Stem Cells and Regenerative Medicine in Animals

Mesenchymal stem cells (MSCs) are a heterogeneous population of fibroblast-like cells that are isolated from a variety of sources, including bone marrow, adipose tissue, umbilical cord blood, and peripheral blood (see MSC image). MSCs make up a very small fraction of the cells present in these tissues.

Mesenchymal stem cells, horse

Image

Courtesy of Dr. Alix Berglund.

MSCs must be processed in a laboratory to eliminate red and white blood cells and to culture-expand the stem cells to obtain sufficient quantities for treatment. Treatments using bone marrow aspirate concentrate (BMAC) and adipose-derived stromal vascular fraction (SVF) do not require culture expansion steps and thus have a shorter interval from collection to treatment; however, evidence from controlled clinical trials is insufficient to support their therapeutic benefit in veterinary patients.

MSCs have been used in veterinary medicine to treat tendinitis, osteoarthritis, desmitis, corneal wounds, and cutaneous wounds. Ongoing investigations are focused on determining the potential benefit of MSCs to treat other diseases.

MSCs have the ability to home to sites of inflammation and are generally injected locally at the site of injury. If the lesion is inaccessible or there are multiple lesions, MSCs can be administered through regional limb perfusion or intravenously, with caution. MSCs are also commonly combined with autologous serum or platelet-rich plasma to treat musculoskeletal injuries.

• MSCs can be stimulated to differentiate into bone, adipose, or cartilage in vitro; in vivo, however, their primary role appears to be to support endogenous healing rather than to engraft and differentiate into new tissue.

• MSCs secrete various cytokines, growth factors, and extracellular matrix molecules that promote tissue healing.

• MSCs have been shown to secrete antimicrobial peptides that inhibit bacterial growth and biofilm formation.

• MSCs from all species secrete cytokines and immunomodulatory factors that inhibit inflammatory cytokine signaling, block lymphocyte proliferation, polarize macrophages to an anti-inflammatory phenotype, and induce the generation of regulatory immune cells. The specific immunomodulatory factors secreted by MSCs differ slightly by species and by tissue source.

• MSCs secrete mediators that inhibit apoptosis of local cells, promote angiogenesis and the differentiation of local progenitor cells, and prevent scar formation.

• Human and mouse MSCs have been reported to produce extracellular matrix molecules, including type I collagen, fibronectin, and elastin; however, such production has not yet been extensively investigated in species relevant to veterinary medicine.

• The secretome of MSCs can be altered and enhanced by an in vivo inflammatory environment or by in vitro treatment with inflammatory cytokines before use.

There is currently no consensus in veterinary medicine on protocols for MSC treatment, and the lack of consistency in the tissue source, timing of injection, dosing, and use of adjunct treatments may explain discrepancies in treatment efficacy reported for various clinical trials.

The most evidence in animals of clinical benefit with MSC treatment pertains to tendon injuries (1, 2), osteoarthritis (3), and desmitis (4). Horses and dogs treated with MSCs for tendon injuries show improved fiber alignment and composition of tissues, increased biomechanical strength, and decreased reinjury rates. There is less clinical evidence for the use of MSCs to treat osteoarthritis in horses; however, some studies suggest a higher rate of return to use for horses or lowering of pain scores for dogs.

Because of their strong immunomodulatory properties, MSCs are being studied in animals to treat immune-mediated diseases. Horses with refractory immune-mediated keratitis reportedly showed improvement in clinical signs or remission after treatment with MSCs (5). In addition, there is ongoing research into the use of MSCs to treat canine atopic dermatitis, feline chronic gingivostomatitis, and inflammatory bowel disease.

Adverse effects have been reported in veterinary patients after the administration of MSCs. These range from inflammatory reactions at the administration site to anaphylaxis and death. Adverse effects of MSC treatment can be attributed to immune responses to fetal bovine serum, a common component of MSC culture media; to alloimmune responses to allogeneic cells; or to formation of microemboli in the lungs when MSCs are injected intravenously.

