The field of regenerative medicine is a unique, innovative and blossoming branch of human and animal healing. By definition, “regenerative therapies” is an umbrella term that includes treatments aimed at regenerating or replacing injured, diseased or defective cells, tissues or organs to restore or establish function and structure. Such therapies have already instilled themselves into virtually all levels of equine practice, and veterinarians can now choose from an array of stem cells, platelet-rich plasmas (PRP), autologous conditioned serums (ACS, interleukin-1 receptor agonist protein/ IRAP) and autologous protein solutions (APS).
“All of these products are thought to exert their therapeutic benefits by increasing the concentration of anti- inflammatory proteins and growth factors at the sight of injury,” said Kyla Ortved, DVM, PhD, DACVS, DACVSMR, the Jacques Jenny Endowed Term Chair of Orthopedic Surgery at the University of Pennsylvania’s New Bolton Center. “This can help direct healing of the injured tissue to yield a superior final product compared to what would be produced by the normal healing process.”
“Products with concentrated interleu- kin-1 receptor agonist (IL-1Ra)—such as APS and ACS—also work through specific blocking of the pro-inflammatory IL-1 signaling pathway,” she added. “Stem cells also have additional therapeutic potential in that they can engraft in injured tissue and have some limited ability to differentiate into tissue-specific cells. They also help recruit endogenous progenitor cells to participate in tissue healing.”
According to a survey published earlier this year in Veterinary Surgery, equine practitioners already embrace each of the four above-mentioned biological therapies, with PRP actually being the most popular (Knott et al. 2022).
As the basic theories and principles related to stem cells, PRP and ACS (IRAP) are relatively widely understood already by equine practitioners, this article instead focuses on some of the newer data and information in the field of regenerative medicine. This includes describing APS and exploring extracellular vesicles (EVs) derived from stem cells that are currently being developed as “cell-less” therapies. “Other” applications for regenerative medicine are also briefly discussed.
Current Use of Available Regenerative Therapies
In the above-mentioned survey by Knott et al., 154 diplomates of the American College of Veterinary Surgery and American College of Veterinary Sports Medicine and Rehabilitation provided information on the biologic therapies they used for musculoskeletal disease. Of those, 87.5% of respondents indicated they had used PRP. ACS was the second-most-popular (79.7%), followed by bone marrow-derived mesenchymal stem cells (BMDMSCs, 72.1%), APS (46.2%) and adipose-derived mesenchymal stem cells (ADMSCs, 19.5%).
“I wasn’t surprised that PRP was the most utilized biologic therapy in the surveyed population,” said Aimee Colbath, VMD, PhD, DACVS, from the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, and one of the authors of the published survey. “PRP is a well-known biologic therapy that is easy to process both in the hospital or stall-side in the field. Plus, several publications support its use in tendon and ligament disease.”
New Kid on the Block: Autologous Protein Serum
The autologous protein serum (APS) used in equine medicine is modeled from a system used in human medicine for the treatment of osteoarthritis. It has been available to equine practitioners for about six years.
“Creating APS, an autologous blood product, is a two-step, patient-side process,” explained Ortved. “The first centrifugation step concentrates platelets to harness the healing benefits of PRP, while the second centrifugation step concentrates IL-1Ra as well as other growth factors and anti-inflammatory cytokines. Thus, many people think of APS as a combination product of traditional PRP and IRAP/ACS.”
According to Colbath’s survey, just under half of all board-certified respondents reported using APS. Intra-articular administration is by far the most popular route of administration, being used for both acute and chronic diseases of high- and low-motion joints, as well as for preventative joint care. APS is also used in postoperative settings
for both cartilage injury and meniscal disease.
In addition, APS can easily be administered intralesionally for tendonitis; however, limited evidence supports this practice.
Ortved and colleagues recently conducted a study evaluating APS in an experimental model of superficial digital flexor tendonitis (SDFT) induced in both forelimbs via collagenase injection. Seven days after collagenase administration, one limb of each of the included eight horses was subsequently injected with APS, while the other was injected intralesionally with saline. A commercial kit was used according to the manufacturer’s instructions to generate the APS.
Ultrasound examinations were performed intermittently throughout the study, starting one week prior to collagenase injection. Histological and biomechanical data were collected 12 weeks post-treatment, and DNA quantification was performed.
“Collagenase injection successfully induced a core lesion in the SDFT of all treated limbs,” said Ortved. “The only statistically significant differences in any histologic, biomechanical, biochemical and genetic analyses was a decreased expression of collagenase III in APS-treated tendons and an increase in total DNA content in saline-treated tendons. The other major finding was that APS-treated tendons had a higher elastic modulus (103 MPa) versus saline treated (79.55 MPa); however, this was not statistically significant.”
