Using Regenerative Medicine to Improve Healing in Soft Tissue Injuries

Kyla Ortved | DVM, PhD, DACVS, DACVSMR | Published: Issue 4, 2023

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Equine athletes of all disciplines are prone to soft tissue injuries. Common injuries include superficial digital flexor (SDF) tendonitis, deep digital flexor (DDF) tendonitis, suspensory ligament desmitis, collateral ligament desmitis, and meniscal tears and degeneration.

Unfortunately, soft tissues including tendons, ligaments, and menisci, have limited intrinsic healing capabilities. Resultant repair tissue is often biomechanically inferior to native tissue leaving the animal prone to degeneration and re-injury. Traditional therapies for soft tissue injuries include systemic non-steroidal anti-inflammatory drugs (NSAIDs), intralesional injections with corticosteroids, cryotherapy, rest, and lengthy rehabilitation programs.  

More recently, regenerative medicine has been incorporated into standard treatment regimens for soft tissue injuries with the goal of decreasing inflammation and promoting healing through the delivery of cells and/or cytokines and growth factors to the injured tissue. Orthobiologics are aimed at producing an end product that more closely resembles native tissue. Currently, platelet-rich plasma (PRP), autologous conditioned serum (ACS), autologous protein solution (APS), stem cells, and amnion are the main orthobiologics used in equine soft tissue injuries. 

Platelet-rich Plasma 

Platelet-rich plasma (PRP) is an autologous blood-derived product defined as a volume of plasma with a platelet count above that of whole blood. PRP can be prepared patient-side following centrifugation or gravity filtration of autologous blood as platelets are smaller and less dense than RBCs and WBCs. Several commercial systems are available including Arthrex ACP® (Arthrex, Naples, FL), GPS® III (Zimmer Biomet, Warsaw, IN), Magellan® (Arteriocyte, Hopkinton, MA), Harvest® SmartPrep® (Terumo BCT, Lakewood, CO), Restigen PRP™ (Owl Manor Veterinary, Warsaw, IN), V-PET™ (Pall Medical, Port Washington, NY), and others. 

There is significant variability in platelet and WBC concentration across products which has the potential to affect efficacy. The therapeutic effect of PRP is due to the degranulation of platelet α-granules which release high concentrations of growth factors including PDGF, TGF-β, FGF, VEGF, and EGF, amongst others, that help modulate the inflammatory and healing response in damaged tissue. PRP also promotes healing by enhancing cell migration, proliferation and differentiation, improving matrix synthesis, and stimulating angiogenesis.¹ Several equine experimental and clinical studies have found that PRP treated tendon and ligaments have improved strength and elasticity compared to control and that re-injury rates are decreased.²⁵ 

The Angel® System (Arthrex) used to prepare platelet-rich plasma (PRP). This system allows precise control over platelet and cell concentrations in the final PRP product. 
Image courtesy of Kyla Ortved

In an experimental model of SDF tendonitis, tendons treated with PRP, when compared to saline treated tendons, had improved quality of repair tissue histologically, significantly more collagen and GAG content, and superior neovascularization. Perhaps most importantly, improved biomechanical properties, including higher strength at failure and elastic modulus, were demonstrated in PRP treated tendons.²³ Several reports have also described successful clinical outcomes following the treatment of horses with SDF tendonitis and desmitis of the suspensory ligament, including lesions in the origin, mid-body, and branches.⁴⁹ Despite seemingly positive clinical outcomes, the lack of a large randomized controlled trial must be considered when interpreting the results of PRP treatments. PRP can be used in the acute phase of soft tissue injuries because it is available patient-side. Ultrasound-guided intralesional injections can be performed at three-to-four-week intervals until healing is satisfactory. 

