Evaluation of Non-invasive Electroarthrography to Predict Cartilage Quality in Equine Osteoarthritis
- 2022-2023 Projects
Adele Changoor and Judith Koenig

Osteoarthritis (OA) is the most common cause of lameness in horses and is characterized by progressive and irreversible cartilage degradation.

Cartilage is the thin tissue that lines the ends of bones in joints and normally enables pain free movement. Early recognition of OA is important to be able to treat and potentially prevent disease progression. Veterinarians could benefit from having a way of identifying cartilage changes that would allow them to objectively monitor OA. Electroarthrography (EAG) is an innovative technology that evaluates cartilage through sensors placed on skin around a joint, such as the equine fetlock. This is similar to the way heart health can be understood using electrocardiography.

The proposed research aims to improve EAG collection to make it easier to use in the clinic, as well as evaluate the ability of EAG to follow OA progression and treatment in an equine fetlock (metacarpophalangeal) model of early OA. We hypothesize that we can streamline EAG collection by creating a read-to-wear device and that EAG will be able to detect cartilage changes over time and show a relationship to magnetic resonance imaging (MRI) and direct observations of cartilage quality using arthroscopy. This research will be the first to demonstrate that non-invasive EAG can be used in a clinical setting to monitor cartilage health and we anticipate EAG will produce comparable results to MRI.

Achieving the study aims would provide a critical link to further develop EAG into a sensitive diagnostic method that could be deployed at the point-of-care and dramatically improve how OA is assessed and treated in horses.

Mesenchymal stromal cells for the treatment of equine tendonitis - 2021-2022 Projects
Thomas G. Koch | Associate Professor | DVM Copenhagen, PhD Guelph

Tendon and ligament injuries are common in the equine athlete. These types of soft tissue injuries result in significant economic loss to the equine industry each year as a result of decreased performance prolonged rehabilitation, and recurrent injury. Even though tendons do heal with rest, the fibroelastic scar tissue formed is functionally deficient in comparison to more flexible tissue of normal tendons. The result is compromised performance of the horse and high re-injury rates.

Currently, tendon injuries are treated in numerous ways including simple lay-off and rest, controlled rehabilitation exercise programs, shock-wave therapy, and biologics such platelet rich plasma and so-called stem cell therapies to name the most common therapies. This range of treatments reflects that no treatment has proven superior, and that adequate tendon repair remains an unmet clinical need.

Culture expanded mesenchymal stromal cells (MSC) have shown promise in reducing the re-injury rate in horses suffering from tendonitis. Improved MSC efficacy may be achieved by extending the dwell time of the cells within the tendon tissue. This has the prospect of healing more horses and extending the careers of more horses suffering from tendon injuries.

Improved MSC efficacy may be achieved by extending the dwell-time of the cells within the tendon tissue. Hyaluronic acid (HA) is used to extend dwell-time of cells within synovial joints. This increased dwell-time is expected to be associated with increased inflammatory modulation within the tissue and improved tissue healing.

Mesenchymal stromal cells for the treatment of equine osteoarthritis - 2021-2022 Projects
Judith Koenig | Associate Professor | Mag Med vet, Dr Med Vet, DVSc, DACVS, DACVSMR

Mesenchymal Stromal Cells (MSC) has been shown to alleviate pain in mild to moderate osteoarthritis (OA) of the fetlock joint. To date, these cells have been from single donor animals which makes the therapeutic response less standardized. We hypothesize that equine umbilical cord blood (eCB) MSC are safe and effective in treating joint pain and inflammation in an injury-induced equine model compared to placebo treatment.

Our objectives are: 1) To compare the clinical safety and efficacy of eCB-MSC with that of saline in an equine fetlock-chip model; 2) To compare joint response to treatment on a molecular level to gain insights into possible mode of action of the eCB-MSCs. These feasibility studies will be conducted in research horses, and the horses will be returned to the research herd upon completion of the study. We expect that the tested cell formulation will not trigger significant adverse reactions following multiple injections.

These results will set the stage for testing in horses with lameness due to fetlock OA.

(EG2021-12) Assessment of safety of encapsulated MSC and pooled MSC in fetlock joints Mesench - 2020-2021 Projects
Thomas G. Koch | Associate Professor | DVM Copenhagen, PhD Guelph

Mesenchymal Stromal Cells (MSC) has been shown to alleviate pain in mild to moderate osteoarthritis (OA) of the fetlock joint. To date, these cells have been from single donor animals which makes the therapeutic response less standardized. Furthermore, the injected cells are often removed from the joint quickly, which likely limits their efficacy. We hypothesize that injection of pooled MSCs from multiple donors within a hydrogel formulation into normal fetlock joints is safe and comparable to injection of non-encapsulated single donor MSCs.

