New Advances In Predicting Wound Healing

By Stephanie C. Wu, DPM

With the preponderance of wounds and the cost of chronic wound healing continuing to soar, this author offers a provocative look at emerging innovations such as medical hyperspectral technology and real-time polymerase chain reaction that may reinvent the diagnostic workup of chronic wounds. Chronic lower extremity skin ulcerations affect millions of people in the United States alone. These ulcerations are defined by the Wound Healing Society as wounds that have “failed to proceed through an orderly and timely process to produce anatomic and functional integrity, or proceeded through the repair process without establishing a sustained anatomic and functional result.”1 The relapsing course of wound healing poses a significant management challenge to healthcare professionals and imposes an astounding economic burden on healthcare.2    The total direct cost of chronic wounds in the U.S. — including wound diagnostic and surgical procedures, pharmaceuticals, wound closure devices and hospital and physician charges — amounts to an estimated $20 billion annually. Medicare expenditures for lower extremity ulcer patients are, on average, three times higher than those for Medicare patients in general.3-5 In addition to the tremendous medical and financial impact, this prolonged and often interrupted healing process also affects the patient’s quality of life and activities of daily living because of impaired mobility and substantial loss of productivity.2 Moreover, chronic ulcers are frequently associated with infections that may lead to some form of lower extremity amputation or induce life-threatening situations.6-7    Chronic ulcerations may be secondary to a plethora of etiologies including pressure, metabolic, trauma, venous, arterial, diabetes or a combination of the above.7 Treatment protocols for chronic ulcerations include adequate and appropriate debridement, nutrition, infection control, ensuring vascular adequacy and correction of the underlying etiology such as compression for venous insufficiency ulcers and mitigation of pressure for plantar and decubitus ulcers.8,9 Further, clinicians often incorporate the use of advanced wound healing modalities such as bioengineered skin equivalents, acellular matrix based materials, negative pressure wound therapy, hyperbaric oxygen therapy and topical growth factors to help accelerate the healing of chronic wounds.6,10-12 Although these efforts show some degree of success in the healing of both simple and complex chronic ulcerations, a good portion of these wounds remain unhealed.    These clinical observations are consistent with and supported by objective healing rates described in a myriad of published and unpublished industry-sponsored randomized trials.6,13,14 It then stands to reason that the chronic ulcerations that are more recalcitrant to treatment may have unresolved pathophysiologic and metabolic conditions that continue to alter and impair the highly integrated cellular and biochemical processes of healing.5    Accordingly, let us take a closer look at just a few of the latest advances in biomedical technology that may help predict ulcer healing and assist in the management of chronic ulcerations.

Medical Hyperspectral Technology: Can It Have An Impact In Vascular Assessment?

Peripheral arterial disease (PAD) is commonly associated with chronic ulcerations and non-invasive vascular tests are part of the routine workup in this patient population. These tests involve inspection of the limbs, palpation of pulses, ankle brachial pressure index, segmental pressures, pulse volume recordings, toe systolic pressures and toe brachial pressure indexes.15-18 Researchers have also advocated optical techniques such as photoplethysmography, which uses infrared light to assess blood flow to the digits and lower limbs, and transcutaneous oxygen tension measurements for the detection of peripheral arterial disease and prediction of wound healing.19-22    One of the newest tools is a novel diagnostic scanning technique that non-invasively quantifies oxyhemoglobin and deoxyhemoglobin in tissue utilizing medical hyperspectral technology. Clinicians can use this diagnostic tool to help identify microvascular abnormalities and tissue oxygenation, which can help in predicting ulcer healing and assist in the management of chronic ulceration.23    Historically, researchers have used spectroscopy, the study of the interaction between radiation and matter, in physical and analytical chemistry to help identify substances through the spectrum emitted from or absorbed by them. Spectroscopy, in the form of pulse oximetry, has also been commonly used in medicine to assess arterial oxygenation saturation.24 Hyperspectral technology is an advanced form of spectroscopy that provides a multi-dimensional gradient map based on chemical compositions of an area of interest.25 The spectrum of reflected light is acquired for each pixel in a quadrant and each spectrum is subjected to standard analysis. This results in the capture and identification of different spectral signatures that exist in a region of interest during a single-pass evaluation, including molecules with overlapping but distinct emission spectra, and constructs a three-dimensional image cube from the compiled spectral data.25    Hyperspectral technology was first developed by the Department of Defense to assess agricultural field conditions, geological features and suspected chemical weapon areas via satellite. Applying this technology within medicine allows one to monitor the metabolic status of tissues and associated physiologic and pathologic changes.26    Tissues have optical signatures called chromophores that reflect their chemical characteristics.27 The two major chromophores of physiological relevance are oxyhemoglobin and deoxyhemoglobin that enable photoacoustic spectroscopy to quantify tissue oxygen saturation with high spatial resolution, and delineate localized oxygen delivery and extraction within the specific tissue microvasculature.27 This variation in the spatial composition of oxyhemoglobin and deoxyhemoglobin is what creates a mottled appearance of an ischemic limb.    Medical hyperspectral technology enables the efficient collection of oxyhemoglobin and deoxyhemoglobin data from over a million points, producing a spatial, two-dimensional map of the state of tissue oxygenation of the localized tissue of interest. In contrast to pulse oximetry, which provides generalized information of arterial oxygen saturation, the spatial oxygenation map generated via medical hyperspectral technology provides unique localized tissue oxygenation data. This may help prevent the inherent variability and unreliability one would see with tissue oximetry when taking repeated measurements at single sites. It will also provide physicians a better understanding of the varying oxygenation status from oximetry depending on where one takes the measurement relative to the wound, and help better predict healing in patients.

