Andy Hoffman, D.V.M., D.V.Sc., D.A.C.V.I.M., Professor, Director, Lung Stem Cell Laboratory and Jose Garcia-Lopez, DVM, DACVS, Associate Professor.

The clinical scenario:

Charger, a 9-year-old Oldenburg gelding, presented to clinicians at the Hospital for Large Animals at the Cummings School of Veterinary Medicine at Tufts University for non-resolving hind limb lameness. During evaluation, Charger displayed excessive toe dragging of the rear limbs and abduction (outward swinging) of the right hind. When trotting, Charger exhibited a 2 out of 5 (0= sound; 5= non-weight bearing) right hind limb lameness, with a positive upper limb flexion test (indicating pain in the upper portion of the leg).

Image I: Ultrasound images of normal joint cartilage and a subchondral bone cyst.

In order to localize the problem better, a regional nuclear scintigraphic evaluation (bone scan) was performed along with radiographs and ultrasound. Results of the bone scan showed a localized and active region of radioisotope uptake (hot spot) at the level of the inner knee joint (medial femoral condyle) of the right hind limb. This was confirmed to be a cyst beneath the knee joint cartilage via radiographs and ultrasound of the region (subchondral bone cyst in the right medial femoral condyle, SEE IMAGE I). Ultrasound demonstrated no damage to the surrounding soft tissue structures.

Based on these findings an arthroscopic debridement of the cyst and injection of autologous stem cells (from the same animal) derived from a fat sample was recommended. Fat was harvested from Charger’s right croup, shipped to and processed by a private company to isolate ‘stem cells’, which were shipped back within 48 hours for subsequent injection.

During arthroscopic surgery, the subchondral cyst was cleaned down to healthy bleeding bone (SEE IMAGE II). The joint was thoroughly cleansed and stem cells were inserted into the defect along with a platelet rich plasma (PRP) clot to ensure position was preserved.  The incisions were closed and recovery was uneventful.

Image II: Endoscopic images of a femoral cyst (a), which was debrided (b) and then packed with 10 million, bone marrow derived stem cells suspended in platelet rich plasma and fixed with fibrin (c).

Following surgery, Charger was placed on short term intravenous antibiotics and anti-inflammatories (Phenylbutazone) until being discharged. The postsurgical examination at months two and four showed adequate healing of the cyst. The horse was returned to his previous level of athletic activity within six months following surgery.

This case demonstrates how autologous stem cells can be derived from fat tissue for use in joint disease.  These same fat-derived stem cells in addition to stem cells derived from bone marrow and even umbilical cord tissue have been widely employed to treat a variety of soft tissue injuries in horses.   The question remains about how to optimize these therapies.  Which stem cell types are best suited for which injuries?    By what methods should cells be handled, processed, even cultured? In this case, cells were directly (enzymatically) removed from fat and shipped back for injection.  Questions remain about how best to address purity, viability and potency of stem cells which are handled, processed and shipped.    However, the case demonstrates that stem cells have potential to be safely transplanted and to contribute to healing of a cyst that is traditionally very difficult to treat.

José Garcia-Lopez – VMD, Diplomate ACVS

Dr. Garcia-Lopez

A fascination with equine athletes and sports medicine drew Dr. Jose Garcia-Lopez to join the faculty of the Cummings School of Veterinary Medicine and its Hospital for Large Animals, where he specializes in lameness, orthopedic and respiratory surgery for its equine patients.

Dr. Garcia-Lopez’s areas of clinical expertise and interests include orthopedic and upper respiratory surgery, as well as innovative lameness evaluation using advanced imaging techniques such as nuclear scintigraphy, MRI and CT. He further focuses and advances novel treatment modalities, which include procedures using Stem-cell, PRP (Platelet Rich Plasma) and IRAP (Interleukin-1 Receptor Antagonist Protein) therapy. In addition, his area of research expertise includes development and testing of new instrumentation and implants used in equine orthopedic surgery. He continues to improve and develop new techniques and instrumentation which aid in management and treatment of fractures and joint injuries.