Our objectives are:

1) To compare and contrast the safety of single donor MSCs and pooled MSCs in normal fetlock joints;

2) To compare and contrast the safety of encapsulated MSCs and non-encapsulated MSCs in normal fetlock joints.

These feasibility studies will be conducted in research horses, and the horses will be returned to the research herd upon completion of the study. Through our industry partner, we have access to a novel way to inject MSCs into joints and trap them there by first mixing them with a gelatin that hardens into microspheres small enough to go through a needle. The product is analogous to a slow-release capsule for medication but developed for cells.

We expect that the tested cell formulation will not trigger significant adverse reactions following multiple injections. These results will set the stage for testing in horses with lameness due to fetlock OA.

(EG2021-13) Assessment of safety and potential of antisense miRNA-181 to treat fetlock OA - 2020-2021 Projects
Thomas G. Koch | Associate Professor | DVM Copenhagen, PhD Guelph

Osteoarthritis (OA) of the fetlock joint is a major equine welfare problem across breeds and disciplines leading to loss of training days, pre-mature retirement, or reduced performance. No therapies exist that delay progression of fetlock OA. MicroRNAs are small molecules that control gene expression; increased microRNA levels reduce expression of selected proteins. MicroRNA number 181 has been shown to be overexpressed in human facet joint OA. Injection of microRNA 181 into lab animal joints resulted in joint disease suggesting a critical role of microRNA 181 in joint health. Moreover, injection of antisense miR-181, which binds to and inactivates the effect of microRNA 181 in mice and rat models of joint disease, prevented the expected joint damage. This suggests that antisense miR-181 may be a useful therapy to treat OA.

We hypothesize that injection of antisense miR-181 into equine fetlock joints is safe and associated with reduced joint inflammation and pain. Our objectives are: 1) To determine the safety of antisensemiR-181 in normal equine fetlock joints 2) To determine the safety, anti-inflammatory properties and analgesic effects of antisense-miR-181 in fetlock joints with documented OA and pain. 3) To determine if antisense miR-181 injected into the fetlock joint is detectable in the bloodstream.

We expect that multiple injections of antisense miR-181 will not be associated with significant adverse reactions in normal or OA fetlock joints. We expect that multiple injections of antisense miR-181 will lead to a reduction of miR-181 concentrations within the fetlock joint, reduced lameness, and reduced joint inflammation.

The Safety and Efficacy of Intra-Articular Therapies - 2019-2020 Projects
Mark Hurtig. Professor | DVM, MVSc, Diplomate ACVS

We intend to compare the efficacy of mesechymal stromal cells (MSCs) - previously known as mesenchymal stem cells - versus steroid injections in the equine fetlock for treatment of joint injury and osteoarthritis. This project also addresses the safety of steroid injections in joints which continue to be widely debated in the racing and sporting industry. We know from human studies that injection of MSCs result in better and longer results in the human knee than steroids. Also, preliminary studies in equine stifle and fetlock lameness seem to indicate the MSC therapies improve the recovery of the horses.

Our own work has shown that we can identify the best MSCs from the specific donors and use these cells in joints to reduce inflammation. In addition, the co-investigators in this proposal have shown that MSCs can be thawed, handled and injected without damaging the cells, and that a single injection of similar MSCs can reduce or stop the progress of osteoarthritis in the knee of sheep. Since chip fractures in the fetlock joint are relatively common source of lameness and contribute to cartilage damage, we will create a small chip fracture in the fetlock joint of research horses and follow the progress of physical and metabolic cartilage injury in horses that receive MSCs or steroids. In order make this model more aligned with real-life management of these injuries we propose to exercise the horses in the form of galloping and jumping during the evaluation of the treatments.

Enhancement of Immune-Modulatory Properties of eCB-MSCs - 2019-2020 Projects
Thomas G. Koch. Associate Professor | DVM Copenhagen, PhD Guelph

Horses are multi-use livestock that contribute more than $19 billion annually to the Canadian economy. Joint inflammation due to osteoarthritis or injury is among the most common causes of lost training days or premature retirement in equine athletes. Stem cells hold the promise of novel therapeutic approaches to these difficult-to-treat problems. Mesenchymal stromal cells (MSCs) can be expanded in culture to achieve the desired number for treatment and are capable of modulating cells of the immune system even after long-term storage, which makes them desirable for treatment of immune and inflammatory disorders.

We expect that equine cord blood-derived (CB-) MSCs can be enhanced to have improved anti-inflammatory and immunomodulatory properties for future use as treatment of a variety of inflammatory or immunerelated disorders in horses. Our sole objective of this study is to determine possible in vitro enhancement of equine CB-MSCs using a novel proprietary culture engineering method to improve these immunomodulatory properties. The work will generate pivotal data to support the clinical evaluation of enhanced allogenic equine CBMSCs to treat joint inflammation in research horses. Upon proving the concept in research horses with induced inflammation, the enhanced equine CB-MSC would be tested in a randomized clinical trial for the treatment of joint inflammation in client-owned horses suffering from osteoarthritis.

