Ashlee E. Watts, DVM, PhD, DACVS
Regenerative medicine is the process of harnessing natural healing processes to improve upon tissue repair for a more functional healed tissue. The holy grail of regenerative medicine would be to recapitulate fetal development, resulting in healed tissues that cannot be distinguished from uninjured tissue. Although to date this has not been achieved in musculoskeletal tissues, the potential to substantially improve outcomes with regenerative techniques is considerable. Consequently, there has been much activity in research and widespread clinical use of regenerative therapies for equine orthopedic applications. Some of the tools for regenerative medicine in orthopedics include stem cells, platelet-rich plasma, autologous conditioned serum, growth factors, and gene therapy. Regenerative therapies can be applied by intralesional, perilesional, intra-articular, or intravenous injections.
Stem cells, unlike their somatic cell counterpart, are self-renewing, highly proliferative, and capable of multilineage differentiation. The ultimate stem cell is made at conception. After fertilization, the zygote consists of totipotent stem cells that are able to form all three germ layers as well as placental tissue. Once the zygote becomes a preimplantation blastocyst, the inner cell mass consists of pluripotent stem cells that will give rise to all three germ layers—ectoderm, mesoderm, and endoderm—and can no longer form placental tissues. At that stage, the stem cells are embryonic. After day 8, the cells become either somatic cells (terminally differentiated) or stem cells committed to a specific lineage (multipotent). After that point, the stem cells are considered adult derived, despite their presence in fetal tissues. Local niches of lineage-committed multipotent stem cells remain in adult tissue throughout life for normal tissue remodeling and repair. With increasing age, the number, expansion potential, differentiation potential, and so-called potency of stem cells declines; therefore, there is increasing interest in allogeneic embryonic and fetal-derived stem cells as well as banking of autologous stem cells from postnatal samples.
The initial enthusiasm for stem cells in regenerative medicine was one of tissue-specific differentiation, in that stem cells implanted to a cartilage lesion would engraft, become chondrocytes, and produce cartilage matrix. As both basic science and clinical data accumulates, it appears that stem cell therapy may also be, largely or in part, due to local production of bioactive molecules and immune modulation rather than tissue-specific differentiation and long-term engraftment. What treatment effects stem cells actually impart is an important question. The answers will likely change what conditions are treated with stem cells and by which stem cell source, when they are applied, by which route, how often they are administered, and the dose of cells used. To answer these questions, additional clinical and experimental studies are required.
Because of the difficulty in isolation, expansion, and cryopreservation of equine embryonic stem cells, they have not been investigated in the horse for regenerative medicine and will not be discussed in this chapter. In contrast, adult-derived stem cells (nonembryonic) are generally considered to be safe and to carry little risk of tumor formation, are easy to isolate and expand, and have been used extensively in the horse. Thus, this discussion will focus on adult-derived stem cells.
Adult-derived mesenchymal stem cells (MSCs) are considered an excellent stem cell source for musculoskeletal regenerative therapies, because they are readily available from several tissues, allow for use of autologous cells as well as allogeneic cells because of immune tolerance to non-self MSCs, and are of mesodermal lineage and thus able to differentiate into cartilage, tendon, and bone. The immune privilege of MSCs may be in part due to lack of expression of MHC class II and most of the classical costimulatory molecules of antigen-presenting cells. Recent evidence also suggests that in addition to being immune privileged, MSCs are immune-modulatory, through secretion of chemo-attractants followed by regulation of immune cell activation (T and B cells). Finally, MSCs may also be anti-inflammatory through inhibition of IFN-γ and TNF-α and stimulation of metalloproteinase inhibitors and anti-inflammatory interleukins, such as IL-10. The most exciting element of the MSCs is their apparent exquisite responsiveness to their microenvironment, in that they behave according to the environment in which they are placed. In this manner, MSCs would respond appropriately to the degree of disease and modulate the local environment in favor of reduced inflammation, reduced apoptosis, and/or enhanced matrix synthesis of endogenous progenitors and tissue-specific cells.
