New therapies for the treatment of equine lameness (Proceedings)

Stem Cell Therapy

Stem cells are primitive cells present in almost every tissue that have the ability to differentiate into different types of tissue. There are basically two types of stem cells that can be utilized: embryonic and adult stem cells. The source of embryonic stem cells is the inner mass of the embryo, so that in the process of recovering the cells, the embryo is destroyed. These cells have the ability to differentiate into all tissues (toti-potent). When used in clinical situations, the use of embryonic stem cells introduces foreign DNA, so some form of immunosuppression would be necessary. Experimentally, embryonic stem cells are unpredictable and can form teratomas in-vivo. The source of adult stem cells is any adult tissue. These cells can differentiate into many, but not all tissues (multi-potent). The tissues that are of most interest to equine practitioners include bone, muscle, tendon, and ligament. These cells have never been seen to form teratomas or tumors in vivo.

Stem cell therapy has been coined "regenerative medicine" because the goal is to regenerate tissue and provide tissue homeostasis, rather than just healing by fibrosis. Tissue regeneration should maximize the strength, range of motion, and performance of the tissue. At the same time regeneration of tissue reduces scarring, pain, and re-injury. Regenerative medicine supplies the three major components necessary for tissue repair: regenerative cells, extra-cellular matrix, and growth factors. The regenerative cells also recruit other cells necessary for tissue healing.

The two major sources of adult stem cells include bone marrow and adipose tissue, although muscle, skin, brain and other tissues contain adult stem cells. It is estimated that 1 in 50 cells (2%) in adipose tissue are stem cells, whereas 1 in 100,000 cells (0.001%) in bone marrow are stem cells. In addition to the high percentage of stem cells in adipose tissue, fat tissue is easy to access in the horse, is a renewable resource, and because fat floats, it is easy to separate the cells. In addition to providing mesenchymal stem cells, adipose tissue can also provide endothelial precursor cells, growth factor producing cells, B and T lymphocytes, macrophages, natural killer cells, preadipocytes, fibroblasts, smooth muscle cells, and endothelial cells. It is not known what role these other cells play in tissue regeneration. Currently, the FDA does not regulate the use of autologous adipose derived stem cells if they are minimally manipulated. Culturing of stem cells to propagate a cell line is not considered minimally manipulating cells so therefore is regulated by the FDA.

The potential uses for stem cell therapy in equine medicine include soft tissue disorders (tendon/ligament), osteoarthritis, osteochondrosis, fractures, or other degenerative processes such as laminitis. In my practice, I will use adipose derived stem cell therapy primarily for tendon, suspensory, or suspensory branch lesions. I have used these cells for other indications including osteoarthritis, navicular disease, and osteochondrosis, but the results are not as favorable for bone or joint problems as they are for soft tissue disorders.

Fat tissue can be harvested from any site on the horse, but lateral to the tail head seems to be most appropriate. This can be readily done through a small lipectomy incision in the standing animal with local anesthesia. This is the most consistent method of collecting fat, but will leave a scar and a small depression at the surgical site. If cosmetics are of major concern, the inguinal area is also rich in adipose tissue, but this requires general anesthesia. More recently, I have been performing liposuction at the tail head using liposuction cannulas designed for human patients. This technique is most appropriate for horses having at least 1 cm of tail head fat as measured by ultrasound. Liposuction is performed bilaterally through very small punctures in the skin, so the post operative cosmetic outcome is excellent. I use ultrasound to guide my liposuction cannulas so that the contamination with muscle tissue is minimized.

The tissue is sent overnight on ice to the laboratory, where the fat is digested, leaving regenerative cells and their supporting cells and matrix. The dose volume can be requested based on the lesion being treated. Typically, a dose of approximately 5 million cells are recommended to treat a single lesion. If a large quantity of cells is recovered, additional doses can be frozen for later use. The turnaround time from fat harvest to arrival for injection is 3 days. When the cells arrive, they are typically injected into the desired lesion using ultrasound guidance following aseptic preparation of the area. Depending on the site and condition, I often combine the stem cells with platelet rich plasma at the hospital just prior to injecting. The aftercare is minimal, as the cells are autologous so there is no detectable tissue reaction. Follow-up examinations and rehabilitation are tailored to the individual and problem being treated.

