Excelling at equine wound healing

Managing wounds in horses is challenging for horse owners and veterinarians alike. In many cases, equine wounds are slow to heal with the potential for unsightly scars and the production of an excess amount of granulation tissue, commonly known as “proud flesh.”

For the past five years, researchers from the University of Saskatchewan (USask) have been working to determine whether stem cells can help enhance healing for equine wounds.

One team member is Dr. Suzanne Mund, a combined Master of Science candidate and large animal surgical resident at the Western College of Veterinary Medicine (WCVM). Her graduate supervisor, research mentor and the research project’s principal investigator is Dr. Spencer Barber — a WCVM specialist in equine surgery. He has conducted research, written book chapters and spoken at international meetings on equine wound healing for decades.

Barber and Mund are collaborating with WCVM researchers Drs. Ali Honaramooz, Bruce Wobeser, Daniel MacPhee and John Campbell. These team members bring advanced and sophisticated technological skills and knowledge to the partnership.

Previous research has shown that there is a difference between leg and body wounds in horses, and between those of horses and ponies, which is believed to be attributed to the differences in inflammatory reaction. Research in laboratory animals shows that stem cells can alter the inflammatory reaction. Based on these past findings, the WCVM team’s research focuses on differences in wound healing on the horse’s thorax (chest area) compared to equine limbs after stem cell administration.

“We know that in horses, if you take a wound that’s the same size on the leg versus on the body, the one on the leg is always going to heal much slower and it’s not going to be as nice a repair,” says Mund. “What we wanted to do was to give stem cells and see if it could enhance the rate and quality of healing.”

Putting stem cells to the test

Stem cells have potential for improving wound healing because they can alter the inflammatory response, which may be important since the body’s healing response is linked to inflammation. Stem cells can also replicate into cells of repair and produce growth factors that could enhance the speed and quality of wound healing.

The researchers used multipotent mesenchymal stromal cells, which can be easily extracted and are found throughout the body of adult horses. This type of stem cell can differentiate into fat cells, cartilage cells and bone cells. They can also produce various proteins that affect the inflammatory reaction and enhance wound repair and wound closure rates. The team also decided to use allogenic stem cells (cells extracted from another horse).

As Mund explains, using stem cells derived from other horses could allow clinicians to treat a wound during acute inflammation when it may be most responsive to stem cell therapy. Waiting to grow stem cells from the patient may take too long, causing veterinarians to miss a “window of opportunity” for ideal treatment.

The team wanted to explore if stem cells would have a favourable effect on the wounds’ inflammation process. If they did, this would help to decrease abnormal inflammation that may be contributing to slow and irregular wound healing.

The researchers surgically made small skin wounds on the thorax and legs of horses involved in the study. Then, they administered stem cells through intravenous (IV) injection and monitored the horses to see if the cells would travel to the wound site and assist in healing.

“Most other studies have always used stem cells topically or injected them locally,” says Mund. “We gave our horses the most cells through IV that have ever been given to horses, by far. The other highest amount was 50 million, and we gave up to 250 million.”

As the wounds began to heal, the researchers continued to monitor the horses and collected biopsies from both the wounded and non-wounded sites to determine if the stem cells were gathering at the wounded area.

There are risks associated with administering foreign cells into a patient, so the researchers closely monitored the horses after the stem cell treatments for any type of allergic reaction.

“The horses tolerated the cells really well, and we found that the cells did go to the wound site,” says Mund. She adds that no one studying equine wound healing has ever shown this outcome.

In September 2018, the research team published a secondary paper in the Journal of Equine Veterinary Science detailing their work in measuring the gene expression of five inflammation-associated proteins — CXCL8, CXCR2, CXCL10, CXCR3, and β-arrestin-2 — in equine wound tissue.

These proteins have been shown in other research animals to be critical in determining the inflammatory reaction, but they had never been identified in horse wounds before. Barber and Mund’s research team wanted to measure the gene expression of these proteins.

“We’re pretty proud that we were the first ones to do that,” says Mund.

The researchers discovered that gene expression is different for CXCL8, CXCR2, and β-arrestin-2 between limb and thorax wounds, but it remains the same for CXCL10 and CXCR3 proteins. This finding could point to why wounds heal at different speeds in different locations. As well, their identification and the differences seen could open the door to further research in this area.