Because of their broad overlap with other cell populations, MSCs cannot yet be sorted accurately by cell surface markers. Therefore, many labs select and isolate MSCs by expanding the tissue culture plastic adherent population of colony-forming cells. This translates to a culture period of 2–3 weeks, in vitro, to isolate and expand MSCs from clinical samples for autogenous therapy. In the horse, MSCs have been isolated from bone marrow, adipose, tendon, muscle, umbilical cord blood and tissue, gingiva and periodontal ligament, amniotic fluid, and blood. The different tissue sources vary in the ease of harvest, expansion potential, and differentiation capacity. Several academic and commercial laboratories provide for the isolation, expansion, and cryopreservation of stem cells from several different tissue sources—namely bone marrow, fat, and umbilical cord or blood. Directions for collection and shipping procedures are available from each lab. To date, bone marrow-derived MSCs from both the horse and human have been the most thoroughly studied and have the most evidence for ability to undergo chondrogenesis, tenogenesis, osteogenesis, and contribute to cartilage, tendon, and bone repair as well as modulate inflammation and soft-tissue repair within the joint.
Autologous or Allogeneic
Autologous (self) therapy has been used most in horses to date. Use of autologous cells is considered safe, with minimal risk for disease transmission. A major disadvantage of autologous cells is that, unless cells have been banked prior to injury, their use dictates a delay of 2–3 weeks for isolation and expansion. Although many labs are offering banking of autologous MSCs, the long-term viability of cryopreserved MSCs has not been fully elucidated. One way to avoid the culture delay for autologous MSCs is to use patient-side kits to concentrate stem cells. Several commercial kits are available that enrich for the nucleated cellular portion resulting in a higher concentration of MSCs in a small volume. Another method to avoid delay would be to use allogeneic (non-self) cells.
Because MSCs are immune privileged (see above), allogeneic cells can be used in nonrelated individuals and without immune testing. Although this has been demonstrated in most species, it has not yet been thoroughly reported in the horse. Use of an allogeneic stem cell line would allow use of an ‘off the shelf’ stem cell product and would have several advantages. First, it may reduce the variability between treatments, as different cultures between and amongst patients have different characteristics. Second, it may shorten the time between diagnosis and treatment. Third, it will allow for younger stem cells from fetal, adolescent, or young adult tissues to be used in aged horses, increasing stem cell potency and possibly enhancing the treatment effect. Finally, it may reduce costs by minimizing procedures, patient visits, and cell preparation time; however, allogeneic stem cells are considered a drug by the Food and Drug Administration (FDA) and, as such, are required to undergo the same safety and efficacy trials and manufacturing processes that are required of pharmaceuticals. Such trials are expensive and time consuming; therefore, commercial allogeneic stem cells are not yet available. In contrast, the use of autologous stem cells in veterinary patients is not currently regulated by the FDA.
Current Use in Orthopedics
Stem cell use in tendon and ligament injury provides the most evidence to date for improved repair. In a report of 105 National Hunt horses with over 2 years of followup, there was a lower recurrence rate of bowed tendon in tendons treated with stem cells (∼25%), compared with traditional therapies (approximately 55%; historical controls). Other tendon injuries that are being treated are lesions of the deep digital flexor tendon in the pastern and foot and ligament injuries, including suspensory and collateral ligaments. Most often, stem cells are suspended in serum, plasma, platelet-rich plasma, bone marrow supernatant, or culture medium for direct intralesional injection under ultrasound guidance 3–6 weeks following injury.
Intra-articular stem cell injection is used in horses for the treatment of acute articular injuries after surgical debridement and for the minimization of osteoarthritis (OA) progression. Several animal models of OA have shown promising results across several different research groups with reduced cartilage degeneration and OA progression and improved soft tissue healing. In the horse, there is experimental evidence for improved healing of cartilage defects treated by microfracture and anecdotal evidence for improved lameness resolution in stifle injuries (particularly those with meniscal injury) after intra-articular MSC injection. Occasional joint flare has been noted following intra-articular stem cell injection and may be related to contaminating foreign substances from the culture medium or injection of dead cells. For intra-articular injection, stem cells are suspended in plasma, serum, or bone marrow supernatant—with or without hyaluronic acid, but not with antimicrobials, as the doses routinely used in joint injections can be toxic to cells.