Autologous Conditioned Serum 

Interleukin-1 receptor antagonist (IL-1Ra or IRAP) is an endogenous protein produced by immune cells, mainly monocytes. It is a competitive antagonist of interleukin-1 (IL-1), which is a central mediator of inflammation. Autologous conditioned serum contains high concentrations of IL-1Ra in addition to other immunomodulatory cytokines. ACS is made from the horse’s own blood and requires a 24-hour incubation period during which blood is stored in a specialized syringe containing borosilicate (glass) beads that stimulate the production of IL-1Ra from white blood cells. Following incubation, the serum with concentrated IL-1Ra, is collected in preparation for injection into the patient. Additional aliquots can be stored at -20oC for future injections. IRAP II (Arthrex Vet Systems) and Orthokine (Dechra Veterinary Products) are two commercially available products. ACS is most commonly recommended for use in inflamed joints; however, a recent study found that tendon lesions treated with intralesional ACS in horses with naturally occurring tendinopathies had significantly smaller cross-sectional areas on serial ultrasonography and better collagen alignment following treatment compared to controls.¹⁰ 

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Sterile collection of blood in order to prepare a hemoderivative or autologous blood-based product.
Image courtesy of Kyla Ortved

Autologous Protein Solution 

Autologous protein solution (APS), or Pro-Stride, is touted as a combinatorial product as it contains concentrated platelets (PRP) and increased concentrations of IL-1Ra, similar to ACS.¹¹ The two-step centrifugation process does not require incubation such that it can be used patient-side. Few studies have evaluated the effect of APS on soft tissue injuries. A recent study demonstrated that collagenase-induced SDF tendonitis treated with intralesional APS had a higher elastic modulus and significantly decreased expression of collagen type 3 twelve weeks after treatment compared to controls.¹² More research is needed on the effect of APS in soft tissue injuries, especially in horses with naturally occurring disease. 

Autologous conditioned plasma (ACP) prepared in the Arthrex ACP® Double-Syringe System prior to injection into a soft tissue lesion.
Image courtesy of Kyla Ortved

Amnion 

Equine amnion-based products are available for the treatment of tendon/ligament injuries (AniCell Biotech). Amnion is collected and decellularized such that an off-the-shelf bioscaffold is available. At this time, support for the use of amnion is mainly anecdotal. 

Stem Cells 

Stem cells are undifferentiated cells that are capable of self-renewal and able to differentiate into different cell types (potency). The potency of stem cells varies from totipotent (able to give rise to all cell types e.g. early embryonic cell), pluripotent (able to differentiate into any of the three germ layers e.g. embryonic- and early fetal-derived stem cells), multipotent (able to differentiate into a number of closely related cells e.g. adult-derived stem cell), to unipotent (able to give rise to only one cell type e.g. progenitor cell). Adult-derived mesenchymal stem cells (MSCs) are used most commonly in equine regenerative medicine as they have the ability to differentiate into osteoblasts, chondrocytes, myocytes, and adipocytes i.e., they are multipotent. Adult MSCs can be obtained from a variety of sources including but not limited to bone marrow, adipose, synovium, umbilical cord blood and tissue, tendon, and muscle. Bone marrow and adipose are the most common source of MSCs used in equine medicine as they can be easily obtained from the sedated, standing horse. Specifically, bone marrow can be harvested from the sternum or tuber coxae and adipose can be harvested from either side of the tail head.¹³¹⁴ The therapeutic effect of stem cells is attributed to their ability to promote healing and limit scar tissue formation, modulate the inflammatory response, and stimulate tissue regeneration.¹⁵ 

Following the collection of bone marrow or fat, MSCs can be immediately concentrated for subsequent use, or they can be culture-expanded in the lab. Bone marrow aspirate concentrate (BMAC) and adipose-derived stromal vascular fraction (ADSVF) are produced without any culture expansion. BMAC can be generated patient-side via centrifugation, while ADSVF requires collagenase digestion of fat prior to concentration. ADSVF is commercially available through VetStem Biopharma. The major downside of these techniques is the limited number of cells that are present in the final product such that many clinicians choose to culture-expand MSCs in a dedicated lab. A two to four-week culture period will typically yield > 100 million cells, which can be frozen and re-expanded as needed. 

