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BY CHARLOTTE BUTLER
RESEARCH PAPER BASED ON EXTENDED PROJECT QUALIFICATION
This paper focuses specifically on equine tendon injury and explains the composition and formation of equine tendon; linking to the science of stem cell therapy following trauma. This paper describes how stem cells are harvested and how the stem cells provide a regenerative treatment. The paper will also explore the current practise of stem cell therapy and the potential of stem cell therapy for equine tendon injury in the future. During the discussion of the topic I shall be reviewing studies and statistics from traditional treatment and stem cell therapy, contrasting and comparing the findings and finally concluding on whether stem cell therapy is an effective and worthwhile treatment for equine tendon injury.
Introduction ‘Tendon injury’ are two words that unquestionably send shivers down any horse owner’s spine; and unfortunately they are two words that the equine vet is all too familiar with. For the working horse, a tendon injury can inflict an end to their ridden career, furthermore is not uncommon to euthanize the horse based on the nature of the injury which results in a long recovery time, high re-injury risk and extensively reduced athletic ability. The Structure and Formation of Equine Tendon Tissue Tendon tissue connects and transmits forces from muscle to bone to move the equine skeleton; tendons act as a buffer by absorbing the forces which could lead to damage of the muscle tissue . Most tendons are described as being extensor: allows a joint to extend Or flexor: allows a joint to bend in towards the body . Tendons are described as being viscoelastic , with high flexibility and strength; the commonly injured digital flexor tendon provides support for the distal region of the limb. Tendon tissue is formed from around 70% water and the remaining 30 % is mainly type-1 collagen (65%-80%)  A hierarchical organization of subunits which become sequentially smaller. 
Type-3 collagen is a weaker type of collagen in the tendon fibre as it forms smaller, poorly organised fibrils which are more susceptible to trauma . Proteoglycans , elastin and other cells such as tenoblasts account for about 2% of dry mass . Type-1 collagen is distinguished by its two alpha-1 chains and one alpha2 chain of amino acids, compared with type-3 collagen, which has three alpha-1 chains .
(Smith and Schramme, 2003)
Collagen is synthesized in-vivo by selfassembly : three peptide chains form on the ribosomes along the rough endoplasmic reticulum (RER); these chains are sent to the lumen of the RER for procollagenases to cleave peptide terminals to form procollagen molecules. Amino acids are hydroxylated  before being glycosylated  without delay 3 of the monomer procollagen alpha chains are combined to give a fibril in the Golgi apparatus. Tendon structure of fibrils is useful as it ensures that minor tendon damage only extends to the fibril, and not the whole tendon. Procollagen consists of an alpha helix chain and amino-terminal (end of the polypeptide chain with NH3+) and carboxyl terminal extensions (end of the polypeptide chain with COO- group) which are cleaved . Deep indentations house the collagen fibrils at the myotendinous junctions , the indentations are a product of myocyte processes at myotendinous joints; collagen fibrils are enclosed in deep indentations produced by myocyte  (muscle cell) processes. The purpose of the indentations is to allow the tension generated by intracellular contractile proteins of muscle fibres to be transmitted to the collagen fibrils  therefore lowering the stress exerted on the tendon throughout muscle contraction. The function of the tendon, and subsequently the athletic ability of the horse, is determined by the arrangement and cross linking of fibrils, as determined by noncollangenous proteins. The second most abundant protein in young tendon (after collagen) is cartilage oligomeric matrix protein (COMP)  which has been shown in-vitro in COMP accelerated collagen formation to collect five collagen molecules in the ‘quarter
stagger’ arrangement of collagen, thereby organizing the fibril structure throughout growth and formation. Following the formation of a fibril, COMP no longer functions in binding and is terminated from the fibril.  During the development of tendon, the tissue seemingly adapts to loads placed on it whilst COMP is copious in the tendon, however on the mature horse, age and exercise promotes a loss of COMP. A study investigating the correlation of COMP in equine tendon and mechanical characteristics of locomotor tendons  analysed two groups of 12 horses, group 1 had restricted age of 2 years (+/- 2 months) and Group 2 consisted of mature horses which varied with age. Group 1 data showed correlation between both ultimate tensile stress modulus of elasticity and stiffness, with COMP; in notable comparison, Group 2 which varied with age did not show correlation due to age and exercise induced loss of COMP. The data from this study suggests that COMP is an important arbitrator in tendon growth, and that low COMP levels in tendon formation predispose horses to tendon injury. Non collagenous proteins in tendon comprise of small proteoglycans (deconin, fribomodulin, lumican and mimecan), and large proteoglycans (aggrecan and versican) these are intensely glycosylated (glycosaminoglycans) glycoproteins . They construct a major part of the extracellular matrix surrounding the tenocytes and collagen, providing support between cells. Due to their negative charge, small proteoglycans attract cations such as calcium potassium and sodium and bind water to aid water transport through the extracellular matrix