IRAP

The acronym "IRAP" stands for interleukin receptor antagonist protein, and is sometimes referred to as "ACS" for autologous conditioned serum. IRAP was first used in human joints following arthroscopic surgery for osteoarthritis. The concept behind its use is that cytokines and growth factors are produced in joint tissues and released into the synovial fluid. These chemicals are necessary at low levels for normal joint homeostasis, but this balance is disrupted when there is trauma or a disease process such as osteoarthritis. Interleukin 1 (IL-1) is the major pro-inflammatory cytokine in joints as well as in all tissues.

To counteract the effects of IL-1, IRAP is the major inhibitory cytokine. IRAP exerts its effect by occupying the receptors of IL-1, thus not allowing it to exert its inflammatory effect. IRAP is secreted from monocytes, macrophages, neutrophils, and other cells.

It was found that the production of IRAP in the blood can be amplified by a process called physicochemical induction. This process involves incubating whole blood in the presence of chromium sulfate treated glass beads. During this process, it was found that there was a vigorous, rapid increase in the synthesis of IRAP and some other anti-inflammatory mediators (IL-4 and IL-10), with no increase in the pro-inflammatory mediators IL-1 or TNF-α.

The kits for IRAP harvesting come with a 60 cc syringe containing the etched glass beads, and a butterfly catheter. The jugular vein is aseptically prepared, and blood is slowly aspirated into the syringe using the butterfly catheter. I prefer to use sterile gloves during the blood collection and then later for harvesting the IRAP. The blood is allowed to mix with the glass beads, and then it is incubated for 18–24 hours at 37ºC. Following incubation, the syringe is centrifuged at 4000 rpm for 14 minutes. The IRAP rich serum is then aspirated through an eighteen gauge spinal needle into a 20 or 35 cc syringe. The serum is then filtered with 0.2 μm syringe filters into 3–5 cc aliquots. Typically 20–25 cc of IRAP can be harvested from a single blood collection. What is not utilized immediately can be stored in the standard freezer for later use.

It was found that a minimum IL-1:IRAP ratio of 1:10 is required to inhibit the IL-1 activity. Therefore it is recommended to drain as much fluid as possible or to lavage the joint prior to injecting the IRAP. IRAP can also be used prophylactically, however it does not stay in the joint or tissue very long. In addition to its anti-inflammatory use in joints, IRAP has also been shown to have important tissue properties in a soft tissue contusion model in laboratory animals.

Clinically, I think the most effective use of IRAP has been for the peri-ligamentous injection ofsuspensory ligaments, particularly the proximal suspensory ligament in the rear limb. This alone or in combination with other therapies has been able return some horses to performance that have not responded to other therapies. I also use a lot of IRAP in joints, but I prefer to have as healthy a joint environment as possible prior to injecting. This may mean injecting an inflamed joint with hyaluronic acid and a corticosteroid 2–3 weeks prior to or surgically lavaging a joint prior to injecting with IRAP. I also like to use IRAP in joints prophylactically. Many of my clients that have horses with problem joints will keep IRAP from those horses stored frozen at the hospital for use before and after major events. I have found that combining IRAP therapy with standard joint injections have kept the joints healthier and prolonged the intervals between standard joint injections.

Extracorporeal Shock Wave Therapy

Much confusion exists in the equine veterinary literature concerning the use of extracorporeal shock wave therapy, as the term "shock wave" has been loosely applied to several different therapies. A true "shock wave" is a high energy pressure wave that is characterized by high positive pressures with a rapid rise time of short duration, followed by a smaller, slower negative aspect. True shock waves have the ability to be focused, and can travel through tissue without losing energy until they reach the desired focal depth, where the energy is released. Other so called "shock wave" machines are in-fact pressure pulse machines that produce unfocused pressure waves. These pressure waves lose energy exponentially (to the 4th power) as they travel through tissue so therefore have only superficial effects. It is important to know the difference between these two types of therapies because the tissue effects are entirely different.