Fully implemented, this therapy would provide a safe, efficacious, and technically simple treatment for selected inflammatory or immune-related disorders in horses.

Vitrified Equine MSC Cartilage for Cartilage Repair - 2019-2020 Projects
Thomas G. Koch. Associate Professor | DVM Copenhagen, PhD Guelph

Horses are multi-use livestock that contribute more than $19 billion annually to the Canadian economy. Injuries to joints are among the most common causes of lost training days or premature retirement in equine athletes. Stem cells hold the promise of novel therapeutic approaches to these difficult-to-treat problems. Large cartilage defects >4cm2 are a challenge to treat, but so-called cartilage chips (1x1x1 mm) have shown significant clinical promise in human medicine, regardless of whether the cartilage was sourced from juvenile human cadavers, unrelated adult cadavers, or from the patient.

Preclinical work in the horse model has also shown promising results using cartilage harvested from the joints of horses. However, current cartilage chip methods are hampered by a limited supply of donor material, risk of disease transmission, suboptimal graft tissue, and donor site morbidity. Donor age, comorbidities, and harvest site may also negatively influence the biological potency of present cartilage and cell grafting methods.

We propose to solve these issues by using cartilage generated in the laboratory from equine cord blood mesenchymal stromal cells (eCB-MSC). We will vitrify (cryopreserve) the cartilage tissue for indefinite storage and availability as an off-the-shelf live-cell cartilage graft that can be implanted using standard surgical methods. We are seeking support to establish a robust vitrification protocol for eCB-MSC-derived neocartilage.

The work will generate pivotal data to support the clinical evaluation of cryopreserved allogenic eCBMSC cartilage chips to repair focal cartilage defects in research horses. Fully implemented, this therapy would provide a safe, efficacious, and technically simple treatment for horses as well as provide an opportunity for a Canadian biotechnology business to bank and distribute vitrified cartilage tissue in unlimited quantities to the world market.

Development of Osteochondral Constructs Using Equine Umbilical Cord-Derived Mesenchymal Stromal Cells for Treating Joint Cartilage Defects - 2017-2018 Projects
Thomas G. Koch Associate Professor | DVM Copenhagen, PhD Guelph

Co-investigators’ names and Departments: Rita A. Kandel, M.D., Department of Pathology, Mount Sinai Hospital, Toronto, ON, Mark Hurtig, Clinical Studies, OVC, Judith Koenig, Clinical Studies, OVC, Ms. Sarah Lepage, PhD Candidate, Biomed Sci, OVC

Orthopedic injuries, constituted mainly of trauma to joint cartilage, are the most common cause of lost training days or premature retirement in the equine athlete. As cartilage tissue has a low intrinsic capacity to heal, repeated injury will eventually result in post-traumatic osteoarthritis in the joint. Though current regenerative therapies for the treatment of focal cartilage defects are showing promising preliminary results, they are wrought with potential complications from secondary surgical sites to isolate patient-specific cells or tissue. Therefore, we propose to investigate the potential of a novel cell type in the generation of osteochondral-like plugs without requiring a secondary surgical site on the patient. We have successfully and reproducibly isolated mesenchymal stromal cells from umbilical cord blood (CB-MSC) and demonstrated their ability to generate cartilage in vitro. We found that by subsequently growing the engineered cartilage on a bone substitute, we could generate an osteochondral construct that could serve as an implant. In 2006, Dr. Rita Kandel’s group in Toronto generated similar constructs and implanted them into sheep with induced cartilage injuries; the implants successfully contributed to cartilage repair at 9 months post-surgery. We believe that with further optimization, we can generate an osteochondral-like implant using CB-MSC with superior properties for in vivo repair of focal cartilage defects. The biomechanical properties of cartilage generated in vitro are historically poor, making the graft susceptible to damage upon transplantation. We propose to mechanically mature the cartilage prior to implantation in order to better withstand the large loading forces within the equine joint upon transplantation.

Effect of extracorporeal shockwave therapy on umbilical cord blood mesenchymal stromal cells - 2017-2018 Projects
Judith Koenig, Dr Med Vet, DVSc, Diplomate DACVS, Diplomate ECVS

