of regenerative medicine in horses - Future Medicine

2 downloads 0 Views 2MB Size Report
therapy in regenerative medicine in horses and is based on presentations from a ... selected from leading authorities in both equine and human regenerative ...

Review For reprint orders, please contact: [email protected]

The current ‘state of play’ of regenerative medicine in horses: what the horse can tell the human

The horse is an attractive model for many human age-related degenerative diseases of the musculoskeletal system because it is a large animal species that both ages and exercises, and develops naturally occurring injuries with many similarities to the human counterpart. It therefore represents an ideal species to use as a ‘proving ground’ for new therapies, most notably regenerative medicine. Regenerative techniques using cell-based therapies for the treatment of equine musculoskeletal disease have been in use for over a decade. This review article provides a summary overview of the sources, current challenges and problems surrounding the use of stem cell and non-cell-based therapy in regenerative medicine in horses and is based on presentations from a recent Havemeyer symposium on equine regenerative medicine where speakers are selected from leading authorities in both equine and human regenerative medicine fields from 10 different countries.

Roger KW Smith*,1, Elaine R Garvican*,1, & Lisa A Fortier2 1 Department of Veterinary Clinical Sciences, The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire, AL9 7TA, UK 2 College of Veterinary Medicine Cornell University, Ithaca, NY 14853, USA *Authors for correspondence: [email protected] rvc.ac.uk [email protected] rvc.ac.uk

Keywords:  horse • mesenchymal stem cell • regenerative medicine

The relevance of the horse for the development of regenerative medicine in humans Many preclinical in vivo studies of new treatments utilize small laboratory mammals, largely because of cost and having established inbred strains with full genome determination and, hence, comprehensive molecular tools. By contrast, while the horse carries greater regulatory restrictions for its use as an experimental model and is very expensive to purchase and keep, it is an animal that has greater similarities to humans with respect to musculoskeletal disease. Physiologically, it is a species that shows an initial growth phase where its musculoskeletal tissues show a capacity to adapt, most specifically to mechanical loading, while, once skeletal maturity is reached, there is a progressive aging similar to humans, leading to an array of age-related diseases [1] . As horses are frequently used as athletes, this species demonstrates naturally occurring exerciserelated musculoskeletal disease similar to that seen in humans. Thus, horses develop

10.2217/RME.14.42 © 2014 Future Medicine Ltd

stress fractures related to exercise-stress adaptation mismatch in young animals (e.g., flat racehorses), osteoarthritis and over-strain injury of its highly loaded tendons. The size of the animal and the unfortunate necessity for euthanasia of animals that cannot be returned to the jobs for which they were purchased, means that there is cadaveric material available for characterization of pathology and investigations of pathogenesis. The presence of naturally occurring musculoskeletal disease in a large animal that ages and exercises makes the horse an ideal species in which to test new treatments and this is of particular relevance to translating regenerative techniques to humans. A shift in focus from repair of damaged tissues towards the regeneration of healthy tissue has opened up many new avenues in veterinary medicine. In the horse, the focus of regenerative medicine lies primarily in the musculoskeletal system, in particular for the treatment of over-strain tendon injuries, where the consequences of injury (altered limb biomechanics, reduced performance

Regen. Med. (2014) 9(5), 673–685

part of

ISSN 1746-0751

673

Review  Smith, Garvican & Fortier and a high reinjury frequency) along with physical features of the disease (in the provision of a ‘receptacle’ for intralesional treatment, assisting ease of administration and therapeutic retention) make tendons a particularly desirable target for such interventions. The injury in this tissue has particular relevance to humans because of the absence of a good laboratory model of human tendon disease [2] and because of striking similarities with the human counterpart disease (Figure 1) . Thus, equine tendons have a similar structure and composition to human tendons, with strong functional correlates that may not be in identical anatomical locations because of the differences between bi- and quad-rupeds. Thus, the weight-bearing tendons with the greatest similarities in the two species are the superficial digital flexor tendon (SDFT) of the metacarpal region in the horse and the Achilles tendon in the human, while those tendons suffering intrasynovial compressive lesions in the human rotator cuff have similar pathology in the deep digital flexor tendon within the digital tendon sheath of the distal limb in the horse. In both species, the age-related accumulation of exercise-induced damage appears to be an important pathogenetic mechanism for tendon disease [3,4] . This has been explained for many years in horses by the apparent minimal adaptive capacity of its tendons in the adult animal, based on a number of experimental studies [5,6] . It is only recently that evidence has come to light to suggest that the same situation exists in human tendons [7,8] . Moreover, as a further remarkable demonstration of similarities between the two species, specific polymorphisms in certain extracellular matrix proteins, tenascin-C [9] and collagen V [10] , related to tendon disease in humans have also been recently implicated in horses [11] . Advances in regenerative medicine technology have come from both human and veterinary fields but, with a more permissive regulatory environment, the treatment of veterinary diseases with stem cells is, in some instances, at a more advanced stage than that of human therapy for a comparative disease. The seeking of improved effectiveness in many of the equine musculoskeletal diseases has encouraged the development of regenerative treatment modalities, which fall broadly into two categories. Non-stem cell approaches (scaffolds, growth factors and platelet-rich plasma)