More research on the safety of MSCs in healthy animals is needed to improve treatment.

Platelet-rich plasma (PRP) is a conditioned serum product, produced via centrifugation or filtration, that contains higher concentrations of platelets than are normally found in blood plasma. When platelets are activated in vivo via inflammation, calcium chloride, thrombin, or lysis, they release numerous growth factors and immunomodulatory cytokines. The growth factors in PRP promote the proliferation of mesenchymal and epithelial cells, production of type I collagen, angiogenesis, and differentiation of local progenitor cells to accelerate the healing of injured tissues.

The two major classifications of PRP are leukocyte-poor PRP and leukocyte-rich PRP. Leukocyte-poor PRP is generally considered superior for treating musculoskeletal injuries. Leukocytes in PRP, particularly neutrophils, can promote inflammatory reactions and inhibit healing; however, they can also improve the antimicrobial properties of PRP.

Platelets in PRP can also be lysed through freeze/thaw cycles to generate PRP lysate, which is acellular and has allogeneic applications. Equine PRP lysate has strong antimicrobial properties and inhibits the production of proinflammatory cytokines by monocytes, which may make it useful for treating septic arthritis.

In veterinary medicine, PRP has been used primarily to treat osteoarthritis, tendinitis, desmitis, and skin lesions in horses and dogs. There are two possible formulations: liquid PRP and coagulated PRP (also termed platelet gel). Liquid PRP can be injected into an injury site; platelet gel can be applied topically or injected into cartilage defects during surgery.

Both PRP formulations can be combined with MSCs to improve healing. In horses, PRP has been reported to promote formation of excessive granulation tissue and slow wound healing of surgically created distal limb wounds (6); thus, PRP may be better suited for wounds with extensive tissue loss or for chronic wounds.

When injected into superficial digital flexor tendon lesions, PRP has been found to increase collagen content, biomechanical strength, and elasticity, and to decrease reinjury rates (7, 8). Only a limited number of randomized, controlled clinical trials have been conducted using PRP in dogs; in these studies, PRP markedly improved wound healing, lameness, and pain scores in treated dogs compared with control animals (9, 10).

The effectiveness of treatment with PRP preparations is extremely variable because of differences in the concentrations of platelets and leukocytes they contain, the diversity of activation techniques, and the variation in platelet and leukocyte concentrations in individual patients.

Autologous conditioned serum (ACS) products are used primarily to modulate inflammatory cytokine signaling in osteoarthritis. Several commercial devices generate ACS by incubating whole blood with glass beads for 24 hours, which stimulates leukocytes in blood to produce concentrated quantities of cytokines and growth factors. The blood is then centrifuged to isolate protein-rich serum for injection.

Several aliquots can be made from one preparation of ACS and stored frozen for future use. Typically, ACS is injected into an affected area every 1–2 weeks for three to five treatments.

The major cytokine mediator produced in ACS is interleukin-1 (IL-1) receptor antagonist, or IL-1RA, which is also referred to as IL-1 receptor antagonist protein, or IRAP. IL-1RA inhibits the activity of the proinflammatory cytokines IL-1-alpha and IL-1-beta.

The proposed therapeutic benefit of ACS relies on generating a relatively high ratio of IL-1RA to IL-1 during centrifugation and activation. IL-1 is produced after traumatic tissue damage or infection and initiates an inflammatory cascade. In joints, production of IL-1 leads to increased pain and cartilage degradation and calcification.

The use of ACS to inhibit IL-1 has been found to decrease clinical signs of lameness, synovial membrane thickness and hemorrhage, and cartilage fibrillation in horses with osteoarthritis (11).

Because ACS inhibits inflammatory responses rather than directly promoting tissue regeneration, it is most effective for treating acute injuries. Although commercial ACS products are specifically labeled for use in dogs, no clinical trials have been performed to evaluate their efficacy in dogs.

ACS is currently recommended only for intra-articular administration because the effects on soft tissue are not well understood. One study in horses found that a single dose of ACS decreased the size and increased the echogenicity of superficial digital flexor tendon lesions and improved the expression of type I collagen compared with control horses (12). However, more studies are needed to confirm the efficacy and safety of ACS for soft tissue injuries.