Shockwave has been used as a treatment to decrease the healing time and improve the quality of the repair in bone and soft tissue injuries, but the exact mechanism is not completely understood. Intra-lesional (into bowed tendons) administration of stem cells in combination with shockwave has been used as an alternative treatment for orthopedic diseases like tendon and ligament lesions in horses, without scientific evidence, but anecdotally has resulted in faster healing times and return to function. There is a need to evaluate the effect that shockwave has on stem cells. One in vitro study showed that equine fat derived stem cells grow more rapidly and differentiate faster into different tissues, but nothing is known about the secretory function of these cells or the effect of shockwave on bone marrow or umbilical cord blood derived stem cells. Labelling these cells allows tracking of these cells also in vivo. Therefore, we hypothesized that shockwave will enhance the progenitor (their ability to differentiate into different tissue) and nonprogenitor (their anti-inflammatory and secretory) functions of stem cells. Our objectives are to firstly evaluate if shockwave increases umbilical cord blood derived stem cell growth rate, proliferation rate and differentiation in comparison to untreated cells in vitro. And secondly, to evaluate the effect of shockwave on topically injected allogeneic (derived from unrelated foals) umbilical cord blood derived stem cell and follow the cells in a neck wound model in vivo.

Comparing loading between the forelimbs of racing Thoroughbred to investigate possible causes of laterality in catastrophic injury - 2017-2018 Projects
Jeffrey J. Thomason, Professor | BA, MSc, PhD Toronto

The U.S. Jockey Club’s Equine Injury Database (EID) has information on more than 1.6 million racing starts by Thoroughbreds in North America. A recent survey of the approximately 3000 catastrophic injuries (CI) appearing in the database confirmed that the frequency of CI is related to the surface type, being greater on dirt, intermediate on turf and least on synthetics. In addition the study found that the left fore and right hind limbs were significantly more prone to CI on dirt tracks than the opposite limbs, but this effect was not evident on synthetic or turf tracks. The researchers speculated that this laterality of injury might be due to uneven forces acing on the left and right limbs in turns combined with relative inconsistency of support during the stance on dirt. These findings are significant for Ontario’s TB racing industry because races are on turf or synthetics while most training is on dirt. The aims of this study are to assess whether laterality of biomechanical loading exists on dirt and synthetic surfaces, and whether epidemiological data identify that as an issue in Ontario. We will tackle this project in 4 ways: (a) We’ll collaborate with the researcher currently studying the Ontario Death Registry, to look for evidence of injury laterality. The emphasis will be on the forefeet, which sustain higher rates of injury. (b) We will take another look at data we have from previous experiment on forces acting on the feet of Thoroughbreds galloping on the straight and turns of different tracks, and will look for evidence of laterality in loading at all stages of the stance for both front feet. We have data from dirt, turf and synthetic surfaces, using the same horse but on different days. (c) We will record new biomechanical data from the front feet of horses galloping on a training dirt track and a racing synthetic track within a few minutes, to find whether and how much laterality of loading exists on each surface. (d) We combine the analyses from all of the data from parts a-c to assess whether laterality is a problem in Ontario, and to categorize which parts of the complex mechanical loading acting on the horse’ hoof during galloping might be at the root of the problem. The results should identify whether turning or the surface induces the higher risk for injury, from a biomechanical perspective.

Comparing loading between the forelimbs of racing Thoroughbred to investigate possible causes of laterality in catastrophic injury - 2016 - 2017 Projects
Jeffrey J. Thomason, Professor, BA, MSc, PhD Toronto

The U.S. Jockey Club’s Equine Injury Database (EID) has information on more than 1.6 million racing starts by Thoroughbreds in North America. A recent survey of the approximately 3000 catastrophic injuries (CI) appearing in the database confirmed that the frequency of CI is related to the surface type, being greater on dirt, intermediate on turf and least on synthetics. In addition the study found that the left fore and right hind limbs were significantly more prone to CI on dirt tracks than the opposite limbs, but this effect was not evident on synthetic or turf tracks. The researchers speculated that this laterality of injury might be due to uneven forces acing on the left and right limbs in turns combined with relative inconsistency of support during the stance on dirt. These findings are significant for Ontario’s TB racing industry because races are on turf or synthetics while most training is on dirt. The aims of this study are to assess whether laterality of biomechanical loading exists on dirt and synthetic surfaces, and whether epidemiological data identify that as an issue in Ontario. We will tackle this project in 4 ways: (a) We’ll collaborate with the researcher currently studying the Ontario Death Registry, to look for evidence of injury laterality. The emphasis will be on the forefeet, which sustain higher rates of injury. (b) We will take another look at data we have from previous experiment on forces acting on the feet of Thoroughbreds galloping on the straight and turns of different tracks, and will look for evidence of laterality in loading at all stages of the stance for both front feet. We have data from dirt, turf and synthetic surfaces, using the same horse but on different days. (c) We will record new biomechanical data from the front feet of horses galloping on a training dirt track and a racing synthetic track within a few minutes, to find whether and how much laterality of loading exists on each surface. (d) We combine the analyses from all of the data from parts a-c to assess whether laterality is a problem in Ontario, and to categorize which parts of the complex mechanical loading acting on the horse’ hoof during galloping might be at the root of the problem. The results should identify whether turning or the surface induces the higher risk for injury, from a biomechanical perspective.