Collagen type III is typically appreciated during the early phases of natural tendon healing. Compared to collagen type I, the typical form found in normal healthy tendons, collagen type III has less elasticity. Ortved thinks that the increased collagen III and decreased elasticity might translate into inferior biomechanical healing, which helps explain why reinjury is so common and why tendon healing takes a prolonged period of time.
In terms of the increased total DNA content in saline-treated tendons, Ortved suggested that those tendons were still in the active phase of healing.
“Increased DNA content correlates to increased cellularity,” she explained. “Tendons that are still in the healing phase will be hypercellular. As healing moves toward completion, they return to a more hypocellular (normal) state.”
Further studies on APS are warranted to determine the role of this orthobiologic in tendon healing. Those studies should also assess whether or not APS offers any advantage over other regenerative therapies, such as PRP, which
is more commonly used for soft tissue injuries. Ortved said she feels those studies would ideally be performed in horses with naturally occurring disease; however, she acknowledges the inherent challenges with such models.
Regenerative Therapies: Limitations and Concerns
Colbath and colleagues relayed in their survey that the most common adverse effects and limitations to use of regenerative therapies were local inflammation at the injection site and cost of the products, respectfully.
The survey reported that between 55% and 86% of diplomates experienced local inflammation in patients when using regenerative therapies. Infections at the injection site also occurred; they were reported by 2.5-8.9% of survey respondents. (The range is because infection rates varied between different therapies.)
In reality, the process of collecting and administering any of the various regenerative therapies can present safety or health concerns anywhere in the process from collection to administration.
“I think the most common complication of regenerative medicine is a lack of beneficial effect,” noted Ashlee Watts, DVM, DACVS, equine orthopedic surgeon and director of Texas A&M University’s Equine Orthopedics and Regenerative Medicine Lab in College Station. “While the horse is not harmed, they also have not benefited from the therapy. We could therefore argue that the chance for other complications, such as infection, is not worth the risk.
“In other words, the chance of benefit is lower than the admittedly low chance of infection. Infections at the site of collection—for bone marrow aspiration, for example—can occur just like an infection can occur at any site where there is a needle puncture or incision; but it is rare.”
In terms of discomfort, Watts said that she assumes there is “some pain” associated with collecting samples, even if it is just the mild discomfort one would expect during and possibly even after venipuncture.
“We performed a study to assess discomfort during bone marrow aspiration and could not find signs of pain or distress, even based on salivary cortisol levels,” said Watts, referring to her 2018 article published in Frontiers in Veterinary Science. “So, despite the widespread belief that bone marrow aspiration is exquisitely painful, we could not confirm this allegation in the horse.”
For practitioners having some anxiety about trying bone marrow aspirates for the first time, particularly due to potential adverse effects, Watts assured veterinarians that the technique is “ridiculously easy once we’ve learned it.”
Allogenic vs. Autologous: According to Watts, another major safety factor to consider when using regenerative therapies pertains to allogenic versus autologous treatments.
“Regenerative therapies have the unique added risk for adverse reaction when they are not autologous because the body may react negatively to foreign (non-self) proteins,” said Watts.
In addition, Watts’ research has shown that allogenic stem cell therapy might fail due to lack of cross-matching between donor and recipient. This is similar to an organ transplantation in people, where the donor and the recipient must be a match for the organ transplant to succeed.
“MSCs have low levels of major histocompatibility complex (MHC) II and secrete immunomodularity agents that regulate B and T cell functions,” explained Watts. “These features led to the belief that they could be used allogenically without cross-matching. However, my research shows that allogenic MSCs are recognized by both the innate and adaptive immune systems, leading to rejection and destruction of the allogenic MSCs.”
Within the first few days of transplantation, about 90% of MSCs due. Watts said this is because of the presence of donor-specific antibodies in the recipient that kill the allogenic MSCs.
“MSCs were once thought to be immune privileged,” Watts said. “This widely accepted theory may in fact not be true, and this lack of donor/recipient ‘match’ may explain why we have yet to prove efficacy of MSC therapy.”
The importance of cross-matching stem cells was demonstrated in a recently published study by Watts and colleagues (Rowland et al. 2021). In that study, horses were treated with either matched (i.e., MHC haplotype) or unmatched MSCs in a metacarpophalangeal joint. For control, horses received either saline or autologous MSCs.
Evaluation of synovial fluid samples from treated joints demonstrated increased periarticular edema and synovial effusion, increased concentrations of chemokines and cytokines, and the presence of donor-specific antibodies in mismatched recipients. Further, recipients receiving matched MSCs had increased MSC concentrations in synovial fluid, demonstrating survival of transplanted cells.