Direct arthroscopic implantation of MSCs into joint defects when treating osteochondritis dissecans (OCD), osteochondral injury, or cyst-like lesions has been used in the horse experimentally and clinically, with improved outcomes. In this application, stem cells are implanted within a scaffold, or a three-dimensional matrix such as an autologous fibrin clot, to maintain them within the articular cartilage defect. One scenario that would not necessitate a scaffold would be injection of stem cells under an OCD flap that is being salvaged by arthroscopic pinning.
Stem cells, especially from bone marrow, have robust bone-forming potential and may prove to be an important breakthrough in equine fracture fixation and arthrodesis. Through an increased rate of bone production, stem cells may help to achieve adequate healing prior to implant loosening or fatigue failure. Application of stem cells to fracture sites is most often done in a scaffold, to maintain the cells at the site of fracture.
Following wounding, circulating platelets accumulate and become activated when exposed to a basement membrane. Activation causes platelet degranulation and release of many bioactive substances that promote healing, stimulate angiogenesis, recruit endogenous stem cells, and regulate inflammation. Specific growth factors released from activated platelets at high concentrations include platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), and vascular endothelial growth factor (VEGF). Platelet-rich plasma (PRP) is a fraction of blood with an increased platelet concentration 2–4x baseline, and it is used largely for its anabolic properties. Other components of PRP are plasma proteins dissolved in water (adhesive proteins, clotting factors, fibrinolytic factors, proteases and antiproteases, basic proteins, and membrane glycoproteins), varying concentrations of leukocytes, and sporadic erythrocytes and stem cells.
The principal advantage of PRP is that production can be performed patient-side for immediate use, relatively inexpensively. Platelet-rich plasma is produced through centrifugation or filtration of venous blood, and the procedure can generally be accomplished within 15 minutes. The blood collection and preparation procedure vary from manufacturer to manufacturer and will influence the composition and volume of the product (platelet and leukocyte fold change, for example). It is likely that the varying reports of efficacy in clinical outcomes are influenced by PRP composition, and the ideal concentration of platelets and leukocytes within PRP remains undefined. Certainly, as platelet concentration increases, so does growth factor concentration. Therefore, higher platelet concentrations may be desirable. In support of this view, in vitro tendon explant data shows that tendon and ligament gene expression was improved with increasing platelet concentration. In the same study, increasing leukocyte concentration increased gene expression of collagen type III, the protein composition of scar tissue, which is undesirable.
Extra doses of PRP can be stored frozen (-20°C) for later use. It is important to note that leukocytes within the PRP will be lysed, and platelets will be activated by this storage process. While some have recommended platelet activation of fresh PRP with varying additives (calcium chloride, thrombin) or by freezing, it is probably not necessary, as the local environment should be sufficient to activate platelets for growth factor release. If PRP is to be used as a clot, addition of thrombin and calcium is required. Clinical evidence suggests that PRP is useful for acute tendon and ligament injury when injected intralesionally under ultrasound guidance into acute to subacute lesions. Platelet-rich plasma has also been used for arthropathy and delayed bone healing. Anecdotally, joint flares have been reported following PRP injection and may be related to the platelet-to-leukocyte ratio and leukocyte concentration.
Autologous Conditioned Serum
Autologous conditioned serum (ACS) therapy was developed to counteract the inflammatory mediator, interleukin-1, with a naturally occurring antagonist protein, interleukin-1 receptor antagonist protein (IL-1Ra; IRAP). Inhibition of interleukin-1 provides an analgesic as well as an anti-inflammatory effect, and thus ACS is used for its anticatabolic properties. Kits are commercially available for production of ACS, in which blood is incubated overnight in the presence of medical-grade glass beads. This incubation leads to the de novo synthesis and release of stored endogenous substances, including IL-1Ra, by leukocytes and platelets in the blood. Twenty-four hours later, the sample is centrifuged and the supernatant (serum) is collected, sterile filtered (0.2 µm filter), and separated into several aliquots. A portion (usually about 2 ml) of the ACS is injected into the affected region, and the extra doses can be stored in a freezer (-20°C) for approximately 1 year. Although IL-1Ra is the target protein made in this process, ACS probably contains a large and diverse set of factors that make it effective.