True focused shock wave therapy will be the only therapy discussed in this presentation. The most common use of shock waves is to break down kidney stones in human urology (lithotripsy). This use stimulated research into the effects of shock waves on other tissues. In research animals, it was shown that shock waves have a stimulatory effect on bone formation. Experimental studies have also shown a dose dependent effect of shock waves on wound and soft tissue healing, as well as enhanced neovascularization at bone-tendon interfaces. These findings prompted the use of shock wave therapy for non-urologic conditions in human medicine, including non-union fractures, heel spurs, and enthesopathies. Despite the positive effect of shock waves in various disorders, the exact mechanism of action of shock waves on tissue is still unknown.

The potential indications for the use of shock wave therapy in horses include suspensory desmitis, tendonitis, osteoarthritis, navicular syndrome, subchondral or other bone cysts, stress fractures, dorsal metacarpal periostitis, and wounds. The main advantage of using shock wave therapy is that it is noninvasive and only requires sedation in the standing animal for treatment. When used properly, there should be no deleterious effects on the tissue. I have never seen a side effect from the use of shock wave therapy in thousands of horses. The main disadvantage is the cost of the therapy if it is not effective.

Shock wave therapy should not be used around active growth plates, at soft tissue/gas interfaces (lung or intestine), in infected areas, around the spinal cord or large peripheral nerves, or in areas with suspected neoplasia.

The main indication for shock wave therapy in my practice is for the treatment of suspensory origin, body, or branch desmitis where there is no fiber disruption evident on ultrasound. This therapy is highly effective if case selection is appropriate. I typically use the highest energy level on the machine, and use between 1000–3000 shocks depending on the area of concern. For a stimulatory effect on tendon or suspensory healing where there is evidence of tissue disruption on ultrasound, I will lower the energy to mid-level, and lower the number of shocks to around 500. For assistance in wound healing, 100–200 shocks are used at the lowest energy levels. For bone diseases, such as osteoarthritis and stress fractures, I have found the best results with using the highest energy levels and increasing the number of shocks dramatically (up to 6000 shocks/site) in repeated treatments, modeled after the protocol for non-union fractures in human orthopedics.

Tildren

The active drug in Tildren is tiludronate, which is in the bisphosphonate class of drugs that is used to inhibit bone resorption in the human diseases osteoporosis, Paget's disease, malignant hypercalcemia, bone metastasis, and periodontal disease. This drug can also used as a marker of bone for nuclear scintigraphy.

The basis of the use of Tildren in equine medicine is that there is a continuous remodeling of bone throughout life. In times of intense work or injury, excessive bone remodeling can occur and can lead to osteoarthritis. This excessive bone remodeling can also cause pain and lameness.

Bone resorption is prompted by the activation of osteoclastic precursors. This is followed later by osteoblastic bone formation to repair the defect. Osteoclast activity and osteolysis is never balanced with the new formation of bone, as the bone resorption rate by osteoclasts is approximately 50 μm per day whereas the apposition rate of bone by osteoblasts is only 0.86–1.35 μm per day.

The main indications for the use of Tildren in equine medicine are for diseases with the potential for osteolytic lesions, including navicular disease, bone spavin, bone cysts, insertional desmopathies, osteoarthritis, fractures, and pedal osteitis. The dose rate is 1 mg/kg diluted into 1 liter of fluids administered IV over a minimum of 30 minutes. Because a transient hypocalcemia can occur following administration, the potential for colic due to hypomotility is possible. I therefore administer a dose of flunixin meglumine post-treatment, and restrict feed for 4–6 hours. This treatment can be repeated in 1–2 months, and thereafter every 6 months if necessary.

Following administration, Tildren is rapidly distributed to the bone, and preferably bound to hydroxyapatite crystals at the sites of active bone remodeling. The duration of action is weeks to months, as tildren is slowly eliminated. The use of Tildren as the sole treatment in horses with navicular disease and bone spavin which had not responded to conventional therapies was reviewed, and it was found that approximately 60% of the horses in both groups were sound at the 6 month follow-up.

Tildren can be used as the sole treatment or in combination with other treatment methods. I often combine its use with other treatment modalities to maximize my efforts. I will recommend its use any time that there is evidence of osteolysis on radiographs, but primarily use it in cases of navicular disease, distal tarsitis, proximal sesamoiditis, proximal suspensory desmitis, and pedal osteitis.