“Clinically speaking, using allogenic stem cells—which is a common practice—without cross-matching may be the Achilles’ heel for reliable and predictable MSC efficacy,” summarized Watts. “Until we have the ability to cross-match donor and recipient stem cells, autologous MSCs would be safer and, likely, more efficacious.”
In the meantime, if allogeneic MSCs are being used, Watts recommended only using allogenic stem cells from a particular donor or donor type one time.
“The recipient’s innate immune system will recognize and attack the MSCs, but at least it will be unlikely that the recipient will already have antibodies ready to attack the MSCs,” she said. “Or simply stick with autologous regenerative therapies.”
Donor Horse Factors: Regenerative therapy researchers also suggest that the overall health of horses for either autologous or allogenic regenerative therapies also needs to be considered. For example, Alicka et al. (2020) relayed that the reparative/regenerative potential of stem cells can be diminished by both age and metabolic disorders. They noted that senescence causes lower cell proliferation and viability, dramatic mitochondria deterioration and insufficient protection against oxidative stress. Senescent cells also secrete proinflammatory and matrix-degrading factors.
After collecting subcutaneous fat from the bases of the tails of horses aged 1-23 years old, stem cells were evaluated for proliferation, morphology and structure, mRNA and miRNA expression profiles, and reactive oxygen species (ROS) accumulation. Results were compared between horses <5 years of age, 5-15 years of age, and >15 years of age.
Adipose-derived stem cells from middle-aged and older horses exhibited typical senescence phenotypes—an increased percentage of cells in G1/G0 cell cycle arrest, binucleation, increased expression of proinflammatory cytokines and miRNAs, and apoptotic markers. In addition, lower mRNA levels of genes involved in stem cell homeostasis and homing were appreciated, as well as reduced levels of glucose transporter-4 and insulin receptor, indicating impaired insulin sensitivity.
That research team therefore concluded that horses >5 years of age have reduced functions of adipose-derived stem cells, including cell survival, homeostasis and proliferation activity. These limit their regenerative capacity. Similarly, Marycz et al. (2016) reported that compared to healthy horses, adipose-derived MSCs from horses with equine metabolic syndrome had:
- senescent phenotypes and increased apoptosis;
- increased nitric oxide and ROS as well as reduced superoxide dismutase activity; and
- impaired mitochondria.
While these and similar studies bring to light important point to consider regarding donor selection, Watts said that we do not have enough data at this point to state which horses are good donors or that old horses are bad autologous donors.
“Given the immune recognition data we do have, it is likely safer and more effective to stick with autologous cellular therapy even if the horse is older,” said Watts. “When deciding whether to use autologous or allogenic transplants, it
is important to consider whether the potential benefits outweigh the potential risks. The veterinary practitioner should ask themselves, ‘If an allogeneic cell is going to be destroyed and ineffective, is it prudent to risk the potential complications such as infection and financial costs when there is a very low chance of benefit?’”
Extracellular Vesicles: The Future of Stem Cell-less Therapy?
Inflammatory environments stimulate the release of extracellular vesicles (EVs) from stem cells. These microscopic particles—membrane-bound vesicles containing “bioactive cargo”—pack a mighty punch, playing important roles in tissue regeneration by:
- limiting tissue injury;
- participating in regenerative processes;
- enhancing cell proliferation and apoptosis; and
- playing a role in cell-to-cell communication by fusing with target cells and delivering their contents, including proteins and genetic information.
“It is the stem cell ‘secretome’—the cytokines, growth factors and contents of EVs, such as microRNAs and messenger RNAs—that are largely responsible for the pro-regenerative properties of MSCs,” explained Nicole Ehrhart, VMD, DACVS, director of the Laboratory of Comparative Musculoskeletal Oncology and Traumatology at Colorado State University’s Flint Animal Cancer Center. Exosomes, one of several types of EVs, are produced within the stem cell and secreted in response to various stimuli, such as tissue damage or inflammation.
According to Ehrhart, “The exosomes can travel between cells and throughout the bloodstream to target damaged tissues. Exosomes fuse with the plasma membranes of cells in those tissues and release their contents, thereby influencing target cell activities and promoting a pro-regenerative tissue response.”
One very exciting advantage of exosome-based therapies, Ehrhart said, is that exosome cargo can be enriched for certain pro-regenerative content. This creates opportunities to formulate exosome therapies for very specific conditions.
“Modulating exosome content will give us the ability to achieve a predictable and specific biological effect that can be harnessed for customized, precise therapeutics,” said Ehrhart. “EVs may also have the potential for being collected and stored at room temperature after lyophilization, which mitigates the need for cold storage shipping and can lengthen the shelf life of EV-based therapies.”