What The Literature Reveals About Medical Hyperspectral Technology

Researchers have also advocated medical hyperspectral technology to help clinicians assess pathologic conditions of localized microcirculation, irritant-induced inflammation, ischemia-reperfusion injury, optically detected cancer and peripheral arterial disease.23,26,28    Khaodhiar and colleagues tested the usefulness of medical hyperspectral technology in 10 patients with diabetes with 21 foot ulcer sites, 13 diabetic patients without ulcers and 14 non-diabetic controls.23 They assessed patients up to four times over a six-month period. Researchers performed measurements of oxyhemoglobin and deoxyhemoglobin at or near the ulcer site as well as in the upper and lower extremity distant from the ulcer. The study authors noted that hyperspectral technology was able to observe changes in the oxygenation/deoxygenation ratio of the immediate peri-wound tissue in ulcers that did and did not heal (P < 0.001).23    The researchers also noted that vascular symptoms correlated significantly with values observed with hyperspectral technology (p<0.01). Sensitivity, specificity and positive and negative predictive values of the hyperspectral technology index for predicting healing were 93, 86, 93 and 86 percent respectively when researchers evaluated images they obtained at the first visit.23    Medical hyperspectral technology may be a useful adjunctive tool to help assess oxygenation of a localized area of interest and help better predict ulcer healing. Completion of further trials in this area will provide further insight into the role of this technology in wound healing.

Are Routine Culturing Techniques Enough?

Chronic ulcerations are often contaminated with bacterial pathogens. These pathogens may form biofilms and prevent ulcers from healing.29,30 Biofilms are ubiquitous, complex structures that consist of microbial-associated cells embedded in a self-produced extracellular matrix of hydrated extrapolymeric substances, which are irreversibly attached to a biological or nonbiological surface.31,32 Bacteria that reside as biofilms are often resistant to antibiotic and immune therapy. Researchers have postulated that the presence of biofilm in chronic wounds may contribute to the intractable inflammatory process and secrete matrix metalloproteinases and their tissue inhibitors to cause local tissue destruction, resulting in wounds that are refractory to healing.31-33    Ngo, et al., examined 12 chronic wound samples they obtained during routine debridement from eight different patients using various microscopy techniques. The authors observed bacterial biofilms in about 60 percent (7/12) of the wounds. The authors also found the necrotic, superficial surface layer of the wounds to be more conducive to biofilm formation than deeper viable tissues. Accordingly, they noted that biofilms may then serve as niduses for continual bacterial seeding that perpetuate the inflammatory process one sees in chronic wounds.32    Bacterial contamination can also develop into bacterial infection, a prime cause of lower extremity morbidity, and frequently leads to wet gangrene and subsequent amputation. The Infectious Diseases Society of America (IDSA) has emphasized the importance of differentiating infection from colonization.34 However, the definition of infection is not an easy one. Cultures, laboratory values and subjective symptoms are all helpful but not definitive, and the diagnosis of an infection’s genesis and resolution has been and continues to be a clinical diagnosis. When suspicious of bacterial contamination and infection, clinicians often take aerobic and anaerobic cultures. These qualitative cultures help identify the bacterial species and antibiotic susceptibility and resistance. Unfortunately, they do not provide much information on the quantity of bacteria. Clinicians rarely pursue quantitative bacterial cultures because counting bacteria is tedious and time consuming.    It is generally accepted that one must treat infection in order to facilitate optimal healing. However, because the definition of infection is arbitrary in many borderline cases, practitioners are therefore basing clinical decisions on culture reports that categorize the bacterial count as scant, moderate or heavy. Edmonds and colleagues demonstrated in a study of clinically uninfected diabetic foot ulcers that antibiotic therapy helped reduce hospitalization and amputation in wounds that lacked overt signs and symptoms of infection.30 Further, Xu, et al., showed that the quantitative measurement of bacterial load correlates with the rate of diabetic wound healing.35

Real Time PCR: Can It Improve The Assessment Of Bacterial Contamination In Wounds?