Adverse reactions are reportedly rare with ACS treatment.

Autologous protein solution (APS) is another regenerative treatment containing higher concentrations of platelets, growth factors, and anti-inflammatory cytokines than are normally found in blood plasma. APS is produced through a process of centrifugation and activation involving polyacrylamide beads.

APS combines the beneficial components of PRP and ACS; unlike ACS, however, APS can be administered patient-side, because it does not require a 24-hour incubation period and is recommended as a single injection.

APS is thought to inhibit inflammatory cascades within injured joints, but this effect has not been confirmed by in vivo studies. In vitro studies suggest that ACS and APS have similar concentrations of IL-1RA and a similar IL-1RA:IL-1-beta ratio; however, APS also has considerably more transforming growth factor (TGF) beta-1, an anti-inflammatory cytokine, compared with serum or ACS.

Horses and dogs given a single intra-articular injection of APS in randomized, controlled clinical trials had decreased lameness and improved pain, force plate, and gait analysis scores by 12 weeks after injection (13, 14).

Alpha-2-macroglobulin (A2M) is a regenerative treatment used for osteoarthritis and synovitis in horses. A2M is a naturally occurring broad-spectrum protease inhibitor found in plasma and synovial fluid. It is proposed to bind and neutralize proteases and cytokines that promote cartilage degradation, thereby slowing this process in arthritis.

A2M can be isolated and concentrated from whole blood through centrifugation and filtration using a patient-side commercial system. This process creates a large volume of product that can be frozen and stored for up to 12 months.

Because A2M is an autologous product, adverse reactions are reportedly rare.

Most veterinary regenerative treatments, including MSCs, PRP, ACS, APS, and A2M, meet the legal definition of a drug. These products are therefore regulated in the US by the FDA as “cell-based products.”

There are currently no FDA-approved regenerative treatments for veterinary medicine; however, guidelines for cell-based products to use in animals and for good manufacturing practice have been released to provide recommendations to manufacturers and veterinarians. Researchers can enroll client-owned animals in clinical studies through an FDA investigational exemption that allows a legal pathway for further clinical development and research of regenerative treatments and requires, among other things, reporting of adverse events.

Additional clinical trials are necessary to determine the safety and efficacy of veterinary regenerative treatments before they can receive FDA approval.

The FDA also has regulatory oversight over devices that generate regenerative products; however, these products do not require premarketing approval or postmarketing reporting. Veterinarians are therefore encouraged to report to the FDA any adverse reactions and product defects associated with these devices, using Form FDA 1932a.

• US Food and Drug Administration: Cell and Tissue Products for Animals

• American Veterinary Medical Association: Regenerative Medicine, Including Therapeutic Use of Stem Cells.

• Voga M, Adamic N, Vengust M, Majdic G. Stem cells in veterinary medicine—current state and treatment options. Front Vet Sci. 2020;7:278. doi:10.3389/fvets2020.00278

• Carr BJ. Platelet-rich plasma as an orthobiologic: clinically relevant considerations. Vet Clin North Am Small Anim Pract. 2022;52(4):977-995. doi:10.1016/j.cvsm.2022.02.005

• Garbin LC, Morris MJ. A comparative review of autologous conditioned serum and autologous protein solution for treatment of osteoarthritis in horses. Front Vet Sci. 2021;8:602978. doi:10.3389/fvets.2021.602978

• M'Cloud WRC, Guzmán KE, Panek CL, Colbath AC. Stem cells and platelet-rich plasma for the treatment of naturally occurring equine tendon and ligament injuries: a systematic review and meta-analysis. J Am Vet Med Assoc. 2024;262(S1):S50-S60. doi:10.2460/javma.23.12.0723

• Mayet A, Zablotski Y, Roth SP, Brehm W, Troillet A. Systematic review and meta-analysis of positive long-term effects after intra-articular administration of orthobiologic therapeutics in horses with naturally occurring osteoarthritis. Front Vet Sci. 2023;10:1125695. doi:10.3389/fvets.2023.1125695