EVs essentially mimic the pro-regenerative effects of MSCs and might, therefore, replace live stem cells to achieve the same effect.
EVs are not yet available for clinical use, but they are being actively explored in research settings.
For example, Arévalo-Turrubiarte et al. (2021) collected and cultured MSCs from bone marrow, adipose tissue and synovial fluid. Culture media were collected and analyzed via transmission electron microscopy to evaluate the “expression particles” released from the MSCs. EVs collected from the culture media were then added to chondrocyte cultures after those cells were treated with the inflammatory cytokines IL-1b and tumor necrosis factor-a.
EVs were successfully collected from the various MSCs, and those EVs significantly reduced the expression of metalloproteinase-13 in chondrocyte cultures (metallopro- teinase-13 contributes to cartilage degradation).
These results therefore confirm that EVs can be isolated from MSCs sourced from various sites, and that those particles indeed have an anti- inflammatory effects.
The study also revealed that EVs isolated from synovial fluid were “not as efficient as EVs derived from BMMSCs in modulating the expressions of genes related to inflammation and EVM degradation.”
Thus, while EV therapy appears promising, like other aspects of regenerative medicine, the processes and protocols require some tweaking. According to the study authors, “the effect of EVs in our in vitro inflammation assay seems to point to different effects according to the origins of the EVs. The choice of single cell type to produce and isolate EVs might result in poorer outcomes in treatment in vivo.”
Additional research to further characterize the content of EVs is needed.
In her review article on what Ehrhart calls a “nascent but rapidly expanding area of research,” she relayed data from several other studies. Key findings relevant to equine medicine included:
- Equine MSC-derived EVs are enriched in pro-regenerative miRNAs that modulate immune and inflammatory processes. Those EVs also stimulate the proliferation of chondrocytes and simultaneously inhibited their death.
- Injecting conditioned media containing EVs from equine MSCs into injured tendons and ligaments of horses resulted in enhanced healing-related neovascularization and a lower rate of reinjury compared to the placebo (nontreated) group. Further, no adverse effects were appreciated following treatment.
“It’s important to note that while these preliminary results are promising, it will be important to develop larger, more complex studies that involve placebo controls to show if these early results still hold true,” Ehrhart added.
In summary, EVs offer a promising tool for future use, particularly as “precision medicine initiatives” become more widespread and popular in equine medicine.
In order to bring this therapy from bench to barn, strategies to efficiently produce large amounts of EVs, standardize their production from various MSC sources, devise isolation and purification methods, and conveniently store EVs need to be identified.
Beyond the Muscoloskeletal System
Regenerative therapies are by far most commonly used for managing musculoskeletal conditions in horses. In the past, regenerative therapies have been used either in conjunction with conventional therapies or in the case of treatment failures as a “Hail Mary.” But, according to Prządka et al. (2021), regenerative therapies such as MSCs are “slowly becoming the gold standard in the treatment of musculoskeletal diseases.”
In this review, Prządka listed various conditions for which stem cells have been studied in horses, including osteoarthritis of the fetlock stifle, pastern and coffin joints, as well as suspensory ligament and SDFT lesions.
Given that we now know that stem cells do not in fact exert their effects by differentiating into the target cell type but instead stimulate the body’s own repair mechanisms, a world of possibilities lies before us.
According to Barrachina et al. (2021), regenerative therapies could play a valuable role in various ophthalmologic, reproductive, integumentary and neurological conditions. One particularly interesting and potentially invaluable indication for stem cell therapy is in the arena of equine asthma.
“The traditional management of SEA (severe equine asthma) only temporarily controls disease exacerbations
in most, but not all, horses,” noted an Adamič et al. 2022 study. “Based on the data available to date, it was considered plausible that MSC treatment could have a positive effect without significant side effects.”
In the Adamič et al. 2022 study, 20 horses with SEA were randomly divided into two groups. One was treated with intrabronchial ADMSCs and the other with oral dexamethasone. Clinical examinations were performed on all horses after three weeks, and bronchoalveolar lavage fluid was analyzed for various pro- and anti-inflammatory cytokines. Non-inferiority of ADMSC treatment was not established, but horses did have significant clinical improvements and long-term improvements in clinical signs (one year following treatment). Decreases in IL-1b, IL-4 and tumor necrosis factor-a protein levels were also appreciated.
Take-Home Message
Veterinary practitioners appear to be enthusiastically embracing regenerative therapies for a wide array of conditions—not just injuries to the joints and soft tissues. While positive results are certainly reported in the literature and anecdotally, those results need to be interpreted with some caution, and additional studies are needed in a large variety of areas as noted in this article.
The future of regenerative medicine in equine practice appears almost limitless, much like the characteristics of a pluripotent stem cell.