[1, 4]. The large proteoglycan aggrecan is an important component of cartilage, versican is found in the blood vessels and skin . Large proteoglycans are found predominantly at areas of tendon compression at change of direction over bone.  Tenocytes and tenoblasts are cellular components which form 90-95% of the cellular elements in the extracellular matrix ; they present the cytoplasmic extensions to split the fibres; spindle shaped tenoblasts are immature tendon cells with high metabolic activity, these mature into elongated tenocytes which have a lower metabolic activity (demonstrated by the lower nucleus: cytoplasm ratio) . Tenocytes are the source of collagen synthesis, and all other extracellular matrix constituents. Tenocytes generate energy via the Krebs cycle, anaerobic glycolysis and the pentose phosphate shunt for their synthesizing role . Collagen fibres are grouped in larger subunits which are separated by fine strands of epitenon  (a looser connective tissue) the epitenon supplies the tendon with blood vessels, lymph and nerves. The epitenon continues between tendon fibres as endotenon. The endotenon inhabits a round-shaped cell which is thought to be the source of tendon mesenchymal progenitor stem cells , these cells are the progenitor cells of adult tissue and under correct stimuli, proliferate to grow tendon tissue. The larger collagen subunits are known as fascicles, and in relaxed tendon these form the ‘crimp’ waveform characteristic of tendon . Endotenon is continuous with the epitenon: a layer of connective tissue surrounding the outside of the tendon. Exterior to the epitenon is the paratenon  this provides a synovial 4
environment on the inner cells to reduce frictional forces within a sheath. Friction forces are reduced between tendon and soft tissues and the paratenon supplies blood vessels for repair . Additionally the paratenon has highly stretchable properties and as a result is seldom ruptured. The paratenon is made of an outer layer parietal, middle mesotenon layer and inner visceral layer continuous with the epitenon; it is said to be areolar  which means it is a tissue with a gel matrix. Tendon sheaths are comprised of an outer fibrous wall and inner synovial membrane to provide a smooth environment over a joint. [20, 1] The blood supply for the tendon arises from three focal sources, the intrinsic systems at the myotendinous junction, [21, 22] where perimysial (connective tissue sheath grouping muscle fibers into bundles) vessels continue between tendon fascicles; these blood vessels are unlikely to extend beyond the proximal third of the tendon . Another blood supply to the tendon is at the osteotendinous junction, where blood supply is weak and limited to the insertion region of the tendon ; and lastly, the extrinsic system via the paratenon or synovial sheath . However upon investigation tendon tissue did not present signs of ischemia upon removal of the paratenon , therefore suggesting that the intratendinous blood supply is of high importance. Within a sheath, branches from major vessels pass through the vincula (mesotenon, supplying blood source ) and reach the visceral sheet of the synovial sheath ; here a plexus is formed to supply the superficial region of the tendon . Some vessels from the vincula penetrate the epitenon to course into the endotenon septa forming a connection between pertitendinous and
intratendinous vascular networks [25, 26]. For tendons without a true synovial sheath such as the human Achilles tendon , the paratenon provides the extrinsic element of the vasculature; vessels enter the paratenon and route transversely, branching to form a complex system of vessels. Arterial branches from the paratenon penetrate the epitenon to course in the endotenon septa; here an intratendinous vascular network comprising of abundant anastomoses is formed. [1, 26] Innervation of tendon tissue consists of cutaneous, muscular and peritendinous nerve trunks. At the myotendinous junction, nerve fibers cross and penetrate the endotenon septa ; this forms excellent plexuses within the paratenon with branches entering the epitenon. Usually nerve fibers do not enter the tendon body, terminating as nerve ending at the surface . Nerve endings of the myelinated nerve fibers identify pressure and tension changes in the tendon (mechanoreceptors). The Golgi tendon organs  are thin pieces of connective tissue which contain groups of branches originating from large myelinated nerve fibres; the fibres end with a flare of endings between collagen fibres of the tendon . Golgi tendon organs are most abundant at muscle-tendon junctions  and unmyelinated nerve endings behave as nociceptors (sensing and transmitting the pain). Sympathetic and parasympathetic fibers are present .
