Veterinary student investigates equine stem cell therapy


University of Georgia veterinary doctoral student receives AAEP grant for work on regenerative medicine.

Lindsey Helms Boone, DVM, a University of Georgia veterinary PhD candidate and surgery resident, received a $5,000 fellowship during the 57th annual American Association of Equine Practitioners (AAEP) convention this past November. The grant will support Boone's endeavors in equine research.

According to the AAEP, Boone's doctoral research involves using equine allogenic bone marrow-derived mesenchymal stem cells (BMSCs) given intra-articularly to treat acute and chronic degenerative joint disease in horses. Regenerative therapy is increasingly used for many equine musculoskeletal diseases, but its precise mechanisms of action, the full potential of its application and the potential for deleterious side effects have not been fully investigated. Boone's research aims to answer many of these questions.

Supported in partnership with the AAEP Foundation and the EQUUS Foundation, the $5,000 grant emphasizes the importance of assisting equine researchers in their exploration of horse healthcare topics.

Boone received her bachelor's degree in animal and veterinary sciences in 2004 from Clemson University and her veterinary degree in 2008 from the University of Georgia. She did a one-year large-animal rotating internship at Texas A&M University in 2009 and is completing a combined large-animal surgical residency and doctoral program in physiology and pharmacology at the University of Georgia.

Regenerative therapies

Regenerative therapies are gaining prominence at veterinary schools across the country, including the University of California-Davis School of Veterinary Medicine (see "Calif. researchers collaborate on animal, human health" in the August 2011 DVM Newsmagazine); the Equine Orthopedic Research Center, Colorado State University; and Cornell University College of Veterinary Medicine.

And the establishment of the North American Veterinary Regenerative Medicine Association has thrust this therapy into the forefront of veterinary medicine. Previous researchers have advocated that "the application of stem cell-based therapies in the horse should be done cautiously, and treatment outcomes (good and bad) should be recorded and reported."1

Equine regenerative medicine has the potential to treat damaging musculoskeletal injuries that lead to limited equine athletic performance, such as tendonitis or desmitis, joint-related pathologies, equine fracture repair and laminitis. The use of stem cell therapy has also shown promise for treating wounds, muscle-related injuries (skeletal and cardiac muscle) and neurologic disease.

Currently, both bone marrow-derived and adipose-derived mesenchymal stem cells are favored in equine regenerative medicine because of the ease of tissue acquisition for cell extraction. Despite the therapeutic potential of these stem cells, there's little objective evidence supporting their benefit to tissue repair, necessitating the need for further research into the optimal cell source and proper treatment regimen (i.e., timing, dose, dosing frequency and route of administration), as well as efficacy and safety of the procedures.

Mesenchymal stem cells for orthopedic injury

Although there's much work being done regarding the use of mesenchymal stem cells for orthopedic injury, most studies are in vitro with relatively few in vivo studies evaluating the use of mesenchymal stem cells in surgically created or naturally occurring musculoskeletal disease. Some concerns that clinicians may have regarding the use of stem cells include their viability, proliferation and ability to renew and regenerate into viable tissue after injection, allowing horses to heal with little scar formation and enabling their return to previous levels of performance.

Successful use of intra-articularly administered mesenchymal stem cells has shown promising results in tissue healing of experimentally produced lesions in dogs and sheep. According to one study of experimentally induced equine femoropatellar joint lesions, in vivo implantation of mesenchymal stem cells within autogenous fibrin plugs yielded increased fibrous tissue deposition that contained predominantly collagen type II.2 However, this lasted only 30 days. But seven months later, the control and treated lesions were indistinguishable.

Researchers at Louisiana State University's School of Veterinary Medicine found that equine BMSCs had superior chondrogenic potential compared with adipose-derived cells in the presence of stimulatory growth factors.3

Besides the viability of stem cells and their ability to convert to various tissue cells for repair (i.e., their pluripotent, self-renewing capability toward tissue regeneration), there's data as to their ability to directly affect the inflammatory process since it appears that stem cell therapy in horses may also provide impact as an immunomodulator.4 Tissue repair and disease improvement as assisted by mesenchymal stem cells has been shown to be related to an individual's immune system. It's been found that mesenchymal stem cells have effects on various immunomodulators (e.g., B cells, T cells, natural killer cells, dendritic cells, macrophages, neutrophils) that can affect the humoral immune response. As they regenerate tissue, stem cells could also help by reducing the immune response, making it easier for them to be effective as opposed to classical tissue grafting or tissue transplantation regarding a concern for tissue rejection.