Mund says the most exciting part of the study was seeing the cells come alive.

“We tagged [the cells] with red fluorescent protein, and then we also transfected [tagged] them with a green fluorescent protein. So, red fluorescence seen when looking at a wound biopsy under the microscope would tell you that the cell made it to the wound site. But then once you start to see green fluorescence, that would tell you the cells were actually alive,” says Mund.

“We saw some green fluorescence on day two, but when you see it definitely on day seven — wow,” says Mund. “Those cells are actually alive, they’re not just dead cells that are stuck there.”

One Health link

Mund says there’s significant interest in this research for human medicine. Part of the research involves exuberant granulation tissue (an excessive amount of granulation tissue), which grows along the edges of a wound and blocks new, healthy skin from growing across it. This is like keloids (raised scars) that develop in the skin of humans — the only other species to suffer from the disorder. Both are related to abnormal inflammatory responses.

“Human medicine has a lot of interest in using stem cells [for wound healing] too,” says Mund. “So, they’re really interested in seeing if this treatment can ‘reset’ the rampant granulation tissue.”

So far, the team has analyzed the data collected from the limbs of horses in the treatment and control groups. They found that stem cell therapy neither improved the speed nor the quality of the wounds’ healing.

Mund points out that negative results are significant, and the team plans to publish their findings once they complete their analysis.

“Publishing negative findings is incredibly important …. It’s putting a reality check on something that may not be the silver bullet that we always thought it was,” says Mund. “No one has done in horses what we have just done, so we have provided substantial information about the use of stem cells administered intravenously to horses as a foundation for further research by us or other researchers.

“Advances in medicine are usually small steps rather than gigantic steps — and our work definitively advances the field of equine wound healing.”

The study’s other highlights included the following:

  • Stem cells decreased inflammation during the early stages in healing. While this finding didn’t make a long-term difference in this project, the study did show that stem cells can modify the inflammatory response at certain time periods. For optimal healing the inflammation needs to be high, while at other times during healing, it needs to be lower. Learning when and how to administer stem cells needs to be determined.
  • Stem cell-treated horses had a decreased amount of new epithelium at the wound sites compared to horses in the control group, and a tendency to have increased wound contraction — suggesting that the stem cell-treated horses may potentially have smaller, less noticeable scars. This area needs further research.

Working on this project has helped Mund discover a love of immunology. She’s also gained a respect for inflammation and its role in healing.

“I’ve learned that inflammation isn’t necessarily a bad thing. So, I don’t think we should always be so quick to stop inflammation — it’s there for a reason. But we also have to balance the horse’s welfare at the same time.”

The Mark and Pat DuMont Equine Orthopedics Research Fund supported this research project, while the preliminary gene expression study and Mund’s salary are backed by the Townsend Equine Health Research Fund (TEHRF).

REFERENCE: Mund SJK, Corbett C, MacPhee DJ, Campbell J, Honaramooz A, Wobeser B, Barber SM. “Identification of mRNA of the inflammation-associated proteins CXCL8, CXCR2, CXCL10, CXCR3, and β-arrestin-2 in equine wounded cutaneous tissue: a preliminary study.” Journal of Equine Veterinary Science. Sept. 2018. 68:51-54.

PROFILE | Dr. Suzanne Mund

Originally from MacNutt, Sask., Dr. Suzanne Mund studied business and psychology at the University of Regina before attending the University of Saskatchewan to complete her pre-veterinary studies. She was accepted in the WCVM’s Doctor of Veterinary Medicine (DVM) program in 2009.

“When I talk to young vet hopefuls, I ask them if they’ve explored their other options because veterinary medicine is a long, hard road you must be passionate about,” says Mund, who completed her veterinary degree in 2013. “But when I look back at little old me, I was going to be a vet — there was no way you could have told me otherwise.”

Mund left Canada for a one-year surgical and ambulatory internship at Brandon Equine Medical Center, a busy equine practice in Tampa, Fla., then returned to the WCVM and began her graduate program in 2014. The stem cell project is the research focus of her master’s degree.

Mund plans to defend her Master of Science thesis in December 2019, and thanks to her research experiences, she’s considering PhD studies in immunology.

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