The Strength of Tendons And Damage from Exceeding Limits. Tensile strength of the tendon is dependent on the collagen content and ratio of collagen type 1 and type 3; the collagen type 1 is stronger and higher type 1: type 3 ratio provides stronger tendon. The thickness of tendon contributes to the strength and loading ability. An area of 1cm 2 is capable of bearing 500kg-1000kg ; superficial digital flexor tendon has ultimate tensile strength of 12 kilo-newtons (1.2 tonnes) [31.] Injury can be caused by acute or chronic, extrinsic or intrinsic or combinations of both; but in acute trauma extrinsic factors are the more common cause. Degenerative tendinopathy, leading to reduced tensile strength is the most common cause in spontaneous rupture; localized hypoxia may arise in tendons during extensive exercise , which initiates tenocytes morbidity . Ischemia occurs under heavy loading and upon relaxation; reperfusion generates oxygen free radicals  which may damage tendon. Damage can occur within the equine physiological limits, resulting in microtrauma . Micro-trauma is often regarded as positive occurrence in the ‘conditioning’ of equine tendons; however constant exposure to such damage will not allow time for repair and cause degeneration . Micro-trauma can cause uneven loading distribution. Within the fibrils, localised frictional forces arise from the scarring which lead to localized fibril damage [1, 35].
High temperature is another factor which lead to tissue damage in the tendon, during locomotion 5-10% of energy stored is converted to heat, in equine superficial digital flexor tendon , it has been recorded for temperatures to rise to 45°C during the gallop ; prolonged or repeated exposure to temperatures this high may lead to degeneration of tissue as a result of hyperthermia. Visoelastic tissue such as tendon stretches during loading, strain rates have been shown in vivo for equine flexor tendons to reach 3-8% at walk, 7-10% at the trot and 12-16% at the gallop. Rupture occurs once the tendon has elongated past 20% of the original length and permanent damage is present after 16% elongation  so it is clear to see how close to the limits horses work at. When tenocytes are subject to strain, stress-activated protein kinases trigger tenocytes apoptosis; it is thought that oxidative stress may contribute to induced apoptosis . Horses are more susceptible to tendon injury when predisposing factors such as genetic, blood type, chronic disease, certain drug use, obesity and poor conformation contribute, horses which participate in high intensity work such as racing or jumping are at greater risk as this increases the loads placed on the tendon. When tendon injury occurs it is a result of macro-trauma or microtrauma[35,41]; macro-trauma is when a singular overstrain of the tendon’s loading capacity occurs, also referred to as acute trauma, micro-trauma is when repetitive sub-maximal overloading damages the tissue, also referred to as chronic trauma[35,41].
The strength of tendons is directly related to the ratio of type 1 and type-3 collagen in the tendons ; type 1 is a more organised, stronger collagen and is the primary component of tendon tissue. If the ratio of type 3 collagen is raised then tendon is susceptible to injury . Response to Injury and Healing When the tendon responds to trauma there are three overlapping phases ; the first stage is inflammation. Erythrocytes and inflammatory cells such as neutrophils invade the site of damage; monocytes and macrophages are most abundant at this point to eradicate the necrotic tendon bundles and damaged cells by phagocytises . Fibroblast proliferation is stimulated and type III collagen synthesis (scar tissue) is triggered. Glycosaminoglycan, fibronection, water and DNA content are also high ; this is initiated within the first 24 hours and continues for approximately 10 days post-trauma, resulting in demarcation of the lesion to lower chance of infection. The second stage of the response to injury is the proliferation stage  which is initiated at around 4 days post injury and continues until approximately 45 days post injury . This stage begins with the formation of a fibrin clot  and proliferation of fibroblasts  .Synthesis of collagen type-III fibrils peaks in this stage as fibroplasia occurs and vascularity increases, also water and glycosaminoglycans are at high levels during this stage. This lasts for a few weeks leading to the formation of a fibroproliferative callus or ‘scar tissue’  and formation of type-1 collagen beings to increase at this stage .
