By Chris O’Sullivan, BVSc, Dipl VCS, MS, MACVSC; Dip ACVS

The future looks promising

Stem cell therapy and research has been topical in the media over the past decade. While there is promise being shown for the treatment of a variety of different disease processes in man, the science behind the use of stem cells is still in its infancy. There are no commonplace stem cell therapies available at the moment and most stem cell therapy is still considered experimental. Horses are at the cutting edge of this new emerging science with stem cell therapy being used for the treatment of tendon and ligament injuries. The first cases were treated in Australia over four years ago and since then approximately 130 horses have been treated. Bearing in mind that this is a relatively small treatment group, the results have been promising to date.

Stem Cells What are they?

A stem cell is a type of unspecialised cell that has the ability to be transformed into any number of different specialised cell types. In other words, from a stem cell a variety of different tissues may be created such as muscle, bone, tendon, cartilage, skin or nerve to name a few. Potentially stem cells may be used to reproduce the majority of different tissues in the body.

Stem cells are also typically able to continue dividing and renewing themselves in cell culture (in the laboratory) for a prolonged period of time. They can be stored long term by freezing, allowing thawing at a later date and subsequent multiplication in cell culture.

Stem cells can be obtained from a variety of sources and are named differently as to their source. Embryonic stem cells are obtained from an embryo typically at day 5 after fertilisation when the embryo has about 200–250 cells. This typically means destruction of the developing embryo in order to harvest the cells. Thus, the moral and ethical dilemma created when human embryonic stem cells are used for both therapy and research has been the focus of much debate.

Adult (somatic) stem cells are obtained from adult tissues including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, fat, skin and liver. Adult stem cells are in very low numbers in these tissues, but when samples of these tissues are collected, stem cells can be harvested from the adult, cultured and grown up to large numbers. Stem cells are also present in the umbilical cord blood at birth.

Stem Cells for treatment of ligament and tendon injuries in horses

Currently in Australia, stem cells are obtained from one of two sources. Umbilical cord blood after foaling where the stem cells are harvested from the placenta and are then frozen and kept until later in the horse’s career when they may be required for treatment. More commonly stem cells are harvested from adult horses’ bone marrow when needed to treat a tendon or ligament injury.

Only certain types of tendon or ligament injuries are suitable for treatment with stem cells. A thorough ultrasonographic examination of the tendon or ligament is required to assess the severity and location of the lesion. Based on the ultrasonographic examination and history, the suitability of the lesion for stem cell treatment is decided. This examination should ideally take place within a couple of days of the injury, and a decision for stem cell therapy should be made at this time. Since there is a lag time for collecting and growing the cells of approximately 3–4 weeks and this is the ideal time frame for re-implanting the stem cells at the injury site.

Ultrasound examination is an essential part of assessment of a tendon.

An ultrasound image showing the transverse view of a severe tendon lesion at the mid-level of a horse's
superficial digital flexor tendon. This type of lesion is an ideal candidate for treatment with stem cell therapy.

When a tendon or ligament injury occurs two scenarios exist. In the case of a horse that has had cord blood taken as a foal, the stored cells are thawed and grown up in culture over two to three weeks. Alternatively, bone marrow is harvested from the injured horse and shipped to the lab for culture of stem cells. There are no results at this stage suggesting cord derived stem cells are superior to bone marrow derived cells for the treatment of tendon and ligament injuries. While there is a very marginal advantage in the time to grow up sufficient cells from frozen cord blood, the cost benefits of having stored cord blood cells appears poor for the typical competition horse.

Harvesting bone marrow is a relatively simple procedure and is usually performed in the standing sedated horse. The marrow is obtained from the horse’s sternum (breast bone) using local anaesthesia. Ultrasound is used to identify the ideal area from which the bone marrow harvest is obtained and a bone marrow needle is used. While the procedure has a very low complication rate in the hands of trained experienced operators, it is not without risk. There have been reports of inadvertent damage to the heart leading to death.

Harvesting bone marrow from the sternum (breast bone) of a standing, sedated horse under local
anaesthetic. The marrow is being aspirated into the syringe through a bone marrow needle.

