EQUINE

Author:

Afra Saif Obaid Al Mheiri

Corresponding Author:

Dr. Khaja Mohteshamuddin

BVSc & AH, MVSc, MBA (HR), MRSB (UK)

Review on Mesenchymal Stem Cell Therapy in Musculoskeletal Injury in Equines

ABSTRACT

Stem cells therapy is one of the new fields in regenerative medicine and it is commonly used in equine practice in treating musculoskeletal injuries. Although many studies have been conducted regarding the use of mesenchymal stem cells as a regenerative therapy, the action of mechanism is still not fully understood and requires further investigation but other studies have shown its efficacy is healing damaged tissue without causing any complications. Key Words: Equine, Stem cell therapy, musculoskeletal injury, mesenchymal stem cells

INTRODUCTION

Regenerative medicine is one of the new emerging fields in the medicine world that helps to repair damaged cells and tissues in both human and veterinary medicine. Stem cell therapy a growing field that is considered one of the regenerative therapies, it has gained a lot of interest in recent times due to its ability to regenerate tissue and differentiate into different cells and improve in healing and reduces scar formation (Voga, Adamic, Vengust, & Majdic, 2020). Mesenchymal stem cells (MSCs) is mostly used in repairment of musculoskeletal (orthopaedic) injuries in horses, but further studies has proven that stem cell therapy can also be used in variety of animal species in different types of diseases like cancer.

This review highlights on the definition of stem cells, how mesenchymal stem cells (MSCs) are put to use in treatment of musculoskeletal injuries in horses, and the future of stem cell therapy in veterinary medicine.

BACKGROUND AND MECHANISM OF MESENCHYMAL STEM CELLS

Mesenchymal stem cells are adult stem cells that can be found in all tissues and organs of the body. The main function of the mesenchymal stem cells are repairing and replacing the dead or damaged cells caused by diseases or mechanical traumas without causing an inflammatory response by initiating the regeneration process, moreover, mesenchymal stem cells have the ability to differentiate into different types of cells (Ito & Suda, 2015). The cell proliferation and differentiation influenced and activated by the cell signaling (autocrine, paracrine, and endocrine or intra- cellular) that helps in gaining information about the type of tissue needed in a particular lesion (F. Ferraro, C. L. Celso, and D. Scadden, 2010). The release of a class of small molecules is capable of binding to the specific receptors on the surface of stem cells and cells of the immune system, called chemokines, is one of the ways to replicate these signals. In this way, the greater the activation of receptor and the greater the favoritism for a positive allosteric gradient, the greater the interaction between the ligand and the receptor and the higher the number of ligands is. (G. J. P. Westen, A. Gaulton, and J. P. Overington, 2014). Hence the signaling triggers a cellular response that activates the process of proliferation and/or cell differentiation, in addition to migration. The better the allosteric gradient is the faster the stem cell receive signals to perform different capacities, in the case of cell replacement the best type of stem cell that has a quick response to the chemotactic signals and aids in cell replacement is mesenchymal stem cells (T. J. Kean, P. Lin, A. I. Caplan, and J. E. Dennis, 2013).

Studies have shown that the MSC have the ability to produce immuno-modulatory molecules that inhibits the function of the immune system cells, for example, it inhibits the activation and proliferation of the macrophages and the natural killer cells and regulate the differentiation of the B cells to make the site of injury an anti-inflammatory environment and proliferate and differentiate without being rejected by the

immune system of the body. (Negi & Griffin, 2020).

The process of migration, proliferation and differentiation is called (Cell Homing) which is the mechanism that is important for activeness of cell therapy.

Cell homing is activated and it stimulates the tissue repairing once the injury has occurred. When the injured site turns hypoxic, it causes the activation of hypoxia- inducible factor 1 (HIF-1) which induced the release of (Cytokines) like storms derived factors-1, and the vascular endothelial growth factor (VEGF). These cytokines cross through the blood streams and reaches the injury site and activates the cell differentiation into the types of cells needed for repairing and healing (Fig1). Furthermore, when the cell homing is activated, it activates the immune system where the mobilization of adult stem cells are known as MSC and many other cells. The main concern in the adult stem cells is their ability to recognize the homing mechanism and signaling, in addition to the directions to the injury site and to repair (Markoski. M, 2016). There has been some evidence that states that the MSC does not stay at the site of the injury (C. De Schauwer, G.Van de Walle, A.Van Soom, E. Meyer, 2013). A study was made by Quin- tavalla et al. (2002), fluorescently labeled MSC were injected in cartilage injury in goats and loss of the injected MSC were seen 14 days later, the MSC were found in deeper are of the injury which proved the migration of the injected MSC (Quin- tavalla et al. 2002).

Experiment have been done both in vivo and in vitro to determine mechanism of MSC in the tissue, although there is still a lack of knowledge regarding how MSC functions, In vitro experiments, the ability to differentiate was conformed via formation of tissue using histological staining. But, in vivo, the tissue formation in, for example articular cartilage cannot be compared to the tissue found in vitro, which means that in vitro tissue formation does not indicate the clinical usefulness of MSC.