Sustained release drug formulations for equine joint conditions: Formulation and forensic considerations - 2016 - 2017 Projects
Mark Hurtig, Professor, DVM, MVSc, Diplomate ACVS

In past studies we have studied long acting drugs that were created by a drug company for the human market to control inflammation in joints for up to 90 days. Using the sheep knee as a model we showed that these drug and polymer combinations take up residency in the joint lining where they are slowly broken down to release the drug and control pain and inflammation. The duration and drug levels in joints can be changed by altering the polymer-drug binding, so release times of a few days to several months are possible. Through a collaboration with polymer chemist Dr. Elizabeth Gillies (Western University, Ontario) we will create non-steroidal anti-inflammatory drug (NSAIDs) formulations that will last from a few days to several months after injection into joints. The advantage of this delivery system is that no drug leaks from the joint into the rest of the horse's body to cause the all too common complications of colic and gut ulcers. Dr. Gillies' role will be to create the polymer carriers from safe, food grade chemicals. These are widely used in human sustained release drugs for the eye conditions, cancer therapies etc. Dr. Gillies will modify NSAID drugs so they can be trapped in the polymer carrier system. Laboratory experiments will be used to estimate the rate of drug release into joint fluid over 21 days. With this information we will conduct trials of sustained release NSAID formulations by injecting them into the fetlocks of horses at the University of Guelph Arkell Research Station. By injecting the drug and then challenging the horse joint with an inflammation-causing molecule (lipopolysaccharide) at intervals of up to 90 days, we will be able to determine the duration of the drugs' anti-inflammatory effect. Many horses have low grade inflammation in joints that is difficult to address, and requires repeated, costly treatments. Such medication regimes are expensive and not without risk, particularly when steroids are used since they may promote cartilage loss over time. An additional consideration is the potential for doping with similar drug formulations. By measuring drug levels in plasma, urine and joint fluid we will be able to estimate when drugs would be detected in doping tests. We will share all our data with racing and clean sport/doping jurisdictions and provide samples of our drug formulations for their own use. This project will provide the basis for commercialization of a sustained release formulation that would improve the safety of NSAID use in the horse, and potentially reduce the incidence and rate of osteoarthritis progression in horses.

Development of Osteochondral Constructs Using Equine Umbilical Cord-Derived Mesenchymal Stromal Cells for Treating Joint Cartilage Defects - 2015 - 2016 Projects
Thomas G. Koch, Associate Professor, DVM Copenhagen, PhD Guelph

Co-investigators: Rita A. Kandel, M.D., Department of Pathology, Mount Sinai Hospital, Toronto, ON, Mark Hurtig, Clinical Studies, OVC, Judith Koenig, Clinical Studies, OVC, Ms. Sarah Lepage, PhD Candidate, Biomed Sci, OVC

Orthopedic injuries, constituted mainly of trauma to joint cartilage, are the most common cause of lost training days or premature retirement in the equine athlete. As cartilage tissue has a low intrinsic capacity to heal, repeated injury will eventually result in post-traumatic osteoarthritis in the joint. Though current regenerative therapies for the treatment of focal cartilage defects are showing promising preliminary results, they are wrought with potential complications from secondary surgical sites to isolate patient-specific cells or tissue. Therefore, we propose to investigate the potential of a novel cell type in the generation of osteochondral-like plugs without requiring a secondary surgical site on the patient.

We have successfully and reproducibly isolated mesenchymal stromal cells from umbilical cord blood (CB-MSC) and demonstrated their ability to generate cartilage in vitro. We found that by subsequently growing the engineered cartilage on a bone substitute, we could generate an osteochondral construct that could serve as an implant. In 2006, Dr. Rita Kandel’s group in Toronto generated similar constructs and implanted them into sheep with induced cartilage injuries; the implants successfully contributed to cartilage repair at 9 months post-surgery. We believe that with further optimization, we can generate an osteochondral-like implant using CB-MSC with superior properties for in vivo repair of focal cartilage defects. The biomechanical properties of cartilage generated in vitro are historically poor, making the graft susceptible to damage upon transplantation. We propose to mechanically mature the cartilage prior to implantation in order to better withstand the large loading forces within the equine joint upon transplantation.

MicroRNAs as Equine Joint Health Biomarkers - 2015 - 2016 Projects
Thomas G. Koch, Associate Professor, DVM Copenhagen, PhD Guelph

Co-investigators: Jonathan LaMarre, Biomedical Sciences, Judith Koenig, Clinical Studies

Injuries involving joint cartilage such as osteoarthritis (OA) are some of the most common causes of lameness and pain in horses. Sophisticated means of monitoring joint health status are needed to allow early detection and intervention as well as monitoring the effect of interventions. MicroRNAs are a class of short non-coding RNAs that participate in various biological processes including cartilage development and homeostasis. MicroRNAs have been measured at the tissue level, in synovial fluid and serum, and may reflect some aspects of the health status of the animal. MicroRNAs may therefore be useful biomarkers of joint health that could be evaluated through frequent blood and or joint fluid sampling and analysis. In the present proposal, operating funds are sought to determine robust methods for detecting microRNAs in synovial fluid and blood plasma from horses and to determine cut-off values for selected microRNAs in healthy horses and horses with joint inflammation.