1. Godwin EE, Young NJ, Dudhia J, Beamish IC, Smith RKW. Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon. Equine Vet J. 2012;44(1):25-32. doi:10.1111/j.2042-3306.2011.00363.x

2. Salz RO, Elliott CRB, Zuffa T, Bennet ED, Ahern BJ. Treatment of racehorse superficial digital flexor tendonitis: a comparison of stem cell treatments to controlled exercise rehabilitation in 213 cases. Equine Vet J. 2023;55(6):979-987. doi:10.1111/evj.13922

3. Vilar JM, Batista M, Morales M, et al. Assessment of the effect of intraarticular injection of autologous adipose-derived mesenchymal stem cells in osteoarthritic dogs using a double blinded force platform analysis. BMC Vet Res. 2014;10:143. doi:10.1186/1746-6148-10-143

4. Van Loon VJF, Scheffer CJW, Genn HJ, Hoogendoorn AC, Greve JW. Clinical follow-up of horses treated with allogeneic equine mesenchymal stem cells derived from umbilical cord blood for different tendon and ligament disorders. Vet Q. 2014;34(2):92-97. doi:10.1080/01652176.2014.949390

5. Davis AB, Schnabel LV, Gilger BC. Subconjunctival bone marrow-derived mesenchymal stem cell therapy as a novel treatment alternative for equine immune-mediated keratitis: a case series. Vet Ophthalmol. 2019;22(5):674-682. doi:10.1111/vop.12641

6. Monteiro SO, Lepage OM, Theoret CL. Effects of platelet-rich plasma on the repair of wounds on the distal aspect of the forelimb in horses. Am J Vet Res. 2009;70(2):277-282. doi:10.2460/ajvr.70.2.277

7. Geburek F, Gaus M, van Schie HTM, Rohn K, Stadler PM. Effect of intralesional platelet-rich plasma (PRP) treatment on clinical and ultrasonographic parameters in equine naturally occurring superficial digital flexor tendinopathies—a randomized prospective controlled clinical trial. BMC Vet Res. 2016;12(1):191. doi:10.1186/s12917-016-0826-1

8. Bosch G, van Schie HTM, de Groot MW, et al. Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: a placebo-controlled experimental study. J Orthop Res. 2010;28(2):211-217. doi:10.1002/jor.20980

9. Tambella AM, Atili AR, Dini F, et al. Autologous platelet gel to treat chronic decubitus ulcers: a randomized, blind controlled clinical trial in dogs. Vet Surg. 2014;43(6):726-233. doi:10.1111/j.1532-950X.2014.12148.x

10. Fahie MA, Ortolano GA, Guercio V, et al. A randomized controlled trial of the efficacy of autologous platelet therapy for the treatment of osteoarthritis in dogs. J Am Vet Med Assoc. 2013;243(9):1291-1297. doi:10.2460/javma.243.9.1291

11. Frisbie DD, Kawcak CE, Werpy NM, Park RD, McIlwraith CW. Clinical, biochemical, and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis. Am J Vet Res. 2007;68(3):290-296. doi:10.2460/ajvr.68.3.290

12. Geburek F, Lietzau M, Beineke A, Rohn K, Stadler PM. Effect of a single injection of autologous conditioned serum (ACS) on tendon healing in equine naturally occurring tendinopathies. Stem Cell Res Ther. 2015;6(1):126. doi:10.1186/s13287-015-0115-0

13. Bertone AL, Ishihara A, Zekas LJ, et al. Evaluation of a single intra-articular injection of autologous protein solution for treatment of osteoarthritis in horses. Am J Vet Res. 2014;75(2):141-151. doi:10.2460/ajvr.75.2.141

14. Wanstrath AW, Hettlich BF, Su L, et al. Evaluation of a single intra-articular injection of autologous protein solution for treatment of osteoarthritis in a canine population. Vet Surg. 2016;45(6):764-774. doi:10.1111/vsu.12512