Regenerative Medicine: Stem Cells, Platelets and IRAP
Equine Regenerative Cell Medicine
by Michael W. Conaway, DVM, Reata Equine Veterinary Group.
The recent exciting discoveries about how injured tissue heals itself has led to a revolution in how we treat equine athletic injuries. We can now improve the quality of life for equine athletes by reducing inflammation and restoring tissue function. Equine medicine is rapidly moving into an era of molecular medicine where the use of our new understanding of molecular and cellular biology allows us unprecedented opportunities to successfully treat previously unmanageable athletic injuries.
This new approach can be thought of as tissue engineering. Equine veterinarians are using naturally occurring biological substances or cells that restore, maintain, or improve tissue function. These techniques are most helpful in situations where the horses natural healing is not proceeding at an optimal rate. Intervention in these instances will produce repair that is more complete than would occur naturally. Tendon and ligament injuries as well as joint injuries fall into the categories that benefit the most from these new biological techniques.
Types of Regenerative Cell Medicine:
1. Stem Cells
2. Platelet Rich Plasma
3. IRAP
Stem Cell Therapy:
What is a stem cell?
Some cells found in the body and the embryo can change into cells that are highly specialized such as tendon cells. These progenitor cells are called stem cells. They can be very useful in providing extra help in the repair of damaged tendons, ligaments or joints.
There are two broad categories of stem cells:
• Embryonic
• Adult-derived.
Embryonic stem cells (ES cells) used to be defined as those harvested from day 8 embryos. More recently advances have been made so that ES cells can be generated from adult fibroblasts using many of the same technologies that were used to clone Dolly the sheep. These cells are known as iPS (induced pluripotent stem) cells.
Adult-derived stem cells (MSC) can be obtained from bone marrow, fat, umbilical
cord blood, muscle, and many other tissues including cartilage, trabecular bone and tendon. They are found in varying numbers in different tissue types.
We are not completely sure which tissue contains the best source of stem cells for each treatment application. Much scientific work remains to be done in this area. Because arthritis is a bigger problem in humans than is tendon problems there is much more data available on stem cell application for joint problems than there is for tendonitis.
Embryonic Stem Cells
Embryonic stem cells are still in the experimental stage for use in horses. Research has been centered on laboratory techniques of culture and the development of patient specific embryonic stem cells to avoid tissue rejection problems.
Umbilical Cord Stem Cells:
Umbilical blood can serve as a source of stem cells. The blood can be harvested by a simple technique at birth. It is sent to a commercial lab where the stem cells are removed and cultured in a special media. Once therapeutic numbers of cells are produced they are frozen and stored. If that foal ever needs treatment later in life it will have a source of its own stem cells available.
Bone Marrow Derived Stem Cells:
Bone marrow–derived stem cells have gained attention as a helpful method of treating tendon, ligament and joint injuries in horses. A recent study reported a 70% rate of return to function of joint injuries and 86% rate of return to function of tendon and ligament injuries with the use of bone marrow derived stem cells. There is some indication that the fluid that is harvested with the bone marrow sample has potent tissue building properties (anabolic effect).
Bone marrow stem cells perform better than stem cells harvested from other tissues in terms of changing into known cell types such as tendon cells. Bone marrow is collected from the sternum (or hip) under standing sedation. It then takes 21 days of cell culture to produce the needed 10 million stem cells. The prepared stem cell population is then implanted using ultrasound guidance into the damaged tendon. The cells from the lab are suspended in the original bone marrow fluid to insure that no foreign material is implanted with the cells and to also take advantage of the rich growth factors present in the bone marrow fluid.
Fat Derived Stem Cells
This technique is based on a study which suggested that fat-derived stem cells exhibited a similar ability to change into known cell types as do bone marrow derived stem cells. However in many other studies they do not perform as well as bone marrow stem cells. Research has shown that equine stem cells derived from fat stores are capable of secreting substances (vascular endothelial growth factor) that cause new blood vessels to migrate into the damaged tendon and improve the blood supply and healing. Currently there is a commercial technique that produces stem cells from adipose tissue that is removed from the horse’s tail head. The sample is sent to the lab where the fat is removed from the sample leaving a cell mixture that contains stem cells. This technique has the advantage of a fast turn around (48 hr) because culturing is not needed. The stem cells are implanted into damaged tendon tissue using ultrasound guidance.
Does Stem Cell Treatment of Tendon Injuries Work?
Early scientific studies suggest that there are encouraging aspects to the use of stem cell technology for the healing of tendon and ligament injury, however definitive proof of efficacy is still lacking.
Platelet Rich Plasma
What are platelets?
Platelets (also called thrombocytes) are small fragments of cells found in the bloodstream. They are derived from larger cells called megakaryocytes. They have many important functions including blood clotting, and tissue repair.
Platelets contain many healing related chemical factors including:
• Platelet derived growth factor, a potent agent that draws macrophages to the damaged area.
• TGF Beta: a factor that stimulates the deposition of extracellular matrix to assist in connective tissue repair.
• Fibroblast growth factor
• Insulin like growth factor 1
• Epidermal growth factor
• Vascular endothelial growth factor
How do platelets help tissue repair itself?
When tissue damage occurs and hemorrhage into the damaged area begins, platelets are among the first arrivals. They initiate the formation of a blood clot and are incorporated into the clot. From this position they release all the factors listed above. These chemicals serve to attract fibroblasts to lay down new connective tissue to form a scar and also to mediate the inflammatory response to control infection.