The remodelling phase occurs at about 6 weeks post injury ; it can be subdivided into two stages. Firstly the consolidation stage occurs which initiates the fibrous tendon tissues to be generated from the cellular repair tissues that were laid down earlier in the healing process ; the fibrous tissues such as type 1 collagen begin to align in the direction of stress along with tenocytes . The consolidation stage is active up to ten weeks post trauma. GAG and collagen synthesis decreases, along with water content ; however collagen type-1 is the predominant collagen type being produced. After the ten weeks the second stage of maturation [41, 40] occurs as fibrous tissue converts to scar tissue over the period of a year whilst the previously high tenocyte metabolism falls along with tendon vascularity . The fibroplasia which occurs produces scar tissue, collagen fibres align in a disorganized arrangement, replacing the normal tendon matrix and significantly reducing the elastic properties and tensile strength of the tendon. Tendon fibres fray as a consequence of injury and the arrangement of collagen fibres cannot imitate normal tendon matrix due to the disruption at the area of trauma . As tendon heals, the process can occur intrinsically  or extrinsically; intrinsic tenocytes and adult stem cells from the endotenon and tenocytes from the epitenon can proliferate or extrinsic cells from the synovium and sheath migrate to the injury . There is slow healing of the tendon as there is lack of progenitor cells in the tendon tissue; they are exclusive to the thin layer of endotenon with an insubstantial amount to promote effective healing .
Various contributing factors lead to the slow healing of tendons, other factors include those such as the poor metabolic rate of tenocytes in the extracellular matrix of tendon ; aging shifts the metabolic pathways of tenocytes to favour anaerobic respiration as opposed to aerobic respiration. The low metabolic rate and anaerobic respiration is essential to maintain tension for long periods in the tendon and carry loads, reducing the risk of ischemia (blood supply restriction) and consequential necrosis (tissue death); the blood supply at junctions and areas of torsion, friction or compression is compromised and blood flow decreases with aging and loading of the tendon. Intratendinous blood supply has been shown my microradiographs to be mainly situated at peripheral regions of the tendon, presenting the hypothesis that the tendon core is somewhat hypoxic . Unfortunately the poor healing of tendon results in loss of the characteristics which make it an effective tissue for loading and taking strain; this loss of elasticity, formation of adhesions, scar tissue, and reduction of tensile strength leads to the inevitable loss of athletic ability for the horse, leaving the horse predisposed to reinjury. Intrinsic healing is favourable to extrinsic healing as it results in more effective biomechanics,  in extrinsic healing adhesions are formed between the sheath and the tendon which leads to failure of the gliding tendon sheath. Adhesions also form when the tendon injury is combined with damage to the synovial sheath, adhesions form as tenocytes and granulation tissue from surrounding tissues attempt to repair the damaged region . Clearly this formulates a substantial problem for horse owners- particularly of competition
horses as the healing process is a long and time consuming ordeal combined with a poor prognosis, leaving the owner short of options. Stem Cell Therapy (Current use and future potential) Tendon tissue holds host to adult stem cells, but the response to injury is insufficient, the differentiation of adult tendon stem cells presents poor differentiation upon contrast with stem cells derived from bone marrow . Stem cell therapy is becoming increasingly popular in the treatment of equine tendon injury. The adult tendon tissue does contain mesenchymal stem cells, but they are limited to the small region of the endotenon tissue between collagen fascicles ; this could explain the poor repair progression following tendon injury. Stem cell therapy focuses on enhancing the natural growth of tendon tissue by vastly increasing the amount of mesenchymal stem cells in the local area of trauma. Mesenchymal stem cells (MSC) are described as being multipotent; they have the ability to differentiate in to a diversity of cell types including: osteoblasts, chrondocytes, marrow stroma, adipocytes and fibroblasts  which initiate tendon regeneration. This differentiation in to fibroblasts offers the potential to regenerate the normal composition of tendon matrix, eliminating the issue fibrous scar tissue consequential of tendon healing under conservative management, thus preventing the risk of reinjury associated with loss of elasticity and athleticism.