Andrew Hoffman – DVM, DVSc, Diplomate ACVIM

Dr. Hoffman

Dr. Andrew Hoffman is a Professor in the Department of Clinical Sciences and Director of Regenerative Medicine.   His research interests include the fundamental biology of stem and progenitor cells and their potential application in cell therapeutics.  Projects in the Regenerative Medicine Laboratory involve over 12 faculty and 5 trainees with a focus on the safety and efficacy of stem cells used for treatment of autoimmune diseases and traumatic injuries to tissues. ”

Andy Hoffman, D.V.M., D.V.Sc., D.A.C.V.I.M., Professor, Director, Lung Stem Cell Laboratory and Jose Garcia-Lopez, DVM, DACVS, Associate Professor.

Understanding Stem Cell Potency: Can the Veterinary Profession Do Better?

Stem cells have now captivated the veterinary profession and for good reason.  These cells produce a vast array of molecules that promote repair and regeneration of damaged tissue. The signals produced by stem cells are so numerous and complex that they cannot yet be reproduced in the laboratory.  Harnessing their potential is the central focus for many laboratories around the world. The hope is that stem cells will revitalize tissue in healing wounds, and reduce inflammation, fibrosis, and auto-immunity. It’s a tall order but there is good evidence from animal models that stem cells or their derivatives can accomplish these goals. 

It’s important to keep in mind that veterinary stem cell research is in the beginning phases, every new group of data is exciting and at times there are unforeseen circumstances that may change the anticipated outcome of the research.

Image III: Equine mesenchymal stem cells in culture.

While the science of animal stem cells is in its early stages, stem cell purveyors have emerged. Thus, a veterinarian may procure a kit to harvest bone marrow or fat, and a company will isolate mesenchymal stem cells (MSCs) and send them back overnight, for delivery to the patient. To do so, the stem cell companies have to overcome several logistical barriers in order to deliver efficiently and reliably, a product which is also safe. Recent data supports the clinical benefits of commercially derived MSCs in clinical trials (e.g. superficial digital flexor injury). Although company websites quote that a majority of horses respond to their stem cell therapies, durable benefits directly attributable to stem cells have been very difficult to demonstrate, and trial design has been limited. Nevertheless, there is increasing evidence that MSCs improve healing, in ways that we do not completely understand.

In contrast to the tight regulation of human stem cell commerce, regulatory agencies (FDA, USDA) have not yet prioritized regulation of the development of animal stem cells, nor have they promulgated regulations that address interstate commerce of animal stem cells.  For emerging therapeutics, commercialization ordinarily speeds up progress.  Owing to the lack of regulation at present, incentives are lacking for commercial entities to evaluate potency, or the use of allogeneic (mismatched) versus autologous (patient specific, matched) cells. University and local laboratories are likely to play key roles in the design and development of more effective stem cell therapies, since veterinary development of these products follows that of human products in this class. For example, the National Institute of Health through PACT (Production Assistance to Cell Therapies, www.pactgroup.net) is becoming a preferred venue for human clinical trials.

Finally, as a veterinary profession, we need to better understand the mechanisms of action of stem cells, and thus how to define their potency.  Recent research shows that “whole cells may be inessential” to many of the therapeutic effects of stem cells, i.e., lysed cells or concentrates of conditioned media used to growth the cells perform just as well.  Stem cells release chemicals and ‘exosomes’ (secreted biologically active packets of membrane bound proteins and nucleotides) into the surrounding media or tissues that contain much of the healing properties. Thus we need to consider potency of the cells and their secreted products equally.

In summary, when new and improved stem cell therapies with well-defined efficacy and potency endpoints are developed, the veterinary profession will enjoy an incredibly powerful tool to improve the health of animals.

It is uncanny how some of the smallest, seemingly innocuous wounds can become the most life-threatening. Those located around synovial structures (joints, tendon sheaths and bursas) often fall into this category. Early recognition of synovial structure involvement followed by aggressive therapy wins the battle in many cases, but not all. The statistics are worse when recognition or therapy is delayed. Having owners who are caring but also knowledgeable about limb anatomy is an important first step. Owners are often the first responder – making the decision on whether to contact their veterinarian.  It is an easy decision to make when blood is spurting, flesh is hanging, or bone is visible but not so easy when the wound is a puncture or just an inch or so in length. Perhaps some of the following information will be helpful in highlighting the importance of small but badly placed wounds.