Electro arthrography for non-invasive on-farm assessment of fetlock joint cartilage health - 2014-2015 Projects
Dr Mark Hurtig, Department of Clinical Studies

Co-investigators: Dr. Adele Changoor, Dr. Mohamed Hoba, MSc candidate, Dr. Karen Gordon, Dr. Don Trout, Dr. Lance Bassage.

Cartilage has unique biomechanical properties resulting from interactions among extracellular matrix components, consisting of hydrated proteoglycan trapped in a collagen network, and interstitial fluid. During compression, cartilage produces electrical signals, known as streaming potentials, due to negatively charged functional groups on proteoglycan1. Streaming potentials reflect cartilage composition and structure and are more sensitive to cartilage load bearing properties than purely biomechanical measurements2-4. A new method called electroarthrography (EAG) measures streaming potentials non-invasively5 and has the potential to become a clinical tool that may contribute to the diagnosis and treatment of degenerative joint diseases.

This study aims to develop a diagnostic EAG method suitable for on-farm assessment of fetlock cartilage. Specific objectives include correlating EAG with direct macroscopic, biomechanical, histological and biochemical measurements of cartilage properties, and comparing EAG to current clinical assessments of joint health.

We hypothesize that (1) EAG signals are strongly correlated to cartilage composition and load bearing properties, and (2) EAG can consistently distinguish early cartilage degradation from more advanced erosion.

Testing hypothesis (1) will involve experiments in fetlock explants where EAG obtained during simulated joint loading is compared to direct assessments of normal, enzymatically degraded, and osteoarthritic cartilage.

Testing hypothesis (2) will require clinical application of EAG in normal horses and those exhibiting clinical signs of joint disease.

An EAG-based diagnostic would provide veterinarians with a sensitive tool for monitoring joint health. Detecting cartilage degeneration at treatable stages may prevent horses from being lost to joint disease and may lower costs associated with training these animals.

Effect of allogeneic umbilical cord blood mesenchymal stromal cells on induced synovitis in horses - 2014-2015 Projects
Dr Judith Koenig, Department of Clinical Studies

Co-investigator: Thomas Koch, DVM, PhD, Dept. of Biomedical Sciences

Currently the focus of systemic and intra-articular therapies involves slowing the progression of osteoarthritis since reversal of established osteoarthritis has yet to be demonstrated.

In equine joints with naturally occurring disease intraarticular administration of both, adipose derived and bone marrow derived, MSC were reported to improve lameness in horses unresponsive to conventional treatment. Recently, a comparable physiologic response to the intra-articular injection of autologous and allogeneic stem cells was reported in the horse. This suggests there may be potential for developing frozen allogeneic stem cell products that could be available for treatment of the equine athlete at the time of diagnosis of injury. Therefore, our hypothesis is that allogeneic umbilical cord blood mesenchymal stromal cells have an anti-inflammatory effect in induced synovitis in horses.

The first part of the study will evaluate the in vitro anti-inflammatory potential of allogeneic umbilical cord blood mesenchymal cells. For the in vivo study, inflammation will be temporarily induced with endotoxin in the midcarpal joint of 6 horses, and allogeneic umbilical cord blood mesenchymal cells will be injected and with repeat synovial fluid samples the effect of the MSC on various joint parameters evaluated.

Electro arthrography for non-invasive on-farm assessment of fetlock joint cartilage health - 2013 - 2014 Projects
Hurtig M

Co-investigators: Dr. Adele Changoor, Dr. Mohamed Hoba, MSc candidate, Dr. Karen Gordon, Dr. Don Trout, Dr. Lance Bassage.

Horses intended for competition and racing endure rigorous training, thereby increasing their susceptibility to joint disease. Veterinarians mainly use physical exam, diagnostic injections, x-ray images 38 and ultrasound to evaluate joint health, yet these methods provide no information about the quantity or health of the articular cartilage. Cartilage damage and erosions are associated with pain and lead to chronic lameness. Some therapies are available to stop or slow cartilage damage, but there are no practical methods for monitoring progress or making a long-term prognosis. Electro arthrography (EAG) is a novel method for measuring electrical signals produced by cartilage through electrodes placed on skin and is similar in principle to electrocardiography (ECG) for the heart.