Mesenchymal stem cells can be isolated from non-haemopoietic bone marrow stroma and can change or differentiate into chondrocytes, osteoblasts, adipocytes, fibroblasts and bone marrow stroma (Tuan et al. 2003). (Fig.2). Mesenchymal stem cells can be recovered from a broad range of tissue and organ and from different ages like Bone Marrow, Cartilage, Tendon, Fat, Blood, Dermis, Synovial Fluid and Muscle. (Taylor SE, Smith RK, Clegg PD, 2007) Mesenchymal stem cells can also be isolated from the neonatal organs including lung, spleen, kidneys and pancreas as well as fatal blood and bone marrow and the umbilical cord. (In ’t Anker et al. 2004).

CLINICAL APPLICATION OF MSC IN VETERINARY MEDICINE

Until now stem cells has been applied mostly experimentally in different species of animals, but stem cell therapy nowadays is mostly used in dogs and horses mostly for musculoskeletal system diseases, it has also been used in cats for inflammatory, respiratory and other types of diseases. Although the clinical applications are limited, stem cell therapy has shown a positive effect in the musculoskeletal diseases.

TENDONS

There are a wide range of animals that are used in different sports activities like hunting and bull fighting. Stem cell therapy is usually applied on racing animals because it is the common sport that causes most of the musculoskeletal injuries such as injuries in the tendons (tendinopathies), bones and

Figure 1: Differentiation of adult stem cells into different cells for tissue repairing

Figure 2: Multiple differentiation pathway of the mesenchymal stem cell

cartilages of the animals.

Naturally when an injury occurs, the injury heals by the process of scar formation, which in comparison to a healthy tissue, is much more insufficient, which may affect the structure, mobility and the function of the tendon or the ligament of the horse and may promote re-injury, this can affect the performance of the racing horses which sequentially, causes economic losses due to the extra physical training and long duration of treatment and recovery. The usual approach of therapy in case of tendinopathies in equines are cooling, bandaging the tendon and a long duration of rehabilitation and exercise as well as use of medications like anti-inflammatory drugs, but often these approaches ends up with surgical intervention which does not allow complete tissue healing and effects the performance, therefore, the most suitable approach can be the use of stem cells in order to regenerate the tendon in a better way. Regenerative stem cell therapy helps in forming collagen fibers and recovering the normal process of collagen fibres and achieves the normal tendon activity with lesser chances of re-injuries to the tendon (Voga M, Adamic N, Vengust M, Majdic G. 2020). Mesenchymal stem cells have the ability to differentiate into different cells depending on the site of the injury, it is therefore considered as a useful candidate for regeneration of tissue in equine medicine, especially in musculoskeletal injuries or stress induced injuries.

Tendons are basically fibrous tissue with connective tissue and collagen fibers that are attached to the bones, when a tendon is damaged it require a large influx of cell and require production of new fibrous tissue but with insufficient mechanical characteristics, the tendon performance weakens and increases the risk of re-injuries. Sine the tendons are composed of high levels of collagen fibers, the injury directly induces the process of cell homing which allows rapid tissue healing when the stem cells are injected at the injury site. (Markoski, M. 2016).

Stem cells from adipose tissue or bone marrow are used in case of tendinopathies for tendon regeneration. The first implantation of autologous Bone Marrow Mesenchymal Stem Cell (BMMSC) was reported in 2003 (Smith RK, Korda M, Blunn GW, Goodship AE. 2003). A cohort study was done in 141 racehorses that had superficial digital flexor tendon injury, all of the horses were injected intralesional injections of autologous BMMSC and the results showed a decrease in the rate of re-injuries in all horses within 2 years. It turned out that 44.7% of hunt racehorses and Graph 1: graph showing comparison of re-injury rates of different treatments in comparison with MSC treatment.

And 25% of flat racehorses had returned bask to racing and were still racing. (Graph 2). (E. E. Godwin, N. J. Young, J. Dudhia, I. C. Beamish and R. K. W. Smith, 2011). The rate of the reduction was similar to another study that was done similarly to horses that were treated with hyaluronan, beta aminopropionitrile fumarate or polysulfated glycosaminoglycans (Markoski, M. 2016).

Studies showed that autologous BMMSC therapy in cases of tendinopathies cause formation of tissue that resembled the normal tendon matrix comparing to the natural tissue healing process that causes formation of fibrous tissue. Another study showed very satisfying results regarding the allogenic mesenchymal stem cell therapy in the case of tendon and ligament disorder, for example, tendinitis of superficial and deep digital flexor tendons, however, autologous bone marrow or adipose derived mesenchymal stem cell therapy has poor or little effect on surgically induced injuries to the flexor tendons (Markoski, M. 2016).

Graph 1: Comparison of re-injury rates of different treatments in comparison with MSC treatment.

Graph 2: Comparison between horses traded with MSC returning to racing and participating in 3 and 5 races in comparison with another study where the horses were treated by the normal approach.