There has been widespread use of ACS in horses, primarily via intra-articular injection in the treatment of joint disease, osteoarthritis, or synovitis. Joint flare, or serious adverse reaction to joint injection, appears to be infrequent but has occurred. Practitioners have also used ACS for intralesional tendon or ligament injection. Anecdotal evidence suggests that the majority of horses that receive and respond to ACS are those that have become refractory to intra-articular steroids except on a very frequent reinjection schedule. Timetables employed for ACS therapy vary among practitioners. Some administer each injection weekly for 3–4 treatments, and others administer each injection monthly.
Growth Factors and Gene Therapy
Addition of growth factors, either directly as proteins or indirectly through gene therapy techniques to stimulate their production, has been used in several orthopedic applications. Compared to injection of protein, which has a very short half-life, gene therapy would allow for continued expression of the transgene, increasing the duration of growth factor exposure. Members of the transforming growth factor (TGF) family and insulin-like growth factor (IGF) have been used via proteins or gene therapy in the joint to stimulate synthesis of hyaline cartilage, improve subchondral bone architecture, and inhibit inflammatory responses; bone morphogenetic (BMP) protein and gene therapy has been used in fractures and cyst-like lesions to stimulate bone production; IGF protein and gene therapy has been used in tendon lesions to stimulate repair; growth hormone-releasing hormone (GHRH) gene therapy has been used in the treatment of laminitis; and IL-1Ra gene therapy has been used in the joint to minimize inflammation.
Many methods for gene transfer are available. For satisfactory transduction efficiency and effective protein expression, the best-described gene therapy procedures involve use of viral vectors such as retrovirus, adeno-associated virus, adenovirus, and many others. In the horse, use of adeno-associated virus and adenovirus has been reported. Nonviral methods are also available but have been less studied. Of the virally mediated gene therapy techniques, there is great variability in the genomic integration and subsequently the duration of transgene expression. It is unknown whether a long duration of transgene expression or permanent transgene expression would be required in orthopedic applications. Gene therapy techniques are not yet available for clinical application, but they may become a routine part of practice in the future.
There is still much to learn about the optimal treatment paradigm in regenerative therapies, including indications, technique, route, dose, timing, and frequency. There are several factors that contribute to the lack of evidence. First, most regenerative techniques are unencumbered by federal regulations and, as such, are being employed for a variety of conditions, by differing manufacturing processes and with differing treatment regimens. Second, autologous regenerative products have differing compositions, both between patients and even within the same patient from different collections. Such widespread use of a variable product makes it increasingly difficult to make sound conclusions. Based on the anti-inflammatory effects and the ability of regenerative techniques to orchestrate tissue repair and regeneration via endogenous cell recruitment and trophic factors, early treatment and possibly repeated treatments may be advantageous.
Stem cell therapy has successfully undergone proof of concept testing in equine tendon, cartilage, and intra-articular musculoskeletal applications. After the success of initial studies, the enthusiasm for stem cells in regenerative medicine was one of tissue-specific differentiation, in that stem cells implanted to a cartilage lesion, for example, would engraft, become cartilage cells, and produce cartilage matrix. As both basic science and clinical data accumulates, it appears that the effectiveness of stem cell therapy may also be, largely or in part, due to local production of growth factors and anti-inflammatory factors. What treatment effects stem cells actually impart is an important question, and continued research is warranted. The answers found by continued research will likely change what conditions are treated with stem cells and by which stem cell source, when they are applied, by which route, how often they are administered, and the dose of cells used. Combined with good diagnostics, surgical care when indicated, and a careful rehabilitation program, stem cell therapy is helping equine athletes with musculoskeletal injuries to return to, and stay in, the same level of performance as they were in prior to injury.