This article originally appeared in the Fall 2024 issue of EquiManagement. Sign up here for a FREE subscription to EquiManagement’s quarterly digital or print magazine and any special issues.
From improving healing rates and reducing reinjury to producing extracellular vesicles and identifying new target tissues, research continues to forge ahead in the field of regenerative medicine. Since first using stem cells in the early 2000s to treat equine soft-tissue injuries, we’ve amassed an incredible volume of knowledge regarding the collection, culturing, and clinical use of stem cells—particularly in the realm of musculoskeletal injuries.
“Stem cells have largely been researched in musculoskeletal disease with a mounting portfolio of efficacy in soft-tissue injuries, including desmitis and tendinitis,” says Aimee Colbath, VMD, PhD, Dipl. ACVS, Assistant Professor of Large Animal Orthopedic Surgery at Cornell University. “Other studies have shown promise in the treatment of infected synovial structures and for promoting wound healing. In addition, emerging evidence exists for the treatment of neurological disease in other species with some safety studies completed in horses.”
With 20 years of clinical experience under our girths, let’s review where we’re at in terms of treating tendons and ligaments, musculoskeletal issues, and other conditions; cell-less therapy; and new ways of efficiently producing large numbers of stem cells.
Stem Cells for Equine Soft-Tissue Injuries
In early 2024, Colbath and colleagues conducted a systematic review of published studies using stem cells, platelet-rich plasma (PRP), or both, in horses with naturally occurring tendon or ligament injuries. They identified 17 studies and reviewed and analyzed the data to assess return to performance and reinjury. Ten studies evaluated mesenchymal stem cells (MSCs) from a variety of sources, including adipose tissue, bone marrow, blood, and umbilical cord. Five studies included horses treated with a combination of stem cells and PRP.
“Meta-analysis showed that stem cells and a combination of stem cells and PRP had a protective effect against reinjury,” says Colbath.
“I find these results encouraging! The majority of published studies are in superficial digital flexor tendonitis (SDFT). In general, horses with SDFT injury go back to work even with conservative therapy. The biggest issue, especially for horses that do speed activity, is reinjury. Prevention of reinjury is a key aspect of treatment success.”
The meta-analysis did not evaluate the time to return to performance, so it is unclear whether biologic therapies shorten the recovery time, she adds.
“In general, I don’t believe they should be used for a faster return to performance,” says Colbath. “Instead, I believe their power lies in creating a better biologically relevant repair that will (hopefully) maintain ligament and tendon health for future performance.”
Stem Cells for Equine Neurologic Diseases
According to a review article by Colbath and colleagues scheduled to be published in 2024, neural cells have limited capacity for repair. As such, researchers are focusing on regenerative therapies to “augment or replace natural cellular or tissue repair.” Few studies, however, have investigated cellular therapies for neurologic diseases in horses.
Mesenchymal stem cells normally express very low levels of neural factors. However, you can expose MSCs to certain cytokines, or use viral vectors, to push them toward a neural cell-like morphology.
“Other methods of differentiation include exposure to other neural cells such as Schwann cells or astrocytes,” the authors wrote. “Differentiation of equine MSCs into cells of neural lineage was achieved by Villagrán et al. (2014) using nitrogen-coated tissue plates. In addition, neuronal cells have been induced from equine adipose-derived MSCs and bone-marrow-derived MSCs in culture using lentiviral vectors.”
Mesenchymal stem cells are also immunomodulatory, meaning acute inflammatory conditions might be particularly amenable to therapy.
Colbath et al. described one equine study that had disappointing results. That small study involved injecting stem cells around the left recurrently laryngeal nerve (perineurally) in five horses diagnosed as “roarers.” No improvement was noted one, seven, or 28 days following injection.
Nonetheless, stem cells might have the potential to benefit an array of neurologic conditions in horses that are otherwise challenging to treat, with few surgical or medical therapy options. Examples Colbath provided include:
- Spinal cord trauma in wobblers.
- Degenerative neuropathies such as left laryngeal hemiplegia (large population of horses).
- Equine motor neuron disease.
- Damage from trauma.
- Damage from infection (EPM).
- Infections such as encephalitis.
She also mentioned other neurologic conditions we do not yet totally understand, such as shivers or stringhalt.
Extracellular Vesicles and Navicular Disease
Skipping the “cell” aspect of stem cells, extracellular vesicles (EVs) offer an alternate way to provide the benefits of stem cell therapy. EVs are membrane-bound vesicles secreted from stem cells via exocytosis.
“EVs are storehouses of cytokines, growth factors, mRNA, and microRNA with anti-inflammatory and immunomodulatory benefits,” explains Sushmitha Durgam, BVSc, MS, PhD, Dipl. ACVS, Associate Professor of Equine Surgery at The Ohio State University. “These vesicles can secrete trophic factors and also participate in cell-to-cell interactions and protein/peptide delivery to recipient. EVs are secreted by all cell types. As such, stem-cell-derived EVs have the potential to exhibit all the benefits of stem cell therapy without the immunogenicity concerns and laboratory culture lag time, instead providing an off-the-shelf, cell-free therapy.”
To date, equine stem-cell-derived EVs have been isolated and characterized from mesenchymal stem cells collected from bone marrow, synovial fluid, and adipose tissue.
“Anti-inflammatory effects of EVs have been documented in in vitro experimental studies with equine chondrocytes,” says Durgam.
Equine researchers are also investigating EVs to treat podotrochlosis. Safe and efficacious biologic therapies that have been specifically investigated for improving the degenerative pathologies of deep digital flexor tendon (DDFT) and the navicular bone fibrocartilage (NBF) seen in horses with navicular disease are lacking and critically needed, according to Durgam and colleagues (Quam et al. 2024).