Stem cells have been shown to effectively form tendon extracellular matrix in vitro and in vivo, from bone marrow derived stem cells; suggesting that stem cells offer a promising medical advantage by reducing scar tissue. Multipotent stem cells present specific characteristics, most distinctively the ability to differentiate in to a range of different tissue cells. Stem cells occupy an assortment of tissue types, however the medical usage of stem cells focuses primarily on the bone marrow derived stem cells at the present time, adipose tissue is another source of mesenchymal stem cells used in practise . The administration of stem cell therapy is reasonably straightforward, providing that the injury can inhabit the administered stem cells. A central core lesion will be able to do this, however unclosed lesions will not hold the stem cells and they will leak out. To prevent leaking within open lesions, a biological scaffold can be put in place . A biological scaffold is derived from mammalian tissues or chemical synthesis; they are comprised of mostly type 1 collagen and many include growth factors to promote tendon regeneration . The use of scaffolds can have detrimental effects however, causing restricted action of the gliding sheath  this is a result of their size interfering with the synovial sheath.
The injured horse is locally anaesthetized and bone marrow is collected from the sternum of the horse with an extraction needle, the bone marrow sample is then sent to a laboratory to have the stem cells identified, isolated and proliferated before being resuspended in autologous bone marrow supernatant (5 million mesenchymal stem cells per ml). The stem cells are then sent back to the practise for the vet to insert directly in to the core lesion of the tendon using ultrasound imagery ; the horse then enters a rehabilitation programme with gradually increased exercise to align the tendon fibres as they heal, prompting a greater strength and elastic capacity to reduce risk of re-injury. Stem cells should be implanted in to the injury within one or two months onset of the injury to ensure minimal scar tissue formation and maximum potential for recovery. A more recent and less common practise of stem cell regenerative therapy is the use of adipose-derived stem cells (ASC) . Adipose tissue provides a readily available source and the stem cells can be collected in significantly greater concentrations in comparison to bone marrow-derived stem cells (BMSC). The surface immunophenotype of ASC has greater than 90% identical features of BMSCs; they behave about the same as BMSCs with the same potential of differentiation [46, 48]. The samples of fat lobules are taken via surgical resection or lipoaspiration (this can be tumescent or ultrasound-assisted). The lipoaspirate is then washed, treated with collagenase digest at 37oC for 30-90 minutes under permanent shaking. After this, the sample is filtrated with a nylon mesh and centrifuged for ten minutes; the erythrocytes are removed before it is washed again. The stem cells are
proliferated, separated and plated for use . The implantation of stem cells in to the site of injury is relatively noninvasive, requiring only a minute entry site created by a needle- thus lowering chance of infection and producing a relatively painless and stress-free procedure for the equine patient. The use of allogeneic embryonic stem cells offers pluripotent stem cells rather that the multipotent ones situated in adult tissue; however the use of them usually dismissed due to the ethical issues surrounding it and risks surround embryonic stem cells. Consequentially embryonic stem cells can form tumours that develop early on in the cells undifferentiated development; otherwise known as a Teratoma . Teratoma is an unusual type of tumour as it is composed of recognisable structures such as muscle and nerve, and even teeth. Although usually benign, teratoma can be malignant . Despite the horses own stem cells being used in treatment, the use of autologous mesenchymal stem cells does not appear to initiate an immune response within the body . Use of autologous stem cells could be useful in future treatments regarding donor samples for immediate treatment . Donor samples could provide a useful treatment if the injured horse was genetically susceptible to tendon injury (possibly due to a high type III: type I collagen ratio) due to abnormality within the genetic code . A donor without any abnormality in their genes would provide mesenchymal stem cells that would differentiate to produce tenocytes that stimulate growth of stronger collagen for the patient's future health.
