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The role of microvesicles derived from mesenchymal stem cells in tissue regeneration, a dream for tendon repair?

1. January 2012

Review article
The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?
Ciro Tetta1,
Anna Lange Consiglio2, Stefania Bruno3, Emanuele Tetta4, Emanuele Gatti1, Miryana Dobreva1, Fausto Cremonesi2, Giovanni Camussi5
1 Center of Translational Regenerative Medicine, Frese- nius Medical Care Deutschland GmbH, Torino, Italy
2 Reproduction Section, “Polo Veterinario di Lodi”, Faculty of Veterinary Medicine, University of Milan, Italy
3 Department of Molecular Biotechnology and Health Sci- ences, University of Turin, Italy
4 Faculty of Science of Animal Production, University of Bologna, Italy
5 Department of Medical Sciences, University of Turin, Italy
Corresponding author:
Giovanni Camussi
Department of Medical Sciences University of Turin Corso Dogliotti 14, 10126 Turin, Italy
Tendon injuries represent even today a challenge as repair may be exceedingly slow and incomplete. Re- generative medicine and stem cell technology have shown to be of great promise. Here, we will review the current knowledge on the mechanisms of the regen- erative potential of mesenchymal stem cells (MSCs) obtained from different sources (bone marrow, fat, cord blood, placenta). More specifically, we will devote attention to the current use of MSCs that have been used experimentally and in limited numbers of clinical cases for the surgical treatment of subchondral-bone cysts, bone-fracture repair and cartilage repair. Based on the recently emerging role in regenerative mecha- nisms of soluble factors and of extracellular vesicles, we will discuss the potential of non-cellular therapies in horse tendon injuries.
Key words: horse tendinopathies, microvesicles, regen- erative medicine, soluble factors, stem cells.
Stem cells have evoked considerable excitement in vet- erinary medicine because of the promise that stem cell
technology could deliver tissue regeneration for injuries for which natural repair mechanisms do not deliver func- tional recovery and for which current therapeutic strate- gies have minimal effectiveness. Tendon injuries have represented an area of particular interest since conven- tional treatments often lead to an unsatisfactory healing process that usually results in a relatively high recur- rence rate. In recent years, regenerative medicine has emerged as an attractive field for new cellular and non- cellular approaches to tissue repair. Here, we will review the current knowledge on the mechanisms of the regen- erative potential of mesenchymal stem cells (MSCs) ob- tained from different sources (bone marrow, fat, cord blood, placenta). More specifically, we will devote atten- tion to the current use of MSCs that have been used ex- perimentally and in limited numbers of clinical cases for the surgical treatment of subchondral-bone cysts, bone- fracture repair1 and cartilage repair2,3. However, by far the most frequent clinically use has been the treatment of overstrain-induced injuries of tendons in horses. We will discuss the hypothesis that also soluble factors and ex- tracellular vesicles, also called microvesicles (MVs), re- leased by MSCs may have a relevant regenerative poten- tial and may open new therapeutic perspectives.
The paracrine effect of stem cells
Increasing experimental evidence indicate that the active factors exert effects on neighbouring cells. Indeed, MSCs express high levels of transcripts of hematopoietic stem cells maintenance factors, including CXCL12 chemokine, stem cell factor, angiopoietin-1 (Ang-1), interleukin-7, vascular cell adhesion molecule 1 and osteopontin4. Sup- port for the hypothesis of paracrine action of MSCs de- rives from in vivo studies indicating that, although MSCs exhibit multilineage differentiation potential and can mi- grate to injured sites after systemic administration, the dif- ferentiation of MSCs in cells of injured tissues contributed little to their therapeutic benefits. A growing number of ev- idence indicates that the in vivo effects of MSCs depend primarily on their capacity to secrete bioactive soluble fac- tors. This bioactive molecules may inhibit fibrosis and apoptosis, enhance angiogenesis, stimulate mitosis and/or differentiation of tissue-intrinsic progenitor/stem cells5 and modulate the immune response6.