Once the marrow is harvested, it is shipped to a lab in a specially designed cooled transport pack. A company in South Australia, Vet Biotechnology, provides the service for veterinarians. At the lab, the stem cells are separated from the bone marrow and are then grown up in tissue culture. Once a sufficient number of cells have been grown (i.e. between 6 and 20 million cells depending on the size of the lesion) the cells are resuspended in non-cellular bone marrow fluid (bone marrow supernatant) and shipped back to the veterinarian. The cells are then injected using ultrasound guidance directly into the tendon or ligament injury ideally between 3–6 weeks after the original injury. The implantation is usually performed in the standing sedated horse under local anaesthesia. Occasionally general anaesthesia is required particularly for lesions below the fetlock.

Bone marrow after collection.                                                            Stem cells under a microscope.

Once the stem cells have been implanted a rehabilitation program is implemented. During the rehabilitation period the tendons healing is assessed regularly ultrasonographically prior to any major increases in workload. Stem cell therapy does not increase the speed of healing of a tendon or ligament injury, it is aimed at improving the quality of the repair tissue. Therefore as with most major tendon injuries the rehab time back to competition is still approximately 12 months in the majority of cases.

Results of Stem Cell Therapy in Australia

While stem cell therapy must still be considered experimental there are growing numbers of horses being treated. In Australia to date 133 horses have been treated and the majority of these – 128 – have been racehorses. At this stage only 61 horses have completed the rehabilitation time (i.e. 12 months post treatment) of which 74% have returned to full work and 64% have returned to competition at this stage. A total of 6 horses have re-injured the original tendon or the opposite limb. These figures to date are promising compared with previous treatments. However, the numbers are still low and only time will tell if this promising trend will continue as more horses are treated.

Bone marrow injection is different to stem cell therapy

Bone marrow injection was popularised in the U.S.A in the late 1990’s for treatment of ligament and tendon injuries. It involves obtaining bone marrow and injecting bone marrow directly into a tendon or ligament injury. This technique is a very crude form of stem cell therapy and should be called bone marrow injection rather than stem cell therapy. The numbers of stem cells are very low with this technique with only about 1 in 100 000 cells in the marrow actually being a stem cell. There have been complications reported with the injection of bone marrow directly into tendons, including mineralisation (calcification) of the tendon. Stem cell therapy, on the other hand, discards these other cells and then grows up only the stem cells to much greater numbers than are present in the marrow alone.

What does the future hold for stem cell therapies?

Currently stem cell research in the horse is ongoing, with stem cells being used experimentally for replacement of damaged joint surfaces in OCD like lesions and traumatic cartilage damage. It is likely as with any new therapy that with time the treatments will evolve.

Advances in the use of growth factors and gene therapy combined with stem cells will likely broaden applications and provide greater treatment efficacy in the long term. Based on the current experimental and clinical knowledge the future looks promising.

Chris O’Sullivan BVSc, Dipl VCS, MS, MACVSc, Dip ACVS

Chris is a registered specialist equine surgeon at Randwick Equine Centre in Sydney. His interests include: performance horse lameness evaluation, diagnostic imaging (Digital radiography, ultratonography and scintigraphy), orthopaedic and soft tissue surgery. Chris has written numerous journal articles, proceedings and book chapters.

Captions

Ultrasound examination is an essential part of assessment of a tendon

An ultrasound image showing the transverse view of a severe tendon lesion at the mid level of a horse’s superficial digital flexor tendon. This type of lesion is an ideal candidate for treatment with Stem cell therapy.

Below: Harvesting bone marrow from the sternum (breast bone) of a standing sedated horse under local anaesthesia. The marrow is being aspirated into the syringe through a bone marrow needle.

Above Left: The bone marrow after collection.

Above: Stem cells examined under the microscope.

(These equine stem cells are part of a research project between Randwick Equine Centre and Dr Clive McFarland and Pankaj Godara from the Graduate school of Biomedical Engineering at The University of New South Wales.)