, at the right location, for the right duration of time. Elevated levels of MMPs are often found in wounded tissues of prolonged duration. Unregulated levels of MMPs lead to dysregulation of the wound microenvironment and degradation of the extracellular matrix thus contributing to the chronicity of nonhealing wounds.8 It would stand to reason that rapid, point-of-care testing to allow clinicians to obtain information on the type and level of MMPs present in the wound tissue could be valuable.
These findings have given rise to the development of tools capable of measuring the presence and levels of MMPs in wound fluids. By utilizing this point of care technology to identify wounds with EPA, health care providers can identify potential problems earlier and develop care plans that leverage the most appropriate therapeutic regimens.
The WOUNDCHEK protease status swab (Woundcheck Laboratories) can detect elevated levels of active human protease in chronic wound tissue. This test detects elevated serine proteases such as neutrophil elastase and matrix metalloproteases.10,11 Results are available in 15 minutes. This information can drive patient-specific therapy to modulate harmful protease activity. Evidence is available to support that certain therapies can reduce MMP levels. One such product is a collagen/oxidized regenerated cellulose dressing, which can bind MMPs, thus reducing tissue levels to support tissue regeneration.12 A better understanding of the underlying mechanisms which lead to EPA may give rise to additional innovations such as molecular-based therapies.
How Wound Microbiota Can Predict Healing
Another biological marker that has utility as a predictor of wound healing is tissue bacterial levels. In humans, the microbiome is composed of an array of microorganisms with symbiotic and pathogenic predilections. Once intact skin is wounded, the natural barrier system is compromised, and bacteria from the environment or the surrounding tissue flora can infiltrate and colonize.13 Wound healing becomes potentially compromised once bacteria have invaded. High levels of bacteria in the tissues impair the wound healing process by contributing to sustained inflammation that stalls wound regeneration and epithelialization leading to chronicity.13
To date little is known about the exact mechanism of colonization and development of the human cutaneous (or skin) microbiome. However, it is known that a microbiome consists of a complex mixture of viruses, bacteria, and fungi.14 The complexity of this microbiota has led researchers to believe that wound colonization is mostly polymicrobial.14 It has been theorized that the shift from microbial colonization to clinical infection may not be based solely on microbial load but on other more complex host factors including patient comorbidities such as diabetes. Chronic wounds often contain high levels of polymicrobial bioburden susceptible to forming biofilms.14
Mature biofilms occur when an assemblage of surface-associated microbes becomes enclosed in a self-produced polysaccharide matrix. Identifying and managing bioburden and biofilm have recently become some of the most studied aspects of wound care.15 The US Centers for Disease Control and Prevention and the National Institutes of Health have estimated that biofilms cause between 65–80% of infections.16 Numerous studies have shown that traditional wound cultures only select for microbes that survive under the conditions conducive to a laboratory setting and therefore do not represent the diverse microbiota present in chronic wounds, especial bacteria residing in biofilm constructs.15 Thus, biofilm management is an important aspect of successful wound care regimens. It can, however, be challenging to identify those wounds having high levels of pathologic bacteria due to the paucity of clinical signs and symptoms of wound infection, especially in immunocompromised patients.15
A handheld, non-invasive, point-of-care imaging fluorescence imaging device can instantly visualize potentially harmful bacteria on the wound surface and surrounding tissues not otherwise visible to the naked eye. The device emits a violet light (405 nm) that illuminates the wound and surrounding area, exciting the wound tissues and bacteria and resulting in endogenous production of fluorescence signals,3 without the need for additional contrast agents.17 Optical filters built into the device remove non-informative colors, without any digital processing, and the resulting image is viewed on the display touch screen in real-time.18 The fluorescence signals produced are tissue specific: endogenous tissue components such as collagen will fluoresce green, while clinically relevant bacteria producing metabolic byproducts like porphyrins fluoresce red, and pyoverdine fluoresces cyan (blue-green).18