Pilot studies in people have been successful at distinguishing between normal and osteoarthritic knees. The EAG assessment is simple and completely non-invasive since it requires only surface electrodes the size of a dime. In this study we will focus on the fetlock since it is the most common joint injured, particularly in the racehorse. Our pilot data in cadaveric forelimbs of horses under simulated weight bearing have shown that EAG signals can be easily recorded from the fetlock and are altered by damaged or osteoarthritic cartilage.

The purpose of the first phase of the proposal is to strengthen the diagnostic value of EAG by performing correlative studies on cadaveric fetlock joints before and after inducing controlled cartilage damage. During the second phase of the study, EAG will be applied clinically to normal and lame horses.

Effect of allogeneic umbilical cord blood mesenchymal stromal cells on induced synovitis in horses - 2013 - 2014 Projects
Koenig J

Co-investigator: Thomas Koch, DVM, PhD, Dept. of Biomedical Sciences

Currently the focus of systemic and intra-articular therapies involves slowing the progression of osteoarthritis since reversal of established osteoarthritis has yet to be demonstrated. In equine joints with naturally occurring disease intraarticular administration of stem cells were reported to improve lameness in horses unresponsive to conventional treatment. Recently, frozen stem cells obtained from unrelated horses were used by injection into joints successfully with only a mild transient inflammation. This suggests there may be potential for developing frozen allogeneic stem cell products that could be available for treatment of the equine athlete at the time of diagnosis of injury. Further study is needed to determine if intra-articular allogeneic stem cells modulate inflammation during acute joint inflammation in horses.

Prospective evaluation of MSC isolation from umbilical cord blood and jugular blood - 2012-2013 Projects
Dr. Thomas Koch, Biomedical Science

Studies have shown that peripheral and cord blood stem cells are difficult to isolate. However cord blood-derived cells may be advantageous for a number of reasons. These cells may elicit minimal, if any, immune response if injected or transplanted into an unrelated individual. These cells may also be capable of giving raise to a wider array of cell types than stem cells from fat and bone marrow.

We have recently isolated stem cells from umbilical cord blood of foals and have demonstrated their potential to form bone, cartilage and fat in petri dishes. So far MSCs have only been attempted isolated from the peripheral blood of mature horses and with poor results.

The studies outlined in this application aim to evaluate equine cord blood and peripheral blood-derived mesenchymal stromal cells for the purpose of developing advanced cell-based therapies in the horse.

Equine induced pluripotent stem cells for treating cartilage defects and disease modelling - 2012-2013 Projects
Dr. Thomas Koch, Biomedical Science

The long-term goal of the studies in this proposal is to initiate a research stream on equine induced pluripotent stem cells (eiPSCs) that will compliment our ongoing research efforts to solve the unmet clinical need for treating cartilage defects in horses.

The recent report on the production of eiPSCs by researchers in Montreal and Toronto has received widespread attention in the veterinary research community. These cells hold the promise of developing patient specific cells that can be used for both cell and tissue replacement strategies of that patient with reduced risk of immune rejection, or for evaluating efficacy and safety of new drugs at reduced costs through pharmacological screening platforms, or to treat inflammatory diseases.

A greater understanding of cellular reprogramming events may in the future also be utilized to help the body heal itself by directing resident cells of damaged tissue towards reparative processes. Our research group is part of newly formed international research consortium with the aim to advance our understanding of these cells biology and clinical potential much more rapidly than would be the case by individual research lab efforts. Exchange of graduate students between labs will also enhance the training of highly qualified personal.

We have just been awarded significant funds for a PhD student to explore canine iPS cells for the same purpose of cartilage repair and support of this grant application for another PhD student working on equine iPSCs would provide significant synergy in the lab for the rapid development of a robust research program in domestic animal iPS cells.

Evaluation of techniques to improve stem cell homing in the horse - 2011-2012 Projects
Dr. Judith Koenig, Department of Clinical Studies

Tendon injuries are common in the equine athlete leading to initial loss of training days and significant economic losses. Upon return to strenuous exercise the repaired tendon often breaks down again due to the inferior quality of the repaired tendon. At this time the horse is often retired from athletic performance. Stem cells in combination with soluble biologics injected into a tendon lesion seem to improve the quality of the repaired tendon.

Currently, stem cell therapy does not return the horse to training and racing faster than conventional treatments, but appears to reduce the number of horses sustaining re-injury. The cause of this enhanced healing is unknown. It could be due to the stem cells themselves or be due to growth factors contained in the solution the cells are suspended in prior to injection. Some growth factors, like transforming growth factor ?, appear to stimulate stem cells to transform into different tissues. In addition, extracorporeal shock wave treatment has shown to accelerate differentiation of stem cells into different tissue types.

In an experimentally bowed tendon in horses, shock wave treatment increased the concentration of transforming growth factor ? and healed the tendon faster, which may be caused by an increased influx of stem cells. Similarly, when a bone defect was created in the thighbone of rats, and shock wave was applied, significantly more labelled stem cells settled into the defect ('homing') and exhibited faster healing compared to untreated bone defects.