In human medicine platelets have been used in maxillary surgery, orthopedic surgery and plastic surgery for years. It is felt that when applied at very high concentrations to damaged tissue it may convert the process from forming a scar to actual tissue regeneration.
Platelet rich plasma has been used in equine medicine to promote healing tendon/ligament injury, joint injury and skin injury.
What Do the Scientific Results Show?
There are a plethora of studies that show the advantages of growth factors in the healing of tendon, ligaments, joints and skin in laboratory animals, but the large studies have not been done on humans or horses.
What is currently available for the use of platelets in horses?
There are several commercially available methods for harvesting platelet rich plasma. The best methods are those that:
- Produce a consistent product with 4 fold increase in platelet concentration. More is not better in this case. Greater than 4 fold increase in platelets concentration does not seem to provide any greater benefit
- Produce a product where the white blood cell concentration is kept to a minimum, and at least no greater than venous blood white blood cell concentration.
Most methods of platelet preparation involve separation of the platelets from the rest of the blood by either centrifugation or gravity.
How are platelets used in equine sports injuries?
Usually the platelet rich plasma is injected directly into the damaged structure, be it a joint, a ligament or a tendon. Digital radiography can be used to identify the joints involved and ultrasound can be used to identify the ligament and tendon areas that are damaged. Often intra tendonous or intra ligamentous injection requires ultrasound guidance to make sure the platelets are delivered where they are needed.
Treatment protocols vary but usually the first treatment doesn’t begin until at least 10 days after the injury. Routine supportive care such as rest, cold therapy, NSAIDS, and pressure wrapping are performed during the 1-10 days from injury. This time-delay from injury also allows the lesion to become more organized so one can better determine the extent of the injury and site/s for injection. Our post-injection protocol is presently: After the first injection, horses are box-stall rested with hand walking for 10 minutes twice daily for the first week which increases by 5 minutes each week to a maximum of 30 minutes twice daily until recheck examination at 30 days post-injection. At recheck examination, a lameness and ultrasound evaluation are performed. If the horse is not at least 50% improved in lameness, and the tendon/ligament is not at least 50% improved in both x-section and longitudinal planes, than a second injection is performed.
NSAID drugs such as bute and banamine may actually inhibit the effect of platelets so they should not be used after platelets are injected.
Most platelet products yield 10 ml per attempt. Any extra platelet rich plasma can be frozen and used on subsequent treatments if needed.
Autologous Conditioned Serum (IRAP)
The autologous conditioned serum system uses glass beads exposed to chromium sulfate as a method to stimulate the horse’s own white blood cells to produce an "anti-inflammatory soup."
It has been shown that when horses joints are damaged there is a release of an inflammatory factor into the joint that greatly increases the risk of long term arthritis occurring. This arthritis causing factor is a cytokine called IL 1 (interleukin 1). Luckily there is a natural antagonist in the horse’s body to this mediator of arthritis. That antagonist is called IL 1 receptor antagonist (IRAP). Studies have shown that IL 1 receptor antagonist provides potent joint protection from IL1.
There has been much work done to find methods of increasing IRAP in the joints of horses and people to prevent or treat arthritis. The most practical method developed so far is the use of glass beads that stimulate white blood cells to produced high levels of IRAP. The serum is then harvested and injected into the joint that is damaged.
A study performed at Colorado State University in 2005 showed a measurable improvement in arthritic scores in horses treated with IRAP compared to those that did not receive the conditioned serum.
References:
D.D. Frisbie, C.E. Kawcak and C. W. McIlwraith, Evaluation of Autologous Conditioned Serum Using an Experimental Model of Equine Osteoarthritis. Proceedings of 51 Annual Convention of the American Association of Equine Practitioners - AAEP, 2005 - Seattle
Lisa A. Fortier, DVM, PhD, ACVS. Stem Cells for cartilage and tendon regeneration. Proceedings of the 11th International Congress of the World Equine Veterinary Association, 24 – 27 September 2009, Guarujá, SP, Brazil
Richardson, Dean W. “They fix horses don’t they?” The past, present and future of equine fracture repair. Proceedings of the 47th British Equine Veterinary Association Congress BEVA
Sep. 10 – 13, 2008 Liverpool, United Kingdom
Clegg, Peter. Tissue engineering of tendon. Proceedings of the European Society of Veterinary Orthopaedics and Traumatology 2008 - ESVOT, Munich, Germany
Ferris, Dora J., Frisbie, David D., etal. Clinical Follow-Up of Horses Treated With Bone Marrow–Derived Mesenchymal Stem Cells for Musculoskeletal Lesions. AAEP PROCEEDINGS _ Vol. 55 _ 2009
Prades, M. Current Trends in Regenerative Medicine for Soft Tissue Musculo-skeletal Injury. Proceedings of the Southern European Veterinary Conference & Congreso Nacional AVEPA, 2007 - Barcelona Spain, Southern European Veterinary Conference (SEVC) and Asociación de Veterinarios Españoles Especialistas en Pequeños Animales (AVEPA) (Eds). Publisher: SEVC-AVEPA, 21-Oct-2007.
Fortier, Lisa A. DVM, PhD, ACVS. Medical therapies for tendonitis. Proceedings of the 11th International
Congress of the World Equine Veterinary Association, 24 – 27 September 2009, Guarujá, SP, Brazil