The MolecuLight i:X (MolecuLight) has been extensively validated in preclinical and clinical studies involving patients with chronic wounds.19 Clinical trials have shown that endogenous, red fluorescent porphyrins emitted from bacteria allow the visualization and location of bacteria present at loads > 104 CFU/g.19 The device has been noted to detect these fluorescent bacterial byproducts on and beneath the surface of wounds, up to ~1.5mm depth.19 It should be noted that numerous porphyrin-producing bacterial species can colonize on chronic wounds and cause a red fluoresce, but Staphylococcus aureus is the most commonly found bacterial species.18,19 Pyoverdine is unique to Pseudomonas aeruginosa; thus, it is the only bacteria to fluoresce cyan.19 The information captured in the images can serve as a biomarker of wound critical colonization. Decision making and treatment algorithms can be adjusted throughout the dynamic wound treatment pathway, determining the need for additional debridement or antimicrobial therapy.20
What Wound pH Reveals About Wound Status
The pH value is that of the negative logarithmic scale for the concentration of H+ ions in solution, in this case the extracellular environment (pH+-log[H+]).21 The range of pH is from 0 to 14. At various pH levels, chemical reactions will be optimized. Many factors, both endogenous and exogenous, can affect the pH.21
Wound pH is a valuable biomarker as it serves as a determinant of metabolic processes throughout the wound healing cascade. It also a parameter that can be monitored to prove the effectiveness of therapeutic interventions. Investigators have tracked the role of pH in wound healing and its effects on the ECM and biofilm formation. The pH value of intact skin, acute wounds and chronic non-healing wounds varies greatly. Chronic wounds have been found to have a slightly alkaline pH and healthy skin exhibits a pH that is slightly acidic.22
Wound pH also plays a critical role in tissue repair and regeneration. At various levels, pH affects a host of important cellular processes such as enzyme activity and growth factor production.22 Pathogens such as Staphylococcus aureus and Pseudomonas thrive in basic environments. When pH levels are not optimized, the wound remains in a prolonged state of inflammation, protease levels increase, thus resulting in destruction of the ECM.22 Additionally, pH can affect the ability of antiseptics to decrease microbial contamination, thus leaving these products ineffective in bioburden and biofilm reduction.22
It has been demonstrated that wound pH reduces as healing progresses.22 Therefore, wound pH can be useful in predicting the likelihood of wound healing. Knowing and tracking wound pH could be used as a simple tool to quickly identify nonhealing wounds. This information can support clinical decision making and help health care providers determine what clinical interventions are most appropriate. By using therapeutics that create an acidic wound environment, tissue repair and regeneration could be optimized.
Several studies have shown that certain dressings can alter wound pH values. When non-permeable dressings were used to treat chronic wounds, investigators found that wound secretions became more acidic as compared to those treated with permeable dressings.23 Another trial showed that treating a wound with a solution at a pH of 7.3 was more effective than when chronic wounds were treated with a solution having a pH of 6.0.24 Employing specialized wound dressings to create a slightly acidic pH the healing trajectory of chronic wounds can be altered.
Tissue Oxygen as a Biomarker for Wounds
Despite the critical need for oxygen, levels are frequently insufficient in patients with chronic wounds due to a variety of systemic disease states causing poor circulation, inactivation of growth factors, and cellular senescence.25 Low levels of oxygen in the wounded tissues will prolong tissue regeneration.25 Thus, determining the oxygen level in wounded tissue can be a valuable biomarker to track throughout the phases of wound healing. This data can assist health care providers in identifying the healing potential of the wound.
Healing wounds characteristically have a limited inflammatory phase, high cellular mitogenic activity, robust neovascularization, intact functional extracellular matrix, strong new collagen deposition, and increased tissue tensile strength, whereas chronic wounds display a prolonged inflammatory phase, low mitogenic activity, a dysfunctional matrix, senescent cells, and excessive scarring and contracture.25 Oxygen levels in the tissue are a major contributory factor in determining the wound’s clinical pathway. Hypoxia is a common limitation to healing in chronic wounds of varying etiologies. Maintaining adequate levels of oxygen in wounded tissues continues to be a challenge.