JOINTS

Joint diseases are one of the common causes that causes chronic lameness and ends or destroys the racing career for race horses. The most common joint diseases that effects the horses is Osteoarthritis. When the damaged occurs to the articular cartilage, ligament or the menisci, it usually causes poor prognosis with limited or insufficient repair, especially for the race horses because of the hypo-cellular and avascular environment of the articular cartilage. (Voga, Adamic, Vengust, & Majdic, 2020).

Other than horses, Adipose derived mesenchymal stem cells (AD-MSC) has been used in treating dogs with coxo-femoral joint osteoarthritis. A double blinded study was done in (14) dogs where (AD-MSC) cells were injected intraarticularly in the hip joint of the dogs, after the examination the veterinarians saw signs of improvement in the scores for lameness and pain, and the mobility in comparison with control dogs. (Black LL, Gaynor J, Gahring D, Adams C, Aron D, Harman S, Gingerich DA, Harman R. 2007).

One of the most common disorder in horses in bone spavin which is the final degenerative phase of osteoarthritis. The usual therapeutic approach in case of bone spavin is use of antiinflammatory corticosteroids in order to minimize the pain as well as the inflammation. A study was done on 16 horses which suffered with bone spavin. The horses were injected intra-articualarly with Adipose Derived Mesenchymal Stem Cell (AD-MSC). 180 days after the treatment, the results showed no signs of lameness in comparison of the control group that are untreated. Scintigraphy examination was done to conform the results and it shows no

inflammation in the tarsal joins of the horses in comparison of the untreated control group which had signs of inflammation. In another study that was done in 80 horses suffering with osteoarthritis, all 80 horses were treated with AD-MSC, and all 80 horses showed decrease in score of lameness in the 90 day follow up which confirmed the beneficial effect of the AD-MSC (Voga, Adamic, Vengust, & Majdic, 2020).

APPLICATION OF STEM CELL THERAPY IN THE UAE

The UAE has been recently focusing and expanding its ways to use regenerative therapies and among these improvements, In September 2007, the California-based company Vet-Stem collaborated with Central Veterinary Research Laboratory (CVRL) and offered them adipose derived adult stem cell for animal therapy throughout the Middle East. Not only it is used in race horses and endurance horses but also it has been used in racing camels with joint and ligaments injuries.

DISCUSSION

Although the stem cell therapy has shown promising result, it does not mean that other approaches or methods of treatment are not effective.

In the first study that was discussed in the review where it involved 141 racehorses it showed that the rate of the re-injuries of the superficial digital flexor tendinopathy was reduced after treatment with mesenchymal stem cells and comparing to the other study that was done by (O, Meara et al. 2010) where the study was done to evaluate the re-injury rate with the usual approach of treatment, the number of the

Figure 3: Ultrasonographic imaging of the superficial digital flexor tendon lesion before and after treatment with 2x 10^6 mesenchymal stem cell

re-injuries were higher than the reinjuries that occurred in the horses that are treated with mesenchymal stem cells as it is shown if Fig 3, in addition, after 2 years follow up around 44.7% of hunt racehorses and 25% of flat racehorses had returned back to racing and were still racing.

In the study that was done in case of osteoarthritis, adipose derived mesenchymal stem cell

were injected in 16 horses with bone spavin and it showed that 180 days later there were no signs of inflammation or lameness comparing to the control group that had both of the clinical signs. These results are similar to the other study mentioned in the review where they injected adipose derived mesenchymal stem cells in 80 horses suffering with osteoarthritis. These results were also similar to the study done by (Black LL, Gaynor J, Gahring D, Adams C, Aron D, Harman S, Gingerich DA, Harman R. 2007) in dogs with coxofemoral joint osteoarthritis.

All of the results shown in these studies were promising result that conformed that mesenchymal stem cell therapy can be an effectives method of therapy of musculoskeletal diseases in horses and other animals species as well since it is able differentiate into the needed cell in the tissue and induce tissue healing and preparing without causing any adverse effect like pain and inflammation, also it reduces the scar formation which helps in regaining the normal function of the effected part.

CONCLUSION

This review illustrated that stem cell therapy is one of the promising regenerative therapies that can be used in verity of animals, but specially in treating injuries of the bones, tendons and cartilage of the horses and the most suitable type of stem cell that helps in regeneration and aids in tissue preparing is the bone marrow derived mesenchymal stem cell. Although studies have been done throughout the years, yet it still needs more time and effort to understand the actual mechanism of the stem cell therapy and whether it can be used for other types of diseases and other animal species.

* This is a part of a senior project submitted by the first author to the Department of Veterinary Medicine for the award of Bachelor of Veterinary Medicine Degree.

Authors:

Afra Saif Obaid Al Mheiri is a Senior Veterinary Medicine Student at Department of Veterinary Medicine, College of Food and Agriculture, United Arab Emirates University Dr. Khaja Mohteshamuddin is an instructor at Veterinary Medicine - College of Food and Agriculture, United Arab Emirates University

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For further information and references, contact Dr. Khaja Mohteshamuddin T: +971 37136595 Email: drkhaja707@uaeu.ac.ae P.O. Box No. 15551, Al Ain, UAE