To begin the quest to find such therapies, Durgam’s research group prepared DDFT and NBF explant co-cultures from tissues collected from seven healthy horses. Those explant cultures were exposed to culture media alone (control), bone marrow mesenchymal stem cell (BM-MSCs) derived EVs (BM-EVs prepared using standard techniques), EVs alone, and EVs with interleukin-1B (IL-1B). Culture media were measured for IL-6, tumor necrosis factor-a, metalloproteinase-3 (MMP-3), and MMP-13. In addition, sulfated glycosaminoglycans (sGAG) were measured in the media and explants.
One of the key findings in this study was that the levels of metalloproteinase-3, a measure of tissue degradation, were significantly lower in both cultures with BM-EVs, even with the pro-inflammatory mediator IL-1B.
This finding was “the most promising indictor of BM-EV ECM [extracellular matrix] protective effect on the DDFT and NBF tissues,” the authors said.
Similarly, they explained, the anti-inflammatory mediator IL-6 was significantly higher in both co-cultures with the BM-EVs. But, sGAG content of DDFT explants was significantly higher in comparison to NBF explants, which suggests reduced breakdown of sGAG in the DDFT explants.
Thus, BM-EVs in this experimental setting exhibited anti-inflammatory properties and stimulated matrix synthesis.
“In conclusion, this research lays a foundation for future work to evaluate BM-EV as an ‘off-the-shelf’ intrabursal/intrasynovial therapy for horses diagnosed with navicular pathologies,” wrote the researchers. “Further investigations are needed to optimize the BM-EV dose and to assess their function in in vivo experiments prior to testing in naturally occurring disease.”
While this study was specific to tissues involved with navicular disease, EVs have the potential to be used for all musculoskeletal degenerative conditions that would be amenable to stem cell therapy. Osteoarthritis is one such example.
Improving EV Productivity and Yield
Because of the benefits EVs have over using stem cells themselves, Gaesser and colleagues have focused on improving EV productivity and yield. In their 2024 recent publication, Gaesser et al. reported that using monolayer cultures of MSCs is not practical for producing the large quantities of EVs necessary for in vivo use. They therefore created three-dimensional (3D) cultures of BM-MSCs in a stirred bioreactor. Comparing those EVs to EVs produced by monolayer cultures, Gaesser et al. reported that this technique did not improve EV production. Further, some of the markers of stemness were reduced in the 3D cultures. However, the research team did not examine BM-MSc differentiation capacity in this study, so they can’t confirm whether the change in markers of stemness represented a change in the cells’ multipotency. Further, they did not examine the functional changes in the EVs produced by the two culture systems. Thus, it is possible that the 3D cultures could alter the EVs, and miRNA content, etc., could “improve EV function and therapeutic efficacy.”
While they were not able to increase the overall yield of EVs in this particular study, Gaesser et al. said further optimization could enhance the expansion of BM-MSCs to increase yield of EVs over monolayer cultures.
Also looking to increase EV yield from MSCs, Duysens et al. (2024) obtained MSCs from muscle and expanded those cells in a “functionally closed, automated, perfusion-based, hollow-fiber bioreactor.” This system “greatly increases the number of generated cells,” and the expansion of muscle-derived (md) MSCs is “very efficient in this bioreactor.” Specifically, the bioreactor generated 20 times more cells than the initial seeding in nine days. This means if the initial seeding was between 10 and 25 million MSCs, the system is capable of producing 200 to 500 million MSCs in only a matter of days.
“Moreover, all the generated cells kept their stem properties,” the team reported.
They concluded, “It is possible to generate large quantities of high-quality equine mdMSCs for clinical applications.”
Stem Cells to Treat Equine Bone Cysts
Canonici et al. (2023) recently explored using stem cells to treat subchondral bone cysts (SBC), one of the leading causes of equine lameness. SBCs are clinically relevant because they impair normal athletic activity. Standard therapies for SBCs have a prognosis for return to activity ranging from 30 to 80%, the researchers noted. Hoping to improve these rates, Canonici et al. conducted a pilot study exploring the intracystic implantation of adipose-derived mesenchymal stromal cells (ADMSCs) together with PRP to promote the healing process and bone recovery. The study involved a 4-year-old Thoroughbred racehorse that had been treated twice unsuccessfully for a cyst of the medial femoral condyle via cyst debridement. During his third arthroscopic procedure, the cyst was filled with the AMSC/PRP combination following debridement. Twelve months later, follow-up radiographs revealed almost complete healing of the lesion and a favorable clinical response to the treatment (i.e., the horse was no longer lame and had begun a racing career). The researchers therefore concluded, “The use of AMSCs and PRP suggests promising benefits for treating subchondral bone cysts.”
Future Endeavors
These and other studies are scratching the surface of how and when we can use stem cells in equine medicine.
“My group is currently conducting a study looking at the efficacy of stem cells in an experimental model of meniscal disease (funded by Grayson Jockey Club Foundation),” says Colbath.
In the long term, Colbath says off-the-shelf therapy, either allogeneic cells or cell products such as EVs, would be advantageous because autologous cells have a three- to four-week delay.
“Further, I think a very promising avenue that has been worked on by Drs. Pezzanite, Goodrich, and Dow at Colorado State University and the Schnabel Lab is their use in infection,” she adds. “With antibiotic resistance on the forefront of everyone’s minds, using stem cells to harness the bodies’ ability to clear infection is hugely attractive.”
Related Reading
- Orthobiologic Treatment for Horses
- Systemic and Intra-Articular Therapies for Lame Horses
- Managing Arthritis in Horses Using Autologous Blood Products
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