Stem Cells and Regenerative Medicine in Animals

Mesenchymal stem cells, horse

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Courtesy of Dr. Alix Berglund.

Mesenchymal stem cells (MSCs) are a heterogeneous population of fibroblast-like cells that are most commonly isolated from a variety of sources including bone marrow, adipose tissue, umbilical cord blood, and peripheral blood. MSCs make up a very small fraction of the cells present in these tissues. Processing in a laboratory is needed to eliminate red and white blood cells and to culture-expand the stem cells to obtain sufficient cell numbers for therapy. Bone marrow aspirate concentrate (BMAC) and adipose-derived stromal vascular fraction (AD-SVF) cell therapy do not require culture expansion steps and thus have a shorter interval from collection to treatment; however, there are presently insufficient controlled clinical trials 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, and there are ongoing investigations to determine the potential benefit of MSCs to treat numerous 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 IV. 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 engraftment and differentiation into new tissue. MSCs secrete various cytokines, growth factors, and extracellular matrix molecules that promote tissue healing. 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 also secrete mediators that inhibit apoptosis of local cells, promote angiogenesis and the differentiation of local progenitor cells, and prevent scar formation. Extracellular matrix molecules including type I collagen, fibronectin, and elastin have been reported to be produced by human and mouse MSCs, but this has not yet been extensively investigated in species of relevance to veterinary medicine. The secretome of MSCs can also 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 treatment protocols for MSC therapy, and the lack of consistency in the tissue source, timing of injection, dosing, and use of adjunct therapies may explain discrepancies in treatment efficacy reported for various clinical trials. The most evidence of clinical benefit with MSC therapy is for the treatment of tendinitis, osteoarthritis,1 and desmitis. 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, although some studies suggest improvement in return to use for horses or reduction in pain scores for dogs. Because of their strong immunomodulatory properties, there is considerable interest in the use of MSCs for treating immune-mediated diseases. Horses with refractory immune-mediated keratitis reportedly showed improvement in clinical signs or remission after treatment with MSCs. There is also ongoing research into the use of MSCs for treating canine atopic dermatitis, feline chronic gingivostomatitis, and inflammatory bowel disease.

Adverse events have been reported in veterinary patients after the administration of MSCs. These range from inflammatory reactions at the site of administration to anaphylaxis and death. Adverse reactions to MSC therapy may be attributed to an immune response to fetal bovine serum, which is a common component of MSC culture media, alloimmune responses to allogeneic cells, or the formation of microemboli in the lungs when MSCs are injected intravenously. More research on the safety of MSCs in healthy animals is indicated to improve clinical therapy.

Platelet-rich plasma (PRP) is a conditioned serum product produced via centrifugation or filtration with an increased concentration of platelets versus normal 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 proliferation of mesenchymal and epithelial cells, production of type I collagen , angiogenesis, and differentiation of local progenitor cells to accelerate the healing process 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, may promote inflammatory reactions and inhibit healing; however, they can improve the antimicrobial properties of PRP. The platelets in PRP may 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 production of proinflammatory cytokines from monocytes, which may make it a useful therapy for treating septic arthritis.

In veterinary medicine, PRP has been used primarily to treat osteoarthritis, tendinitis, desmitis, and skin lesions in horses and dogs. Two formulations of PRP can be used: 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 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; thus, PRP may be better suited for wounds with extensive tissue loss or 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. Only a limited number of randomized, controlled clinical trials have been conducted in dogs, but these studies showed that PRP significantly improved wound healing, lameness, and pain scores in treated dogs compared with controls.

The effectiveness of treatment with PRP preparations is extremely variable because of the differences in concentration of the platelets and leukocytes they contain, the diversity of activation techniques, and the variation in platelet and leukocyte concentrations in individual patients. The hydration status and systemic health of the patient are additional contributing factors.

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 the blood to produce concentrated quantities of cytokines and growth factors. The blood is then centrifuged to isolate the 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 total.

The major cytokine mediator produced in ACS is interleukin-1 receptor antagonist (IL-1RA), also referred to as interleukin-1 receptor antagonist protein (IRAP). IL-1RA inhibits the activity of the proinflammatory cytokines interleukin-1a and -1b (IL-1a and IL-1b). The proposed therapeutic benefit of ACS relies on generating a relatively high ratio of IL-1RA to IL-1 during the centrifugation and activation processes. 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. Using ACS to inhibit IL-1 has been found to decrease clinical signs of lameness, decrease synovial membrane thickness and hemorrhage, and decrease cartilage fibrillation in horses with osteoarthritis. Because ACS inhibits inflammatory responses rather than directly promoting tissue regeneration, it is most effective for treating acute injuries. Although commercial ACS products are available for 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 collagen type I compared with controls; however, more studies are needed to confirm the efficacy and safety of ACS for soft-tissue injuries. The preparation technique, patient health, and natural variation between individuals have been found to affect the quality of ACS, but adverse reactions are reportedly rare.

Autologous protein solution (APS) is a more recently available therapy that concentrates platelets, growth factors, and anti-inflammatory cytokines through a centrifugation and activation process with 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 means of in vivo studies. In vitro studies suggest that ACS and APS have similar concentrations of IL-1RA and a similar IL-1RA:IL-1b ratio; however, APS also has considerably more transforming growth factor beta 1 (TGF-B1) 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.

Most veterinary regenerative therapies, including MSCs, PRP, ACS, and APS, meet the legal definition of a drug. These products are therefore regulated by the FDA as “cell-based products.” There are currently no FDA-approved regenerative therapies for veterinary medicine, but researchers may enroll client-owned animals in clinical studies through an FDA investigational exemption. The investigational exemption allows a legal pathway for further clinical development and research of regenerative therapies and requires, among other things, reporting of adverse events. Further clinical trials are necessary to determine the safety and efficacy of veterinary regenerative therapies before they can receive FDA approval.

• Voga M, Adamic N, Vengust M, Majdic G. Stem Cells in Veterinary Medicine-Current State and Treatment Options. Front Vet Sci. 2020 May 29;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 May 10;S0195-5616(22)00028-6. doi:10.1016/j.cvsm.2022.02.005

• Garbin LC and Morris MJ. A Comparative Review of Autologous Conditioned Serum and Autologous Protein Solution for Treatment of Osteoarthritis in Horses. Front Vet Sci. 2021 Feb 19;8:602978. doi:10.3389/fvets.2021.602978