In different pre-clinical animal models, MSCs administra- tion have been shown to improve perfusion and restore cardiac function after myocardial infarction7; MSCs accel- erates recovery in acute kidney injury (AKI) induced by toxic agents or ischemia reperfusion and induces func- tional improvement in chronic kidney disease8-13. In addi- tion, MSCs have been studied in several in vivo models
212 Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221

The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?
of lung disease14,15. For example, in the bleomycin in- duced lung injury and fibrosis, MSCs improve lung inflam- mation and survival when given intravenously. These ef- fects are not accounted to lung engraftment rates (< 5%) but rather to a paracrine mechanism16.
The beneficial effects of MSCs infusion in different animal models are interpreted as not dependent on a direct sub- stitution of injured cells, but rather on paracrine effectors that facilitate endogenous repair processes. In this way, a paracrine role of MSCs in renal tissue repair has been supported by experiments showing that conditioned medium (CM) from MSCs mimics the beneficial effects of the cells of origin, when intra-peritoneal injected in mice with cisplatin induced AKI17. Moreover, intravenous admin- istration of CM from MSCs induces significant survival im- provement in fulminant hepatic failure18,19.
MSCs have been also investigated as a new therapeutic strategy for graft-versus-host disease, Chron’s disease and for the prevention of organ transplantation rejection. The mechanism by which MSCs modulate the immune re- sponse is still under investigation, but it is evident that it involves also the release of soluble factors and not only the cell-to-cell contact. MSCs may suppress several T-lymphocyte activities both in vitro and in vivo and may alter the cytokine expression profile of dendritic cells (DCs), naïve and effector T cells and natural killer cells (NK) to induce a more anti-inflammatory or tolerant phe- notype and to increase the proportion of regulatory T (Treg) cells. Prostaglandin E2 (PGE2) is implicated in the immunomodulatory effects of MSCs. Indeed, PGE2 pro- duction is up-regulated after co-culture of human MSCs with peripheral blood mononuclear cells20 and the in- hibitors of PGE2 production diminish MSC-mediated im- munomodulation in vitro21. Indoleamine 2, 3 deoxyge- nase (IDO), PGE2 and TGF-?1 can represent relevant mediators of MSC inhibition of NK functions21-23. MSCs also secrete IL-6, that is involved in the reversion of mat- uration of DCs to a less mature phenotype24. Blockade of PGE2 synthesis in MSCs reverts the inhibitory effects on DC differentiation and function. PGE2 and IL-6 can me- diate the effects of MSCs on DCs, thus leading to T-cell suppression25.
Regenerative medicine and tendinopathies
Tendon repairs are often weak and susceptible to re-in- jury. Given the frequency and increasing cost of these in- juries, mainly in sport horse, as well as the relatively poor result of surgical intervention, it is not surprising that new and innovative strategies like tissue engineering have become more appealing.
Several lines of evidence suggest that multipotent stem cells are present also in tendons and ligaments. First, both human and mouse tendons develop fibrocartilage and os- sification in response to injury26,27. Second, tendon-derived immortalized cell lines or human tendon derived fibrob- lasts express genes of adipogenic, osteogenic and chon- drogenic differentiation pathways, suggesting that they possess multiple differentiation capacities in vitro28,29. Fi- nally, postnatal stem cells capable of differentiating into adipocytes and osteoblastic cells have been identified in
human periodontal ligaments30 while human and mouse tendons harbor a unique cell population, termed tendon stem/progenitor cells (TSPCs), that has universal stem cell characteristics such as clonogenicity, multipotency and self-renewal capacity31. Recently, Lovati et al.32 iden- tified TSPCs specifically in the horse SDFT with the abil- ity to be highly clonogenic, to grow fast and to differenti- ate in different induced cell lineages as well as bone marrow derived progenitor cells (BM-MSCs). The hy- pothesis that TSPCs possess a mesenchymal stem cell behavior opens a new prospective for tendon regenera- tive medicine approaches because TSPCs could repre- sents an important tool to study basic tendon biology. The exact site for TPSCs cells within tendon is not known, but they are most likely to reside in the endotenon tissue be- tween the collagen fascicles and adjacent to the vascu- lature33. Although this might be true in young growing ten- don, mature equine tendon, however, does not appear to possess a substantial subpopulation of these cells capa- ble of differentiating into multiple cell lines, as reported for adult tissue34,35, and this may explain why this component of the repair process is limited and hence natural repair is inferior to normal tendon.