Boone's AAEP fellow research

Boone explored various aspects of regenerative medicine as a portion of her work, including the research for which she received the AAEP fellowship. As a clinician using various regenerative medicine therapies, Boone had a lot of questions as she injected horses intra-articularly with mesenchymal stem cells.

The basis for her PhD work was her curiosity about what happened to the cells after they were injected into a joint: Where do they go? Do they live in the synovial fluid? Do they develop toward an articular cartilage phenotype? Can they start to produce cartilage if they're exposed to synovial fluid? What's the proper vehicle of delivery?

Boone also became interested in how to manipulate the cells and how to use them in an allogenic manner. She concentrated on two major types of articular disease that the veterinarians at the University of Georgia were treating—osteoarthritis (OA), an ongoing inflammatory process that leads to articular cartilage degeneration, and osteochondritis dissecans (OCD) lesions, or subchondral bone cysts that have abnormalities to their articular cartilage but don't necessarily have the degree of on-going inflammation within the joint.

Originally, Boone wanted to try to meld the two, but she says she is now "very much more interested in the fact that the cells we inject also have immunomodulatory anti-inflammatory properties. Can we use those properties to help modulate the inflammatory environment within the joint and then use them to repair articular cartilage? Is there a way to make them useful in an ongoing OA horse, which I think is much more enticing to people, but also fixing articular defects that are an interest, looking at the inflammatory environment?"

The first project of Boone's PhD work looked at harvesting synovial fluid from horses and examining the survivability, proliferation and chondrogenesis of stem cells in allogenic synovial fluid in vitro.

"If I take cell lines from a group of several horses and add synovial fluid that's not their own, will they survive, will they increase in their numbers and can they promote cartilage?" Boone asked. "We use various levels of synovial fluid, because in vitro in the synovial fluid environment you don't have that ongoing process of replenishment. In vitro, obviously you have a limited amount of growth factors—nutrients that will be depleted over time."

During this first study, she looked at the response during a 72-hour period—in some cases out to five days—to various percentages of media supplementation with synovial fluid up to 100 percent; she introduced only synovial fluid. In this study, she says, "We were using normal synovial fluid, though we're planning on trying to use OA synovial fluid as well."

Originally Boone and her colleagues wanted to look at normal synovial fluid to see if BMSCs injected into that environment would be viable or if some would die or be lost. "Preliminary data seems to show the viability is not altered," Boone says. "The BMSCs survive fine and do seem to slightly proliferate in the synovial fluid. You don't have a sharp die-off of cells."

Boone is now trying to repeat those studies, but it's difficult because you have to harvest quite a bit of synovial fluid, and it takes a lot of time.

"If you inject 20 million cells within a joint, they should have time to find where they need to go and have the nutrients they need until they find another source of nutrients," says Boone. "We think they find those needed nutrients in the synovial membrane due to its vascularity."

The first thing is determining whether they live and whether the synovial fluid pushes them toward a chondrogenic phenotype. "Our data is preliminary," Boone cautions.

In the clinic, Boone and her colleagues tend to inject BMSCs in platelet-rich plasma (PRP) into joints. "I was interested in using allogenic stem cells and using autologous PRP. What I wanted to know: Was there any difference between use of allogenic PRP and autologous PRP and its introduction to stem cells on the viability, chondrogenesis and proliferation?"

The variance of PRP work is the basis for the grant she was awarded—the difference between using allogenic vs. autologous PRP with BMSCs.

Boone's hope is to advance her studies, to take her allogenic work into a model system—either a joint-defect or OA model—and look at allogenic bone marrow-derived stem cells. She's unsure if she'll be able to look at the model studies, though, because of financing issues. Maybe another grant or fellowship is in the future?

Ed Kane, PhD, is a researcher and consultant in animal nutrition. He is an author and editor on nutrition, physiology and veterinary medicine with a background in horses, pets and livestock. Kane is based in Seattle.


1. Koch, TG, Berg LC, Betts DH. Current and future regenerative medicine—principles, concepts, and therapeutic use of stem cell therapy and tissue engineering in equine medicine. Can Vet J 2009;50(2):155-165.

2. Vidal, MA, Robinson SO, Lopez MJ, et al. Comparison of chondrogenic potential in equine mesenchymal stromal cells derived from adipose tissue and bone marrow. Vet Surg 2008;37(8):713-724.

3. Frisbie, DD, Smith RKW. Clinical update on the use of mesenchymal stem cells in equine orthopedics. Eq Vet J 2010;42(1):86-89.

4. Peroni, JF, Borjesson DL. Anti-inflammatory and immunomodulatory activities of stem cells. Vet Clin North Am Equine Pract 2011;27(2):351-362.

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