Puncture wounds are often overlooked as harmless, but objects that create punctures (nails, tree branches, pitch fork tines, etc) usually travel beyond skin level into deeper tissues and may tract considerable distance away from the entry site.  A good example of this is a “street nail.” The “stepped-on nail” can penetrate just the insensitive lamina or extend deeper to the digital cushion, the coffin bone, or one to all three synovial structures (coffin joint, navicular bursa, digital flexor tendon sheath) located within the foot. Although the damage to the tissues is initially limited to the path of the penetrating object, and the horse may be reasonably sound, bacteria carried in by the object can multiply and invade surrounding tissues. When combined with soil particles, only a few bacteria are needed to overwhelm the horse’s ability to fight off a developing infection on its own. Determining the path of a penetrating object through the foot can be simple if the object is still in place, but much harder if the foreign material has been removed.  In the latter case, contrast radiographs may be obtained using either iohexol (a radiographic dye) or a sterile surgical probe if the entrance to the tract is still visible. Other diagnostics which can be useful in more chronic cases include synovial fluid sampling of the coffin joint and tendon sheath, to evaluate the cellularity of the synovial fluid, ultrasound imaging of the coffin joint, navicular bursa, and tendon sheath or the use of MRI.  Ultrasound and MRI imaging can eliminate the risk of iatrogenically spreading an infection into a synovial structure if it is not already present.

Once confirmed that the puncture wound involves a synovial structure, aggressive therapy is required to eliminate  infection and debris from within the space to avoid the destruction of tendons, cartilage and bone and to minimize the development of scar tissue which can alter movement. Treatment options have evolved over the past several years to include more refined surgical approaches to the navicular bursa which minimize further damage to the soft tissues of the foot, provide better delivery of potent antibiotics using regional limb perfusion and reduction of inflammatory mediators within the synovial space through the use of conditioned serum products such as IRAP.  Treatment is also directed at preventative measures against support limb laminitis while the injured foot regains its original soundness.

A successful outcome is dependent on early recognition, a global approach to therapy, and a bit of good fortune. Remember that although we were brought up to believe that good things come in small packages, this is not an adage that should be applied to punctures and small wounds.  The right thing is  always to assume the worst and be appreciative when it isn’t.

Infection of synovial structures may involve joints (septic arthritis), tendon sheaths (tenosynovitis) or a bursa (bursitis), which is a fluid-filled cavity situated within tissues exposed to friction. The infection occurs when microorganisms are given time to colonize the space after a penetrating wound, a joint injection, following surgery or following a spread of bacteria via the blood stream (hematogenous infection).  Although common in the foal, hematogenous spread is rare in the adult horse but has been reported to occur (< 10% of cases). Therefore, synovial infection should be considered as a potential differential diagnosis in any horse with sudden onset of lameness, synovial effusion (fluid buildup) and fever.  Horses with drainage through communicating wounds will generally be less lame than those without drainage.  Therefore, it is important to rule out possible synovial structure communication with a wound at the time of injury rather than wait to see if the horse becomes lame.  Many times, these horses do not become significantly lame until 7-10 days later when the overlying wound has closed sufficiently to prevent synovial drainage.

A diagnosis of joint, tendon or bursa infection is generally confirmed through fluid analysis and culture.  Normal synovial fluid has less than 300 white blood cells/dL and contains less than 2.5 g/dL of protein.  More importantly, lymphocytes are the predominant cell type at 90% or greater.  Difficulty may arise in differentiating periarticular trauma (inflammation surrounding the joint) and joint infection in early cases of infection when total white cell counts are not significantly above the normal range.  However, if the percentage of inflammatory cells (neutrophils) is high infection should be assumed.

Synovial fluid obtained for cell count should also be submitted for bacterial culture. Isolation of the causative agent can confirm the diagnosis of infection but more importantly it can guide antimicrobial treatment. Blood culture bottles with enrichment medium optimize isolation rate (~77 % vs. 37% with agar plates). The medium used in blood culture bottles contain lytic agents which release engulfed bacteria enabling them to be cultured. In addition the medium also contains SPS (sodium polyanethole sulphonate) and/or resins which absorb antibiotics which if they have been previously given to the horse would delay bacterial growth.

The initial treatment of a potentially septic (infected) synovial structure should include broad spectrum antimicrobials directed at both aerobic and anaerobic micro-organisms. The source of the infection should be taken into consideration. In traumatic wounds, members of the family Enterobacteriacaea are most likely to be found but Streptococcus, Staphylococcus, and anaerobes (those bacteria surviving in the absence or oxygen) may also be present. Traumatic wounds generally result in a mixed population of bacteria. Horses which develop infection following synoviocentesis (joint taps) or surgery are likely to be infected with Staphylococcus. It should be remembered though that fungal infections, however rare, do occur in horses and should be considered in patients that are not responding to antibiotic therapy as anticipated.