Experimental and clinical studies support the use of MSCs for SDFT and suspensory ligament injuries, with most studies showing improved tissue architecture, biomechanical function, and decreased re-injury rates.¹⁶¹⁹ Tendon and ligament lesions are best treated in the acute phase when a hypoechoic lesion is present on ultrasound. Chronic lesions with increased echogenicity are difficult to inject and likely too fibrotic to remodel effectively. Tendon and ligament lesions can be accurately injected under ultrasound guidance. The number of cells injected varies tremendously with most lesions being injected with ~10-50 million cells. The injection can be repeated every three to four weeks until adequate filling of the lesion is appreciated on ultrasound. 

Extensive lesions in the SDFT or suspensory are sometimes treated with regional limb perfusion of MSCs, instead of direct injection. 

Collection of bone marrow from the sternum of a horse in preparation for culture expansion of autologous MSCs for future ultrasound-guided injection into a tendon lesion.
Image courtesy of Kyla Ortved

Alternative Stem Cell Sources

Dental pulp has been described as a relatively non-invasive source of stem cells in humans and horses, with cells being isolated from the pulp chamber of extracted teeth,²⁰²¹ although very few studies have evaluated the regenerative potential of equine dental pulp. Recently, Bertone et al. investigated the effect of injection of an off-the-shelf product Pulpcyte® (VetGraft, Columbus Ohio) in horses with osteoarthritis and tendonitis or desmitis.²² This commercially available product, described as dental pulp connective tissue particles by the authors due to the scarcity of cells and relative abundance of ECM, was associated with improved lameness scores following intra-lesional injection. Despite the reported benefit of dental pulp products, further research is required before they can be recommended. 

Embryonic-derived and induced pluripotent stem cells (iPSCs) are also being investigated for potential benefit in musculoskeletal healing. Equine iPSCs cells have been described, although these cells have not been used clinically yet.²³²⁴ Additionally, much interest lies in using allogeneic cells because these would be a patient-side product with no lag time for culture expansion. At this time, it is uncertain whether allogeneic cells are effective or safe due to recognition by the host immune system.²⁵ 

Conclusion

Orthobiologics are being used with increasing frequency and pre-clinical and clinical data continue to be promising. Many different therapies are available with selection often dependent on the injury. Overall, regenerative therapies can improve the body’s natural healing ability such that a superior end product is produced. 

References 

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  2. 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 
  3. Bosch G, Moleman M, Barneveld A, van Weeren PR, van Schie HTM. The effect of platelet-rich plasma on the neovascularization of surgically created equine superficial digital flexor tendon lesions. Scand J Med Sci Sports. 2011;21(4):554-561. doi:10.1111/j.1600-0838.2009.01070.x 
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About the Author

Kyla Ortved, DVM, PhD, DACVS, DACVSMR

Dr. Kyla Ortved is an associate professor of large animal surgery at New Bolton Center, University of Pennsylvania in Kennett Square, PA. She received her DVM degree from the University of Guelph in 2006 and completed her large animal surgical residency training at Cornell University in 2010. Kyla became boarded with the American College of Veterinary Surgeons in 2011. Following her residency, Kyla went on to obtain a PhD in gene therapy for equine cartilage repair at Cornell. In February 2016, Kyla became boarded with the American College of Veterinary Sports Medicine and Rehabilitation. She joined the large animal surgery faculty at New Bolton Center in 2016 as an equine orthopedic surgeon and was named the Jacques Jenny Endowed Term Chair of Orthopedic Surgery in 2019. Her research program focuses on understanding the pathophysiology of equine osteoarthritis and developing gene and cell-based therapies to improve cartilage repair and prevent osteoarthritis.    

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