During the repair process, there is a large influx of cells into the lesion. Kajikawa et al.36 showed that at 24 h af- ter the injury, the wound contained circulation-derived cells but not tendon-derived cells. Tendon-derived cells appeared in the injured area at 3 days after the wound, and significantly increased in number with time and main- tained a high level of proliferative activity until 7 days after the injury, whereas the circulation-derived cells de- creased in number and are replaced by the tendon-de- rived cells. These findings suggest that circulation-derived and tendon-derived cells contribute to the healing of ten- dons in different periods as part of a biphasic process but that the cells mainly involved in the synthesis of new tis- sue are believed to be tendon derived cells36,37. For this reason some authors hypothesized that the implantation of far greater numbers of progenitor stem cells, than are present normally within tendon tissue, would have the potential of regenerating or improving the repair of the tendon. Fibroblasts derived from tendon or other sources could be used38, but the removal of sections of tendon to recover cells leads to the formation of a sec- ondary lesion in the horse that is unacceptably. Alterna- tive cell sources under investigation (Tab. 1) include dermal fibroblasts, which were shown to be capable of functionally bridging a tendon defect and to have simi- lar histological and tensile properties to the tenocyte- seeded scaffold39 although in vitro these cells behave differently from tenocytes40. By contrast, an optimal in vivo regenerative response could be accomplished by MSCs of different sources (Tab. 1).
Stem cell therapies in tendons
MSCs have been implanted into surgical defects in ten- dons in multiple in vivo experiments in laboratory animals with mostly positive outcomes. Most of these models have used surgically created defects in rabbit or rat ten- dons and have variously shown some improvement in
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Table 1 – Sources for cell therapy of tendinopathies.
Cell source Advantages Disadvantages Ref
EMBRYO Embryonic stem cells – pluripotent – teratoma formation 88
Amnion-derived cells
MSCs from umbilical cord tissue
no invasive collection high plasticity and proliferative capacity high number of immediately available cells for therapy well-tolerated by horses

  • no invasive collection
  • greater multipotent than BM-MSCs
  • possibility to obtain more rapidly proliferating cells by cell sorting
  • no immune response
  • strict surveillance [72] of parturition
  • strict surveillance [89-90] of parturition
    Concentrated bone marrow aspirate (BMC)
    Stromal vascular fraction from adipose tissue
  • minimal manipulation – no cell expansion
  • minimal manipulation
  • no cell expansion
  • well-tolerated by horse
    invasive aspiration [91] procedure with risk of pneumopericaridium
    no reports on the use
    of BMC on tendonitis
  • invasive collection [92]
    MSCs from bone marrow (BM-MSCs)
    MSCs from adipose tissue
    Tendon stem/progenitor cells
  • multipotenti
  • no immune response
  • higher proliferative potential and less senescence of BM-MSCs – multipotent
  • possible activation of this endogenous population
  • multipotent
  • invasive aspiration procedure with risk of pneumopericaridium
  • limited potential than ESC in terms of expansion (delay of 2-4 weeks to obtain
    a sufficient number of cells to in vivo implant)
  • invasive collection
  • invasive collection
    (removal of sections of tendons leads to the formation of secondary lesion) – mature equine tendon
    do not posses a substantial population of these cells
    Fibroblasts derived from tendon
    Dermal fibroblasts
  • appropriate tendon matrix synthesis
  • appropriate tendon matrix synthesis
  • easy to recover, with acceptable donor site lesion – similar histological and tensile properties than tenocyte
  • invasive collection [38] – age-related reduction
    in synthesis of matrix ability
  • invasive collections [37]
  • different protein-matrix [39] synthesis than tenocytes
    214 Muscles, Ligaments and Tendons Journal 2012; 2 (3): 212-221 The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?