The oxygen gradient in wounded tissue is unequal. Supply may not meet demand. This is especially true in patients suffering from systemic conditions such as diabetes, arterial disease, and venous disease. Although the etiology of nonhealing chronic wounds is multifactorial, hypoxia is a common component in a vast majority of cases. From inflammation through tissue remodeling, oxygen plays a key role in the essential stages of wound healing.25 The formation of the stable collagen fiber triple helix is O2 dependent.25 Without oxygen, the hydroxylation of the proline and lysine side chains do not allow for the proper assembly of the triple helix structure and the resulting procollagen is non-functional.25,26 It is these extracellular cross-linkages that are ultimately responsible for the tensile strength needed in prolonged wound healing.25,26
Near-infrared spectroscopy (NIRS) is an emerging technology that has been successfully used to evaluate functional tissue oxygen saturation in the management of diabetic foot ulceration.27-29 One such commercially available NIRS device is the SnapShotNIR (Kent Imaging). This non-contact device is hand-held, mobile and offers repeatable immediate images that can be used to determine site-specific quantifiable levels of tissue oxygenation. This diagnostic tool utilizes differing optical signals based on the proportion of oxygenated hemoglobin found within the tissue capillary bed. The images obtained allow clinicians to get a better idea of microcirculation and functional blood flow to the wound as well as the surrounding tissues. When low tissue oxygen levels are detected, it stands to reason that supplying supplemental oxygen to these wounds may promote healing.
How Measuring Tissue Temperature Can Reveal Wound Status
Systemic or local temperature abnormalities can be noted in tissue caused by ischemia, trauma, inflammation, or infection. Using temperature as a biomarker can alert health care providers of clinically significant events prior to systemic indicators.
Thermal energy cannot be detected by the naked eye. It has a much longer wavelength than visible light. Infrared thermography is a diagnostic technology that lets clinicians and patients instantly visualize and verify thermal energy. Objects with a temperature above absolute zero emit heat. Even extremely cold objects such as ice will emit infrared (IR). The greater an object’s temperature, the more intense IR radiation emitted. Infrared allows for the ability to appreciate what the naked eye cannot detect. Infrared thermography cameras produce images of invisible infrared radiation based on heat and provide precise non-contact temperature measurement capabilities. Because heat sensed by an infrared camera can be very precisely quantified, new applications for infrared cameras continue to emerge. One of the largest areas of growth appears to be in the health care sector.
Thermal imaging consists of comparing images obtained on both limbs and perform an asymmetrical analysis by subtracting mean temperature of the nonaffected limb from the corresponding value of the affected one.30 Long-wave infrared thermography can measure radiant heat from a body surface and has long been accepted as a valuable adjunct to standard investigations in the early detection of inflammation and infection.30
Studies have shown that when lower temperatures were found in the periwound skin of pressure injuries versus the wound bed, poor wound healing was noted.31 Other studies have shown that infrared thermography has proven useful as a quantitative measure to detect wound infection.32 With the absence of infection, investigators found the mean temperature difference between the periwound temperature, and the contralateral control site was less than 2ºF.32 When infection was present, the mean periwound temperature became elevated by more than 2ºF.32 There is mounting evidence to support the utility of employing infrared thermography to predict wound prognosis better than visual assessments alone.
In Conclusion
As wound care providers, we are always in search of tools that will allow us to better measure what we manage. The development of diagnostics is slow, and uptake of use is even slower. Early detection of wounds that are not on a healing trajectory and may not have success with standard of care therapies will improve patient outcomes and save health care dollars. There are several promising tools now available to detect and track biomarkers having known predictive value. Access to immediate, bedside, point of care testing tools in offices and clinics will support the development of effective treatment algorithms personalizing care for each individual patient.
Windy Cole, DPM, has practiced in Northeast Ohio for over 22 years. She is an Adjunct Professor and Director of Wound Care Research at Kent State University College of Podiatric Medicine. She is board certified by the American Board of Foot and Ankle Surgery and the American Board of Wound Management. Cole is a member of the American College of Clinical Wound Specialists Board of Directors. Additionally, she holds multiple advisory and editorial positions with various medical and wound care publications, and sits on the advisory board of multiple emerging biotech companies and has been integral in collaborating on innovative research protocols in the space.
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