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Analytical Chemistry

Tracking Wound Healing With Raman And Near-IR

Spectroscopic methods give doctors access to information they couldn’t otherwise see

by Celia Henry Arnaud
January 13, 2014 | A version of this story appeared in Volume 92, Issue 2

Credit: Francis Esmonde-White
Blake Roessler demonstrates how a Raman probe could be used to make measurements on the feet of diabetic patients.
A man being examined using a Raman probe (light spot).
Credit: Francis Esmonde-White
Blake Roessler demonstrates how a Raman probe could be used to make measurements on the feet of diabetic patients.

Doctors can’t always determine whether a wound is healing just by looking. By the time they realize a wound isn’t healing, it can be too late. Drastic measures—possibly amputation—might be required.

Spectroscopic methods such as Raman and infrared could give doctors early warning that a wound, such as a diabetic foot ulcer or a burn, isn’t healing as it should. These techniques could also provide real-time information to surgeons as they make decisions in the operating room about what tissue can be saved and what tissue should be removed to prevent long-term complications.

“A chronic wound is one that hasn’t healed after eight weeks,” says Michael S. Weingarten, a professor of surgery at Drexel University College of Medicine and medical director of the Hahnemann Hospital Comprehensive Wound Healing Program. Otherwise healthy people who injure themselves should be able to heal within that time, he says.

Knowing whether a wound is healing is important because too slow progress suggests a change in treatment strategy is needed. Current guidelines from the Wound Healing Society for the treatment of diabetic ulcers, for example, call for reevaluating treatment if the ulcer does not shrink by 40% or more after four weeks of therapy (Wound Rep. Reg. 2006, DOI: 10.1111/j.1524-475x.2006.00176.x).

Doctors typically track wound healing by measuring the length, width, and depth of a wound, Weingarten says. But such methods depend on where and by whom the measurements are being made. They’re notoriously variable.

Spectroscopists hope that their methods will reduce that variability. The goal is to identify spectral signatures that indicate whether a wound is healing.

“You’re typically looking for spectral changes related to changes in proteins’ secondary structures, for lipids that don’t belong there, or the absence of lipids that should be there,” says Michael D. Morris, a chemistry professor at the University of Michigan who is developing noninvasive Raman methods to assess wound healing. “As healing progresses, you’re looking for a return to normal composition. The changes in trauma are sufficiently dramatic that you don’t have much trouble finding changes of 10% or more.”

Spectroscopic methods could be particularly helpful in monitoring foot wounds in diabetic patients. Weingarten and his collaborators at Drexel University College of Engineering are using near-infrared spectroscopy to do just that.

In a preliminary trial of 46 patients with diabetic foot ulcers, Weingarten and his engineering collaborators used diffuse near-IR spectroscopy to measure total hemoglobin, oxygenated hemoglobin, and deoxygenated hemoglobin (Wound Rep. Reg. 2012, DOI: 10.1111/j.1524-475x.2012.00843.x). The researchers analyzed each wound once per week for four weeks. They found that the change in these spectral data over time, rather than the absolute values, correlated with healing.

The spectroscopic data weren’t used for treatment decisions. But during the data analysis, the researchers found that the spectroscopy caught problems that hadn’t been visually apparent. “In about three or four of the patients, I thought they were healing, but the data said no,” Weingarten says. “The data turned out to be right, because three or four weeks later the wounds fell apart.”

Spectroscopy could also help doctors catch a type of bacterial bone infection called osteomyelitis that’s a common complication of foot wounds in diabetic patients. Although it can develop in other circumstances—at the site of an orthopedic implant or as a result of bedsores, for example—it’s typically the result of infection in foot ulcers of diabetic patients. In extreme cases, osteomyelitis can require amputation of a toe or foot.

Morris’s group at Michigan is using Raman to analyze biopsied tissue from patients with suspected osteomyelitis. The work is being done in collaboration with Blake J. Roessler and Crystal M. Holmes of the department of internal medicine at the University of Michigan Medical School. When they analyzed bone samples, they found acidic calcium phosphate minerals, including brashite and noncarbonated hydroxyapatite, says Karen Esmonde-White, a postdoctoral biomedical engineer who works with Morris.

These minerals “are absolutely not found under normal in vivo pH conditions,” she says, because these unstable minerals rapidly convert to carbonated apatite. “The fact that we observed these minerals in all the patients we got a biopsy from says that the wound environment is chronically acidic.”

Those findings have given them insights into how the bacterial infection works. “Our results suggest that the bacterial biofilm is generating a chronically acidic environment that dissolves some of the bone mineral. Then these acidic calcium phosphate minerals are precipitating on the surface of the bone,” Esmonde-White says. “We have a unique marker for these bone infections. We don’t observe these minerals under normal conditions or in any other disease.”