    structure and strength of defects implanted with MSCs in a biodegradable scaffold (collagen gel, Vicryl knitted mesh or fibrin glue) compared to controls implanted with just the scaffold, as assessed by histology or simple bio- chemical assays41-45. In other studies using a rat patellar defect model, MSCs implantation has been associated with both greater ultimate tensile stress and improved quality of reparative tissue determined by an increased collagen I/III ratio46,47. Thus, MSCs-seeded constructs implanted in vivo have shown the ability to integrate into the tissue and induce the synthesis of tissue-specific ex- tracellular matrix. In the horse, tendon injuries are mostly located in the superficial digital flexor tendon (SDFT), which represents the strongest tendon in the equine body. The SDFT displays several similarities to the human Achilles tendon concerning anatomy, biomechanics and pathogenesis of tendinopathy. In different species, path- omorphology of tendinopathy differs in lesion size. In the horse, one typical so-called “core lesion” is usually cen- trally located within the tendon, extended in length and still surrounded by intact tendon tissue. The equine SDFT in- jury lends itself to cell therapy because provide many of the additional elements required for tendon tissue engi- neering. The lesion manifests within the central core of the tissue provides a natural enclosure for implantation that, at the time of stem cell implantation is filled of granulation tissue, which acts as a scaffold (Fig. 1)48. This enables the application of MSCs without any artificial scaffold mate- rial, merely by injecting a cell suspension directly into the lesion49; thereby, MSCs are exposed to a natural environ- ment providing collagen fibers and growth factors. In ad- dition, during rehabilitation with controlled exercise, there is an ideal mechanical stimulation allowing the newly created tissue to organize itself in the direction of the force application, hence this approach can be referred to as “in vivo tissue engineering”50. Unfortunately, in the horse, the efficacy of these treatments is difficult to determine, since the use of control animals is rarely reported and of- ten the stem cell treatment is combined with other biolog- ical factors, such as bone marrow supernatant, autolo- gous serum, or platelet-rich plasma. In any case, since this treatment regime was first published in 200349 there
    have been several experimental and clinical studies with encouraging results, giving evidence of the benefit and safety of MSCs application for tendon regeneration. Fur- thermore, unfortunately, it is still unclear whether the ma- jor contribution of the MSCs to the healing process is to differentiate into tenocytes and thus produce extracellu- lar matrix molecules, whether it is rather to supply growth factors and thus stimulate the residing cells within the ten- don51,52 or whether a combination of the two mechanisms occurs6,53. Mononuclear cells could represent an exo- genic stimulus for induction of pro-inflammatory mediators in tendon54. In addition, recent studies have suggested an anti-inflammatory role of implanted stem cells. In this context animal model studies have demonstrated that MSCs are hypo-immunogenic and inhibit the activation of T and B lymphocytes and NK cells55,56. The precise mech- anism of the anti-inflammatory effect of these cells is largely unknown. The role of soluble factors and extracel- lular vesicles as effectors in paracrine effect is described below. In essence, the paracrine effect results in the combination of different, biological activities: anti-apopto- sis, additional recruitment of resident multipotent stem cells, stimulation of angiogenesis, and the release of growth factors48.
    The clinical and not experimental nature of the use of MSCs for horse tendinopathies preclude the routine post mortem analyses but some experimental works has been carried out to monitor the fate of injected MSCs in horses and the structural aspect of the healing. Guest et al.57 studied the fate of autologous and allogeneic MSCs trans- fected with green fluorescent protein (GFP) following in- jection into the SDFT and revealed that GFP labeled cells located mainly within injected lesions, but with a small proportion integrated into healthy tendon. Further- more, the authors showed that both autologous and allo- geneic MSCs may be used without stimulating an unde- sirable cell mediated immune response from the host. Other postmortem examinations have shown that MSCs application improved the extracellular matrix structure of damaged tendons. In histological sections of MSC-treated tendon lesions, compared to non-treated tendon lesions, increased tendon fiber densities, increased organization
    Figure 1. Severe SDFT core lesion in a forelimb SDFT. Arrows show anechoic area in transverse (A) ultrasound scans, and slightly ipoechoic area in transverse (B) ultrasound sections, respectively, in the same lesion 50 days after amniotic derived cells implant.
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of the collagen fibers and a reduced vascularity have been found58-60. The beneficial effect of MSCs seems to be due to the improvement of structural organization rather than of matrix composition. However, it has been shown that MSCs treatment can enhance expression levels of cartilage oligomeric matrix protein (COMP)58,59, a glycoprotein that is known to be important for tendon elasticity and stiffness62. Ultrasonographic follow-up ex- aminations showed significant improvements in fiber alignment and echogenicity scores at 1, 3 and 6 months after MSCs treatment63, supporting the histological find- ings in the above-mentioned studies. In these studies, au- tologous adult progenitor cells have been used, either ex- panded bone marrow-derived MSCs60,64-66, or adipose derived MSCs59,67 or adipose-derived mononuclear cells (ADNCs)58,68. Furthermore, the effects of autologous bone marrow derived expanded MSCs and bone marrow-de- rived mononuclear cells on tendon healing have been compared revealing a similar improvement, in both treat- ment groups compared to the control group, which was demonstrated by significantly improved ultrasonography and histology scores, higher COMP expressions and rel- atively lower type III collagen contents61,70.