One of the new modalities one may use to assess the exact degree of bacterial contamination is real time Polymerase Chain Reaction (PCR). Polymerase chain reaction is a commonly used technique to exponentially amplify deoxyribonucleic acid (DNA) via in vitro enzymatic replications. This enables amplification of DNA without any restrictions on the form of the DNA. Accordingly, this allows extensive modification to form a wide array of genetic manipulations to help with the detection of hereditary diseases, paternity testing and diagnosis of infectious diseases.36 One can use PCR to amplify specific regions of a DNA strand. This can be a single gene, a part of a gene or a non-coding sequence.    Quantitative polymerase chain reaction (qPCR) is a modification of the PCR technique to rapidly measure the quantity of DNA, complementary DNA or ribonucleic acid (RNA) present in a sample.37 Since PCR amplifies the DNA exponentially, doubling the number of molecules present with each amplification cycle, one can accordingly calculate the number of amplification cycles and the amount of PCR end product to deduce the initial quantity of genetic material.    The most sensitive quantification methods are done via real time polymerase chain reaction (real time PCR). Real time PCR allows measurement of the amount of DNA after each cycle of PCR with fluorescent markers. Real time PCR enables simultaneous quantification and amplification of a specific part of a given DNA molecule to determine whether a specific sequence is present in the sample. If a specific sequence is present, one can also obtain the number of copies in the sample.    The molecular analysis of bacterial biota that is part of biofilms has recently elucidated thousands of new, previously unidentified bacterial species. While the species found in salt seas and other exotic locations heighten our imaginations, the bacterial flora found on normal skin, normal intestine, the esophagus, stomach, feces and vagina are no less impressive. Real-time PCR allows us to elucidate thousands of species where only a handful were thought to exist previously.33,38-41 A sample of PCR data from normal skin demonstrated that 1,345 clones were readily identified at the species level by molecular methods of PCR amplification of the 16S rRNA genes. When a similar PCR technique was applied to the bacterial biofilms recovered from the human intestine, 1,700 out of the 2,100 clones identified by sequence were previously unknown to have existed and were cultivatable. These data suggest that less than 15 to 30 percent of the bacteria from these sources may be identified or enumerated by routine culture techniques.    The coupling of the quantitative real-time PCR to the problem of enumerating bacterial loads from biofilms will provide a rapid unique tool from measuring the effect of wound treatment. Not only can the time to identify bacterial growth be reduced from three to five days to three to five hours, but the use of the rRNA primers will identify the presence of six times more bacteria than even the best culture techniques.    Real time PCR is carried out in small reaction tubes (0.2-0.5 ml volumes) that typically contain a reaction volume of 15 to 100 µl. These tubes are inserted into a thermal cycler, a machine that heats and cools the reaction tubes to the precise temperature required for each step of the reaction. Unlike northern blotting that required large amounts of RNA and cannot be performed when tissue samples are limited, only a minute sample of approximately 100 µl is necessary for real time PCR. One can easily obtain the sample during routine wound debridement and place it in a vial containing a nucleic acid stabilizer. Further, one may store and preserve the sample at 4°C for up to 30 days.    Scholl’s Center for Lower Extremity Ambulatory Research at Rosalind Franklin University of Medicine and Science is currently quantifying, via real time PCR, the number of bacteria present in samples before, during and after treatment in chronic wounds. Since anywhere from 15 to 30 percent of the bacterial species present on humans have previously been cultivated, this leaves 70 to 85 percent of bacteria that are never cultivated.    While cultivation of these organisms has been problematic, with our current techniques, the enumeration of these organisms is now possible. We coupled the technologies used in the sequence match, which were previously used to identify cloned species to known 16S ribosomal DNA sequences, for assignment to taxon. Using these primer sequences and real time PCR, we can now enumerate 90 to 95 percent of the organisms present in wounds. This allows us to identify 500 to 600 percent more bacteria then previously used techniques.    Real time PCR appears to be a powerful tool for elucidating the clinical microbiology of a chronic disease state. We await the results of our study to help determine the role that real time PCR may play to help assess bacterial contamination and help predict wound healing. If confirmed and widely adopted, real time PCR could be a useful tool in both the clinical and research setting.

In Conclusion

Biological details of the progressive breakdown of the tissue and impediments to healing are not well understood. Researchers have hypothesized that the failure of the chronic wound to heal is multifactorial. Possible factors may include: an overproduction of proteases from inflammatory cells which degrade secreted growth factors that normally drive repair; an inability to adequately respond to hypoxia; defects in angiogenesis; and the apoptosis of cells in the inhospitable milieu of the chronic non-healing wound.    Adequate blood flow to the wound environment, appropriate debridement, controlling exudate and edema, decreasing bacterial burden, and the promotion of healthy granulation tissue are important factors that may facilitate healing.42 However, newer modalities such as medical hyperspectral technology and real time PCR may help give clinicians a closer look at the wound milieu and provide further insight in predicting wound healing.    As technology advances and our understanding of the chronic wound environment increases, we continue to take steps to understanding factors that prevent healing and develop appropriate treatment modalities to heal wounds and improve the lives of our patients. Dr. Wu is the American Podiatric Medical Association/American Diabetes Association Senior Fellow at the Center for Lower Extremity Ambulatory Research (CLEAR) at the William M. Scholl College of Podiatric Medicine at Rosalind Franklin University of Medicine and Science in Chicago.







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