The State of Stem Cell Therapy
Stem cell therapy has received a good deal of attention in both human and veterinary medicine in recent years. It holds theoretical promise for treating conditions ranging from traumatic tendon and cartilage injury to liver failure, Parkinson's disease, and nerve/spinal cord damage, but it is still in its infancy. At the 2007 American Association of Equine Practitioners Convention, held Dec. 1-5 in Orlando, Fla., Lisa Fortier, DVM, PhD, Dipl. ACVS, associate professor of clinical sciences at Cornell University, said, "Stem cells are not yet living up to the hype. We've got a long way to go before we really know what we're doing and can fine-tune these therapies."
Fortier and Roger Smith, MA, VetMB, DEO, MRCVS, PhD, Dipl. ECVS, professor of equine orthopaedics at the Royal Veterinary College in Herts, U.K., presented an in-depth session on the current state of stem cell research for horses. Their presentations described the specifics and challenges of stem cell work along with the evidence that supports its use for equine tendon and cartilage defects.
Stem Cell Background
First, what is a stem cell? Even this seemingly basic answer is unclear, said Fortier. "The definition and identification of stem cells is constantly evolving," she noted. "There is no current consensus on a gold standard assay to isolate or identify stem cells."
Part of the challenge is that once stem cells differentiate into specific cell types (such as tendon or ligament cells), classifying them can be ambiguous. (Is that a stem cell, a tendon cell, or a stem cell that became a tendon cell?) Also, no single cell surface marker can differentiate stem cells from other cells. Instead, one must identify them by seeing what markers are present and absent, much like a combination lock (i.e., only a very specific combination of markers defines a stem cell). Researchers are working to develop an assay combining many different markers to identify stem cells so they can determine absolute numbers of stem cells harvested and used in therapies. This will help clarify research on their use.
"To date, equine studies that have investigated the use of 'stem cells' contain no information regarding characterization of the cells before implantation or data concerning survival or function of the transplanted/grafted cells," noted Fortier.
While researchers aren't yet in agreement on the methods and criteria for identifying stem cells, some are working on more functional testing to find out what stem cells can do. To discuss this testing, we need to know a few additional "background basics" of stem cell therapy. These include whether they are embryonic stem cells (ES, cells derived from embryos or generated using genetic means) or adult-derived mesenchymal stem cells (MSC). The latter are further broken down into the type of tissue from which they were harvested--whether they came from bone marrow or fat (adipose) tissue (BM-MSCs and A-MSCs, respectively). Each type and location of stem cells carries specific nuances; they can't all be lumped together.
Embryonic stem cells carry a few challenges. Traditionally, there has been a lot of political/ethical debate about the generation and destruction of embryos to generate stem cells, and there has been a concern about immune rejection of the cells since they would contain the genetic material of an individual different from the recipient. Very current breakthroughs, however, suggest that embryonic stem cells can be made from adult somatic cells such as skin cells. This methodology involves the introduction of four genes that "re-program" a cell to become an embryonic stem cell. This methodology obviates the need for generation or destruction of embryos and it allows the establishment of patient-specific stem cells that would not be rejected by the immune system.
Bone marrow-derived stem cells can be harvested from the sternum (breastbone) or iliac crest (part of the hip). These stem cells only comprise one in 10,000-100,000 of nucleated cells in bone marrow, said Fortier, but they "have received the most attention scientifically and hence are the best characterized." They are harvested with the horse standing and sedated, then they are cultured for about three weeks to increase their number to 10 million or so. They are then implanted into a lesion along with bone marrow supernatant (liquid), which contains growth factors to help heal the lesion. Since the cells come from the patient, there's no risk of rejection.
Adipose-derived stem cells have not done as well in some cell differentiation studies (which evaluate how well stem cells can be induced to grow into different tissue types, such as bone, muscle, or liver). Harvesting them results in more donor site morbidity (damage) than harvesting of BM-MSC. However, they do have their advantages; they don't have to be cultured for three weeks, so treatment of a lesion can occur quickly--within a couple of days. Fat tissue is harvested from the tailhead, then the fat cells are removed and what's left (about 50 million nucleated cells, about 2% of which are A-MSC) is injected into the lesion. This approach also carries no risk of rejection.
Several questions about stem cell therapy apply to all types of cells, said Fortier. They include the following:
- What's the best approach to using stem cells?
- How many stem cells do you need?
- Do we expect the stem cells to take environmental cues from surrounding tissues and just turn into what's needed, or do they need some guidance?
- How important are growth factors?
- What's the best way to grow stem cells in culture to gain maximum effect?
"There is a lot to learn, so we need to pick specific areas to focus on in order to optimize clinical implementation of stem cells," she commented. "The future of stem cell therapy is limitless for healing tendon, cartilage, laminitis, bones, nerves, etc."
Stem Cells for Tendon Lesions
"The horse is a professional athlete, and tendon injuries are extremely common," noted Smith; one study found that they affect 23% of National Hunt horses in training and 46% of limb injuries at racecourses.