If stem cells are truly immunomodulatory, allogeneic trans- plantations should be possible. Safe and efficacious ap- plications of allogeneic stem cells would imply that off – the-shelf stem cell products could be developed for increased availability and rapid implementation of stem cell therapies early in a disease course54. Indeed, not only autologous progenitor cells but also allogeneic bone mar- row-derived MSCs57, allogeneic adipose-derived MSCs67 and allogeneic amniotic derived MSCs72 have been ap-
plied for treatment of equine tendon injuries and no evi- dence of immune rejection were detected.
Extracellular vesicles released from MSCs as an emerging paracrine mechanism
Recent studies have shown that beside soluble factors small vesicles released from cells, named extracellular vesicles or MVs, are instrumental in cell-to-cell commu- nication73,74 (Fig. 2). MVs are an heterogeneous popula- tion of small vesicles constituted by a circular fragment of membrane containing cytoplasm components which are released by different cell types. The two major classes of MVs released in the extracellular environment are the ex- osomes and shedding vesicles75. Exosomes originate from inward of endosomal membrane, accumulate within multivesicular bodies, are secreted by a process of exo- cytosis and exhibit a 30-120 nm size. At variance, shed- ding vesicles take place from direct budding of plasma membrane surface and are more heterogeneous in size ranging from 80nm to <1mm depending from the cell of origin and on stimuli75. The released MVs can be up-taken by neighbouring cells either as result of surface receptor mediated interaction or by a process of membrane fusion. After interaction MVs can be internalized by the recipient cells and deliver their content73,74. Therefore, MVs have been uncovered as a new mechanism of inter-cellular communication that involves direct receptor mediated stimulation of the target cells and delivery of bio-active lipids, proteins and nucleic acids. The content of MVs and their biological action not only depends on the cell of ori-
Figure 2. Schematic representation of the potential anti-inflammatory action of microvesicles (MVs) released by mesenchymal stem cells (MSC) on horse tendon.
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The role of microvesicles derived from mesenchymal stem cells in tissue regeneration; a dream for tendon repair?
gin, but also on the metabolic state of the cells. Therefore, different stimuli may modify not only the amount of MVs release, but also their content. One of the most exiting findings is that MVs were found to be a vehicle for ex- change of genetic information capable to induce transient or permanent phenotypic changes in the recipient cells73,74. This observation has deep implications in differ- ent physiological and pathological conditions. In the con- test of stem cell biology it has been suggested that sig- nals shuttled by MVs are an integral component of the stem cell niche and may be critical in the differentiation de- cision of stem cells76. In particular, the signals between in- jured cells and stem cells are bi-directional73. Indeed, MVs derived from injured cells are able to induce tissue specific differentiation of bone marrow cells and MVs de- rived from stem cells are capable to activate regenerative programs in cells survived to injury. The first possibility is proved by the observation that MVs released from injured lung cells induce expression of specific lung transcripts and phenotypic changes in bone marrow cells77. The hor- izontal transfer of genetic information from stem/progen- itor cells to differentiated cells was firstly shown for MVs derived from human endothelial progenitors (EPC). These MVs shuttle mRNA to quiescent endothelial cells via in- teraction with specific adhesion molecules (?4- and ?1- integrins) and activate an angiogenic program78. The mo- lecular analysis of mRNA indicate that MVs derived from EPC contain specific subset of cellular mRNA, including mRNA associated with pathways relevant for angiogen- esis such as the PI3K/AKT and eNOS signalling path- ways78. This mRNA are functional as are they are trans- lated into proteins within the recipient cells. Besides mRNA, MVs may transfer microRNAs (miRNAs) to target cells79. Since miRNAs are naturally occurring regulators of protein translation, this observation opens the possibil- ity that stem cells can alter the expression of gene prod- ucts in neighbouring cells by transferring miRNAs con- tained in MVs80.