Very little is known about homing of stem cells in the horse, and if it is possible to use shock wave treatment to attract stem cells to a tendon lesion (similar to other species); therefore, this study is designed to evaluate how equine stem respond to shock wave in vitro and if they grow faster into the different cell types then untreated stem cells. If successful in vitro, we will evaluate the use of shockwave to accelerate the differentiation of stem cells into tendon tissue in horses with experimental tendonitis in vivo.

CD117 positive equine cord blood-derived cells – a fountain of cells suitable for equine cell-based therapies such as cartilage repair? - 2010-2011 Projects
Dr. Dorothee Bienzle, Department of Pathobiology, Co-investigators: Thomas Koch, Department of Clinical Studies; Dean H. Betts, Department of Physiology & Pharmacology, UWO

The novel isolation of mesenchymal stem cells from equine cord blood reported by Koch and colleagues in 2007 from the Ontario Veterinary College has initiated a whole new area of equine veterinary research which is now being pursued at multiple institutions worldwide.

The long-term goal of this proposal is identification of cells suitable for novel cell-based therapies in the horse.

The objective of the proposal is to isolate cells from equine cord blood, which are superior for improving repair of focal cartilage injuries in the horse. Equine cord blood contains cells with potential to produce cartilage in a laboratory setting.

However, differences in cartilage potential were noted between different cell cultures. There is, therefore, a need to establish cell lines with a consistent and reproducible cartilage potential before these cells can be introduced into mainstream equine clinical practice.

The hypothesis of this proposal is that cord blood contains few, but very potent cells, which can be identified on a molecular level, and possess high proliferative and chondrogenic potential. The availability of well-characterized cell lines is the first prerequisite for pursuing commercialization of cell-based therapies.

Effect of mechanical stress on laminar junction remodeling. - Projects 2005-2006
Dr. Jeff Thomason
Mechanical stresses occur in hooves with every footfall, usually leading to adaptive responses over time. That is, the living and inert components of the hoof are remodeled to continue to withstand the imposed stresses. Like bones and muscles, the hoof has a need for some mechanical stimulation, but not too much or too little. Stresses in a beneficial range promote vascular activity and apparently adaptive remodeling. What this study will describe is how the laminar junction remodels in response to stress in a beneficial range. In conjunction with previous work on stresses and strains during different activities and on different substrates, the results will indicate the levels of exercise that are appropriate for good hoof function and structure.

Osteoarthritis: estrogens and cartilage interactions in the horse. - Projects 2004-2005
Dr. J. Raeside
Osteoarthritis is a common joint disease causing lameness in horses. No satisfactory treatment exists for longer-term resolution, though some progress is being made with both surgical and pharmacological approaches. Estrogens are responsible for many changes beyond the reproductive system; and an emerging concept is that some responses are due to special actions of estrogen metabolites formed in the target tissues themselves such cartilage in the joint. The study will address local metabolism of estrogen by cartilage and then examine the actions of estrogens on cartilage with a view to treatment, including cartilage cells in culture in preparation for grafting.

Identification and prediction of canon bone fractures in 2 and 3 year old racehorses. - Projects 2004-2005
Dr. M. Hurtig
This project is part of a more comprehensive program designed to reduce musculoskeletal injuries at Ontario racetracks by improving the monitoring of horses and racetrack surfaces. In previous work, the Comparative Orthopaedic Research Group has established the utility of using accelerometers for monitoring shoeing and track conditions. The current proposal expands the examination of horses presented from the Ontario Racing Commission Death Registry program to establish the incidence and location of microfractures in canon bones. Quantitative Ultrasound (QUS), a non-invasive technology that can be used on living horses for assessment of bone quality, will be correlated with three-dimensional imaging to link speed of sound measurements with bone pathology.

Characterization of nutritional factors affecting the rate of post-exercise muscle glycogen synthesis in horses. - Projects 2004-2005
Dr. R. Geor
The optimization of the energy supply in horses during athletic events is crucial. Muscle glycogen is the most important source and the replenishment of these stores is essential. This study addresses the factors affecting the rate of muscle glycogen depletion as well as dietary and feeding strategies that will assist in recovery post exercise. New information about the ability of the horse to digest different carbohydrates will be acquired and can be used as guidelines in formulating products.

Quantifying the hoof's response to loading during exercise: changes in external shape, growth rate and internal anatomy. - Projects 2004-2005
Dr. J. Thomason
Hooves experience the shock of hitting the ground as well as the increasing force when bodyweight is transferred to the leg. The active response of the hoof to many factors such as gait, speed, substrate etc will be examined. With this knowledge, it should be possible to predict and prevent hoof lameness due to mechanical aetiology.