He focused on the superficial digital flexor tendon (SDFT) in particular, noting that a large part of its function is to store energy via its elasticity and return that energy to the horse for the next stride. "That's what makes the horse an efficient runner," he explained. "The horse is estimated to be 120% energy efficient at the gallop." The downside, of course, is injury when the tendons and ligaments are overstretched.
When a tendon is injured and subsequently healed, the scarred tendon is less elastic than normal tissue, putting it at risk of reinjury; one study of National Hunt horses found that 56% of those with SDFT injury suffered reinjury. "This is the rationale behind use of stem cells to treat tendon overstrain injuries--we need regeneration (of normal tissue) rather than repair (scarred tissue)," Smith explained.
Equine MSC cultured in the laboratory create a matrix of tendon tissue that is "remarkably ordered," he observed. This is one of the keys to healing tendons--the cells must be organized linearly so they can handle the linear stress placed on them, rather than being disorganized so they can't stretch effectively.
One good thing about tendon lesions is that they generally form a closed cavity within the tendon, which helps hold stem cells in place and provides a vascularized scaffold (granulation tissue bed with blood vessels) to help organize healed tissue and provide blood supply, growth factors in the fluids present to help heal the lesion, and a mechanically appropriate environment.
Smith reported that lab animal studies have found that treatment of surgically created tendon and ligament lesions with MSCs results in better tissue organization, composition, and mechanics compared to controls. In addition, an equine study in the U.K. using BM-MSCs found that in 82 of 168 treated racehorses that were available for follow-up after one year, there was a 78% success rate (no re-injury) compared to 43% of horses conservatively treated in another study--a 35% improvement in success rate. More specifically, the success rate in National Hunt horses (in training and racing) was 82% of 71 horses, and the success rate in 11 flat racing horses was 50%. Twenty-four sport horses in other disciplines had an 87% success rate, compared to a 57-77% success rate with conservative treatment in another study.
He noted that horses that re-injured had a significantly longer interval between injury and treatment (83 vs. 44 days), suggesting that delayed treatment resulted in more fibrosis of the lesion. He now recommends earlier harvesting of bone marrow (within one month of injury) and treatment. Pre-injury harvesting and storage of cells may also prove beneficial, as might storage of stem cells recovered from that horse's umbilical cord at birth.
"Stem cells won't remove fibrous tissue once it's there, so treatment will be less effective on chronic cases," he advised. "I recommend it for first-time injury, but sometimes it's also been tried on horses with more chronic presentation or those that have had poor success with other treatments."
Less evidence is available regarding the value of A-MSCs in tendon injuries; Smith reported that a pilot study using the collagenase model of tendon injury found improved tissue organization and increased specific gene expression compared to controls. Although this approach has been used in many U.S. horses, clinical results have yet to be published.
Both treatments are followed by a 48-week protocol of rest and controlled exercise designed to provide appropriate mechanical stimulus to the healing cells without causing further damage. The protocol is adapted based on the individual horse's progress.
"There are some encouraging aspects to this technology, although definitive proof of efficacy is still lacking," Smith noted. "Furthermore, there have been no direct comparisons between the two techniques (BM-MSC and A-MSC). Combining stem cell therapies with other more established methods to prevent re-injury, such as desmotomy of the accessory ligament of the superficial digital flexor tendon (superior check ligament desmotomy), makes a lot of sense and might have value. But it must be remembered that there are still considerable gaps in our knowledge, although the technology is developing rapidly."
He explained the harvesting and treatment procedures in detail for the audience.
Stem Cells for Cartilage Lesions
One advantage of using stem cells to treat cartilage lesions is that the cells are harvested and inserted during a single arthroscopic surgery; no laboratory culture time is required. The technique involves removing any calcified cartilage, using a micropick to perforate cartilage and thus get growth factors from the bone beneath, and filling the defect with a stem cell mixture harvested from bone marrow. The mix includes thrombin to break fibrinogen down into a fibrin scaffold, which holds the stem cells and growth factors in place.
"First you dry the area with helium, then put in tricalcium phosphate (if there is a bone void underneath the cartilage), then put in the graft (stem cell mixture)," Fortier explained. "It clots immediately and sets in 30-45 seconds, and you can sculpt it so it fits in perfectly."
She discussed one ongoing study, funded by the Grayson Jockey-Club Research Foundation, of young (2- to 5-year-old) horses using this technique and BM-MSC in 15-mm full-thickness surgically created defects. Eight months post-surgery, treated sites had significantly more fill of the lesions (more than 75% fill vs. less than 25% fill in control sites on the same horse) and improved texture of the repair. Glycosaminoglycan (GAG) content (which helps hydrate and lubricate the joint) was not normal in treated lesions, but it was better than in control sites.
"Stem cell therapy is an exciting technology, but it's still developing," summarized Smith. "We must have sensible expectations for the therapy; this field is high on emotion and low on science. We're trying to readjust that balance, but certainly for your clients they'll always be attracted to stem cells and you have to temper that enthusiasm with explanation of where the technology currently is."
About the Author
Christy West has a BS in Equine Science from the University of Kentucky, and an MS in Agricultural Journalism from the University of Wisconsin-Madison.
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