Concerning the regenerative potential of MSC-derived MVs experiments have been performed in different animal models of tissue injury81-85. In models of acute renal injury MSC-derived MVs were found to be able to mimic the beneficial effects of the cells. In particular MVs acceler- ate the recovery in models of toxic and ischemia-reperfu- sion injury of the kidney and significantly enhance survival in a lethal model of cisplatin induced acute renal in- jury81,82. The mechanism was related to the delivery of mRNA derived from the MSCs and to its translation in the recipient cells. Through this mechanism MSC-derived MVs can limit the injury by inhibiting apoptosis and stim- ulate regeneration by inducing cycle re-entry of injured tu- bular epithelial cells. Therefore, the recovery for acute re- nal injury promoted by MSCs, mainly take place from the renal resident cells that undergo transient de-differentia- tion, proliferation to reconstitute the loss cell mass and fi- nally re-differentiation. Similar results were observed in a model of ischemic hearts treated with MVs derived from embryonic MSCs84,85.
Based on these observations, we can speculate that MVs released from MSCs may act also in different context of regenerative medicine such as the tendinopathies (Fig. 2). MVs, released by MSCs, may interact with and stimulate
tendon-resident cells to initiate an anti-inflammatory, anti- apoptotic and angiogenic response, and to reprogram so- matic cells toward a regenerative response. In particular, MVs derived from MSCs may counteract the action of in- flammatory cells accumulated at the site of injury.
In recent years, regenerative medicine has emerged as an attractive field for new cellular and non-cellular ap- proaches to tissue repair. The current knowledge on the mechanisms of the regenerative potential of MSCs put at- tention on the role of soluble components released by cells in the conditioned media. Soluble components, or growth factors, are used indirectly in equine medicine, as before discussed, in cases where stem cells are com- bined with platelet rich plasma, bone marrow supernatant, or autologous serum.
Growth factors are peptide signaling molecules that reg- ulate many aspects of cellular metabolism including the cell cycle, cell growth and differentiation, and the produc- tion and destruction of extracellular matrix products. Their effects are mediated primarily via autocrine and paracrine mechanisms, which provides the rationale for local admin- istration of exogenous growth factors to influence cellu- lar metabolism59. Of the growth factors influencing tendon metabolism, platelet derived growth factor, insulin-like growth factor-I (IGF-I), and transforming growth factor β show the most promise for enhancing tendon healing86. Although exogenous IGF-I has been shown to stimulate tendon healing in vivo in an equine model86 it has a short half-life, which necessitates repeated dosing, making clinical application challenging and costly. For this reason Schnabel et al.59 examined the effects of MSCs, as well as IGF-I gene enhanced MSCs (AdIGF-MSC) on tendon healing in vivo showing that both MSC and AdIGF-MSC injection resulted in significant histological tendon healing with minimal added value of IGF-I gene-enhanced MSC implantation compared to native MSC injection. This min- imum added value would confirm the hypothesis that in it- self the stem cells secrete growth factors and that the therapeutic effects of MSCs are mediated by paracrine factors secreted by the cells to stimulate the residing cells within the injured tissue rather than differentiate themselves. These paracrine factors could be exploited to extend the therapeutic possibilities of MSCs for the treatment of a variety of diseases. In this context MVs have a potential therapeutic application, as they mimic several of the biological actions of stem cells and may limit the concern of using of active replicating cells that may undergo mal-differentiation or mutation. In addition, MVs may be engineered to express and deliver molecules that favor reprogramming of resident cells toward regen- eration.
Use of the cells and technologies presented here in the horse are likely to continue and expand in the near future. The horse has been advocated as an animal model of
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tendon and ligament injuries, since many of the sponta- neous injuries seen in horses are similar to those seen in human athletes but other equine tissues and diseases, such as recurrent airway obstruction (asthma) and vari- ous hypoxic ischemic injuries, seem like straightforward candidates for equine stem cell research.
It is hoped that experience gained from treating naturally- occurring tendon injury in horses will provide sufficient supportive data to encourage the translation of this tech- nology into the human field where large randomized con- trolled trials will lead to a higher level of clinical evi- dence87.

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