The treatment of septic arthritis (joint infection), tenosynovitis and bursitis (infection or inflammation of a tendon sheath or bursa) has greatly improved over the last decade which has led to improved outcome. This in large part is due to early aggressive treatment, availability of a greater variety of antimicrobial choices, and improvement in the delivery of antibiotics to the site of infection.

Dr. Patricia Provost

Whether mentoring future veterinarians, treating sick or lame horses and other large animals, or performing research for the advancement of veterinary care, Dr. Patricia Provost joined Tufts to make a difference in the lives of animals. Dr. Provost is a board-certified surgeon (Diplomate of the American College of Veterinary Surgeons (ACVS)

Prior to joining the faculty at the Cummings School of Veterinary Medicine, Dr. Provost spent a brief time first in private general equine practice and then at a thoroughbred racetrack. She left private practice to join Tufts to work with some of the finest horses in the country, doing treadmill lameness and respiratory evaluations.

Dr. Provost’s areas of clinical expertise and interests include musculoskeletal disorders in foals, uncommon lameness, neoplasia, and gastrointestinal surgery. Her area of research expertise includes wound healing, use of growth factors for soft tissue, ligament and corneal wound healing, skin tumors, and back disease. Her current research effort is directed at treatment of Methicillin resistant Staphylococcus aureus, or MRSA, in horses. She continues to dedicate much of her time to the improvement of healing in equine animals because it is an important component to all types of surgery.

Dr. Provost is the author of several textbooks and also lectures on a wide variety of surgical topics in horses, cattle, camelids, and small ruminants.

Meet the HLA Team – http://vet.tufts.edu/hla/faculty/

Hospital for Large Animals

The contrast material fills the navicular bursa.

Nagel, a four year old Quarter Horse gelding, presented to the Tufts Hospital for Large Animals at the Cummings School of Veterinary Medicine after stepping on a nail, which entered the outer aspect of his right hind sole. Initial home treatments included daily foot soaks, phenylbutazone (an anti-inflammatory) and a course of antibiotics. Unfortunately, Nagel’s lameness returned despite his owner’s supportive and diligent home care. Radiographs taken by the referring veterinarian showed a penetrating wound that was tracking to the navicular bursa, with a small volume of gas observed within the bursa. Contrast injection into the site of nail puncture confirmed a communication of the wound with the navicular bursa. At that time, Nagel was referred to Tufts for exploratory arthroscopy.At the time of presentation, Nagel was quite lame (4 out of 5) on the right hind leg. Based on the available diagnostic findings, Nagel was placed under general anesthesia for an arthroscopic examination of the right hind navicular bursa. At the time, a moderate amount of fibrinous material was removed. The wound tract was then thoroughly lavaged with saline using a high pressure system and debrided. In addition, regional limb perfusion (RLP) was performed using the antibiotic Amikacin.

Nagel was hospitalized after surgery for continued broad spectrum intravenous antibiotic and anti-inflammatory therapy. Regional limb perfusion was performed at several time points following surgery with alternating antibiotic use. To further support Nagel during the healing process and to prevent further contamination of the wound, a foot cast was placed on the right hind limb. Following placement of the cast, Nagel ambulated comfortably at the walk. Due to the care and support of Nagel’s owners, referring veterinarian and clinical staff at Tufts Hosptial for Large Animals, Nagel  healed thoroughly  returning to athletic performance.

Regional Limb Perfusion (RLP)

Regional limb perfusion (RLP) is a technique which provides a high concentration of an antibiotic to the soft tissues, joints, and bones of the limbs. The antibiotic used would be very expensive if given at a dose appropriate for the entire horse but the cost of the drug is more affordable since it is delivered into a superficial leg vein below the level of a tourniquet, and the benefits great. Drug concentrations achieved generally exceed that required to kill many types of bacteria and persist locally in the tissues for an extended period of time. This makes RLP a practical adjunctive treatment for septic conditions affecting the extremities (the knee and hock and below).