Success of Stem Cell Therapy (compared to traditional treatment) The nature of stem cell therapy suggests that the equine patient will have an increased chance of returning to full work at the prior level- due to new tendon tissue forming in place of the alternative scar tissue. Scar tissue tendon fibres lack viscoelastic properties and are more susceptible to re-injury upon loading of the tendon. A study was conducted in which 66 horses with tendon injury were treated by adipose derived stem cells , all of the horses were free from any other major disease and racehorses were not considered. The owner followed up the horse’s progress via a survey. Results showed that on average (SDFT and DDFT injury on rear and fore limbs) 77% of horses returned to full work at the previous level, 17% returned to full work at a lower level and 6% were unresponsive. The data showed that SDFT responded better to treatment with 81% fore-limb and 80% hind-limb patients being back in full work at their previous level, and DDFT was less successful with only 70% return to full work on the fore-limb and 50% return to full work hind-limb. When the results are compared against traditional therapy there is a marked difference as only 40 to 60% of horses became sound after a year, compared with 94% of stem cell treated horses were sound after a year . A comparative study focused on the healing and re-injury of tendon trauma in sport horses, comparing conservative therapy and other traditional therapies such as injecting sodium hyaluronate and
PSGAG into the injured tendon . Although this does not consider stem cell therapy it is worth considering the correlation between tendon fibre linear alignment and recovery rates in the 141 horses. In Dyson’s study, 50 horses received conservative treatment, following a controlled exercise programme, 50 horses were treated with high molecular weight sodium hyaluronate, 20 received intralesional and intramuscular injections of PSGAG and 30 horses received just intramuscular injections of PSGAG. Fibre patterns were graded based on their linear arrangement on transverse and longitudinal imaging. It was found that the grading of linear tendon fibre realignment was directly related to the incidence of re-injury in the horses . Stem cells form linear alignment of tendon fibres as opposed to the scar tissue formation, so the correlation suggests that stem cells would provide effective healing with less chance of re-injury. The studies mentioned seem to provide firm evidence that stem cell therapy is a reliable and successful treatment, however there are limitations. An example of a limitation is that stem cell therapy is not always a treatment option, as previously discussed, the stem cells often require a scaffold  to keep them at the location require, if this cannot be achieved then the treatment is not suitable as the stem cells will not function to their medical purpose. Core lesions of tendon offer a suitable closed location for implant of stem cells without them leaking out, however other lesions require a biomechanical scaffold that mimics tendon extra cellular matrix, and this is not always possible due to the location of the trauma.
Not all evidence is in favour of stem cells providing effective healing in tendon tissue. Awad et al  investigated the rabbit patellar tendon and found that the mesenchymal stem cell therapy improved biomechanical properties of the tendon, making more abundant mature collagen fibres and cells, but there was no improvement on the microstructure of the tendon upon comparison with the control. In contrast research by Young et al (1998)  found that when compared with controls, the Achilles tendon in a rabbit presented significantly better structure regarding ability to load and better aligned collagen fibres. When considering equine tendon research, a study by Lacitignola et al (2008)  provides significant findings. Tendon breakdown was induced in horses by an injection of collagenase enzyme in to the superficial flexor tendons. After the induced damage, horses received injections of either: bone marrow mesenchymal stem cells, bone marrow mononucleated cells or controls of fibrin in to the damaged tendon. Histological results upon euthanasia of the horses showed that the stem cell group presented higher collagen type 1 to type 3 ratio and better alignment of fibres, which would result in stronger tendon tissue for loading; therefore suggesting that stem cell therapy is a superior in comparison to other treatments used in the study for promoting a higher ratio of Type-1 collagen and as a result, retaining the viscoelastic properties of tendon tissue more effectively.
Conclusion In conclusion, it can be determined that the use of stem cells in the regenerative therapy following equine tendon injury offers promising outcomes. There is clear evidence that stem cells offer the potential for new tendon tissue fibre to grow in linear alignment as opposed to the criss-cross scar tissue and promote improved chance of recovery. The studies show great statistical evidence in favour of stem cell therapy, with 94% of stem cell treated horses becoming sound after treatment, with a dramatic comparison to the 40-60% of traditionally treated horses. Stem cell research has already made a huge impact on the equine competition industry, with high level competition horses being thrown a lifeline regarding returning to full work, as opposed to early retirement, change to a less active lifestyle or euthanasia. The potential for horses to be administered with allogeneic stem cells which contain preferred genes, which will subsequently produce a stronger tendon, offers a huge advancement in medical treatments. Mesenchymal stem cell use cannot be subject to the ethical scrutiny that surrounds embryonic stem cells, based on the fact that they are derived from adult tissue of the equine patient, which therefore makes them a useful medical tool; and the additional risk of teratoma formation associated with embryonic stem cells provides substantial medical motive to avoid their use in vivo. The use of stem cells in for equine tendon injury is still a new and developing treatment that is not fully understood. As research continues the use of stem cells in equine tendon injury can be evaluated further, but as it stands they appear to provide a medical advantage.
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