Her team is now trying to determine how early the changes can be detected. “The trend is toward noninvasive, early diagnoses of the ulcer itself. In more than 80% of amputation cases, they’re preceded by an ulcer,” Esmonde-White says. “If we can detect this at the ulcer phase, then we can use less-invasive interventions.” For example, doctors might be able to remove just part of an infected bone rather than amputate.

Another type of wound that spectroscopy could help monitor is burns. “The big deal about burns is that you know there’s a burn injury, but you don’t know how bad the burn is just by looking at it,” says Jonathan R. Peterson, a medical student at Michigan who is working with Morris.

Burns are classified by their depth. A superficial burn, which is restricted to the outer skin layer, the epidermis, will heal by itself. But a deep burn, which damages all three skin layers—the epidermis, dermis, and subcutaneous tissue—typically won’t heal. Current practice is to replace the dead tissue with a skin graft. Superficial and deep burns are both “pretty easy to identify just by looking at a burn,” Peterson says.

The problem is in the middle. So-called partial-thickness burns, which damage the epidermis and part of the dermis, can also be divided into superficial and deep burns, but it’s not visually obvious which is which.

“If it’s superficial partial-thickness, it can also heal by itself,” Peterson says. “If it’s deep, at the beginning it might look like a superficial wound, but it’s not going to survive. There’s nothing you can do to save it. The sooner you get that out and graft it, the much better results you’re going to have.”

Raman might be able to distinguish between these types of burns. As with near-IR, Raman can measure hemoglobin concentration, which reveals whether oxygenated blood is reaching a particular area. The researchers are in the early stages of using Raman in mice to differentiate burns created with branding irons. The results so far are encouraging, Morris says. The work is being done in collaboration with Benjamin Levi and Stewart C. Wang, both of whom are in the department of surgery at the University of Michigan Medical School.

Spectroscopy could also be used to inform decisions in the operating room.

Credit: Jonathan Peterson
Raman is used to monitor burns on an anesthetized mouse. Measurements are made in the center of each square formed by four black dye spots.
An anesthetized burned mouse being examined with a Raman probe.
Credit: Jonathan Peterson
Raman is used to monitor burns on an anesthetized mouse. Measurements are made in the center of each square formed by four black dye spots.

Eric Elster, a captain in the Navy and a professor and surgeon at the Uniformed Services University of Health Sciences in Bethesda, Md., is working with Raman spectroscopists to develop methods that surgeons can use to answer questions before, during, and after surgery. For example, does a patient need surgery? How much tissue should be removed? Is a particular tissue viable? Is a wound ready to be closed?

“These types of assessments of the viability of tissue are currently made with visual inspection of the tissue,” Elster says. Spectroscopy allows us “to use information that’s in front of us that we can’t see with our eyes.”

Surgeons typically look for whether blood reaches the tissue and whether the tissue is being oxygenated. “Both of those are linked to the ultimate viability of the tissue, but there’s not always a one-to-one relationship between them. In fact, some data suggest in certain circumstances they’re not linked much at all,” Elster says.

Blood getting to the tissue isn’t enough. The oxygen has to be released from hemoglobin in the blood and then be taken up by the tissue.

“Raman’s nice because it gives you a molecular signature of the tissue itself,” Elster says. “It starts to get at what the tissue is actually doing with the blood and oxygen that’s being delivered.”

Elster is also using Raman to detect heterotopic ossification, a type of mineralization in soft tissue that often develops at the interface between an amputation stump and prosthesis. About 65% of military personnel with injuries to their limbs develop heterotopic ossification, and 20% of those need surgery to remove it, Elster says.

“Raman can give you this vibrational footprint that shows you mineralization,” he says. “It shows you mineralization before you can see evidence of it visually. That’s important because there are potential therapies for heterotopic ossification that you could implement if you knew earlier.”

One of the challenges is figuring out whether tissue is viable. Nonviable tissue needs to be removed so it doesn’t cause additional problems. “But how much do I remove?” Elster asks. “I don’t want to remove so much tissue in a patient with an amputation that their subsequent rehab is even more challenging. But I don’t want to leave necrotic tissue because that will pose serious issues, both for the tissue locally and for the patient systemically.”

All of these technologies need further development before they’ll be ready for widespread use. Doctors don’t want to look at spectra; they want something that interprets those spectra and gives them the equivalent of a traffic light.

“Surgeons and medical people are not spectroscopists,” Morris says. “Translating the information from what a spectroscopist sees—changes in band positions, intensities, and so on—into a red light and a green light is going to be a major issue.”


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