To perform, the horse is lightly sedated and the tourniquet is applied above the level of the injury.  Once the skin over the distended vein is cleaned, the drug, diluted in saline, is slowly injected, via an IV catheter, into the vein. The tourniquet is left in place for an additional 20 minutes, which prevents the antibiotic from quickly leaving the affected area. Once the tourniquet and the catheter are removed, the injection site is temporarily bandaged. The procedure may be performed daily, alternating the type of antibiotic, or every other day using the same antibiotic. Drug selection and frequency of delivery are determined by the clinician based on the results of the bacterial culture and drug sensitivity patterns.

The contrast material fills the navicular bursa.

CONTRAST STUDIES

This radiograph of the foot shows a horse which stepped on a nail. Radio-opaque contrast material was injected into the puncture wound which was located in the medial sulcus (inner side) of the foot.  The contrast material fills the navicular bursa (arrow) confirming that the nail tract had communicated with the bursa.  The horse responded positively to navicular bursoscopy and administration of systemic and regional limb perfusion antibiotic therapy.

IAD is an inflammatory condition of the airways that is triggered by exposure to a high level of particulates in the air, such as dust, mold, pollen and spores. Studies have shown that feeding – especially hay and pelleted feed – is the most important contributor to inhaled dust. Other important sources are cleaning stalls and sweeping the aisles. Exposure can be reduced by using “processed forages” such as Denge, or soaking the hay for 15-20 minutes before feeding to minimize dust levels. Another way to reduce barn dust exposure is to sprinkle the aisles with water before sweeping.

As a general rule, 24-hour turnout with access to a run-in shed is highly beneficial for many patients. If housed indoors, affected horses should be stabled in a well ventilated stall without hay being stored in their vicinity. It is especially important to avoid storing hay overhead. We recommend that horses with IAD stay outside until one hour after feeding time and while the barn is being cleaned, as these are time when the dust levels are highest. During the summer, pollen and mold spores can sometimes be higher outside than inside, which may require horses to stay inside when the air quality was poor.

IAD is a disease of the airways which disturbs normal lung function (most often causing exercise intolerance) in addition to acting as a source of irritation (causing cough). Clinical signs are associated with swelling or obstruction and thus narrowing of the lower airways, which prevents air from reaching the maximum number of air sacs (alveoli) for gas exchange. In horses suffering from IAD, the airways are narrower because of inflammatory secretions, excessive mucus and fibrous tissue. In order to overcome the resulting increase in airway resistance, the horse has to work harder to achieve each breath and ultimately becomes exercise intolerant.

Horses with IAD most commonly present with a history of cough (which may be triggered by exercise), decreased performance, higher breathing rate or nasal discharge (usually clear to mucoid). The clinical signs are usually subtle and can affect horses of any age. In the worst case, long standing airway inflammation may lead to development of “heaves” (also known as recurrent airway obstruction or RAO), which tends to affect mature to older horses. Horses with heaves may even present to the veterinarian in an emergency setting, as affected horses exhibit significant lower airway inflammation and obstruction resulting in markedly increased work of breathing. When horses have an exacerbation of RAO, their whole bodies can be seen to rock with the effort they expend in breathing. Guidelines for the definition, diagnosis and treatment of IAD and RAO were published in 2007 by the American College of Veterinary Internal Medicine, under the co-authorship of Dr. Andrew Hoffman at Tufts University.

Horses can develop inflamed airways in response to particulates in their environments, such as plant pollens, and the molds and spores found in even the best of hay. Subsequently, the lung mounts an inflammatory response with its full complement of cells and mucus. Eventually, the tissues thicken, and permanent obstruction may occur. Airway hyperreactivity may also be triggered by a viral disease, such as equine influenza, for months after the primary disease has resolved. You may note this as a persistent cough, especially during exercise, because inflamed airways are more reactive, or “twitchier” than those of normal horses. This means that the airways, which are already narrowed, constrict even more in response to a number of stimuli. These stimuli may be the same particulates that initiated the original airway inflammation (such as molds and pollens), cold air or gases, such as ammonia.

The diagnosis of IAD is best based on lung function testing and bronchoalveolar lavage (BAL), to direct therapy and monitor treatment success. Although IAD is rarely a curable disease, clinical signs and airway obstruction may be alleviated by administration of inhaled or systemic corticosteroids, bronchodilators and mast cell stabilizers or changing the environment. In this context, pulmonary function testing has been utilized to better monitor and characterize the disease severity, evaluate the treatment response and objectively guide the long term management of horses with airway inflammation, in order to preserve athletic performance and health.