Scaffolds are artificial 3D structures into which cells are either seeded or migrate and which supports their attachment and organization as well as providing mechanical protection and enabling nutritional supply for the cells. Equine tendon lacerations have been repaired using materials such as carbon fiber, terylene

674

Regen. Med. (2014) 9(5)

and poly-L-lactic acid (PLLA) with varying degrees of success [12–15] . A commercial scaffold preparation of acellular, lyophilized pig bladder submucosa (ACell Vet™ Inc., MD, USA) has been used as a suspension for injection into damaged equine tendons and ligaments and persists within the lesion for only a short time. One of its mechanisms of action has been postulated to be the recruitment of local mesenchymal stem cells (MSCs) or growth factors, but no convincing evidence of efficacy has so far emerged in its clinical use. In addition, local reactions to this xenogenic implant have reduced its popularity in recent years. Complex combinations of growth factors are involved in tendon formation and growth. The benefits of therapeutic administration of recombinant growth factors (e.g., IGF-1, rEGH, TGF-b, GDF-5) in isolation or combinations thereof, remains unproven, but a focus of much research, with optimal dosage and treatment intervals still unknown. The use of rEGH in a collagenase model of tendon disease resulted in a significant increase in tendon cross-sectional area along with a reduction in yield and ultimate tensile stress, and reduced tendon stiffness [16] . This was interpreted as a deleterious outcome, but this experimental data represents one of the few examples of maintenance of tendon elasticity after treatment compared with the usual increase associated with more fibrosis. The experimental administration of IGF-1 reduced swelling, increased cell proliferation and collagen content and resulted in a lesion that, ultrasonographically, appeared smaller 3 weeks after cessation of treatment [17] . However, the nature and functional ability of the tissue that fills this lesion remains in question; an increase in the proportion of scar tissue would not align functionality with an echogenic ultrasonographic appearance. Encouraging reports of IGF-1 also exist for the treatment of chondral injuries in horses [18] . Platelet-rich plasma (PRP) is currently one of the most popular biological products for equine regenerative medicine. It can be produced using either centrifugation or filtration. PRP contains a rich mix of growth factors, most notably TGFβ-1, PDGF and VEGF. PRP preparations have been analyzed in vitro where they have shown anabolic effects on both cell  [19] and tissue cultures [20,21] . A filtration device has been evaluated that is practical in the field producing a filtrate containing a sevenfold increase in platelet concentration compared with peripheral blood, which can then be directly injected into the lesion [22] . This method has many advantages, including the recovery of functional platelets that do not prematurely release PDGF and suitability of storage for future use. In experimental lesions, application of

future science group

The current ‘state of play’ of regenerative medicine in horses: what the horse can tell the human 

A

Review

B

C

E

D

F

G

H

Figure 1. The horse as a good model for human tendinopathies. (A) The external swelling of the superficial digital flexor tendon of the horse (arrow), analogous to the swelling of the Achilles tendon in the human (B). There are similarities in both ultrasonographic appearance of hypoechoic areas (arrows); transverse and longitudinal images of horse (C & D) and human (E & F), and histology (horse (G) and human (H), which both show disorganized matrix and increased vascularity (arrows).

centrifuged PRP increased collagen and DNA content (therefore increasing cellularity) as well as increasing neovascularity, ultimate tensile strength and elastic modulus [23] . However, this treatment also increased glycosaminoglycan (GAG)  content, a consistent feature of pathological tendon and most changes could be ascribed in increased inflammation and fibrosis, possibly through TGFβ-1 and its pro-scarring effects [24] . In a subsequent study, however, post-PRP treatment demonstrated significantly better ultrasonographic tissue characterization [25] and the application of PRP to clinical cases of suspensory desmitis has been anecdotally positive. Nine standard-breds treated for midbody tendon lesions were compared with noninjured

future science group

controls; all treated horses returned to racing, after an average of 32 weeks, although with a reduction in earnings in the first year and in starts in the third year [26] . This finding is endorsed by a further report of 11 horses with suspensory ligament branch desmitis, treated with intralesional PRP, in which the ultrasonographic appearance of the lesion resolved within 3 months [22] . Stem cell approach

Although the mechanism of action of stem cells has yet to be fully elucidated, stem cells are being explored as a treatment for many musculoskeletal disorders in the horse, most notably tendinopathies.

www.futuremedicine.com

675

Review  Smith, Garvican & Fortier Types & characterization of stem cells

MSCs have traditionally been defined broadly by their ability to continually self-renew and to differentiate into multiple specialist cell lines. In practice, this generally takes the form of demonstrating osteogenic, chondrogenic and lipogenic differentiation. Some reports describe tenogenic differentiation, although the lack of well-defined, relevant outcome measures still limit its confident representation of true differentiation into this cell type. Although it is generally deemed both necessary and relevant to demonstrate trilineage ability, as a definition of ‘stemness’, this approach carries many frustrating inadequacies, as well as proving expensive and time-consuming to implement. In addition, a population of MSCs, even those derived from a single parent cell, can display a surprising degree of heterogeneity. This raises additional concerns regarding the ‘maintenance’ of a population of stem cells, throughout the requisite multiple passages needed to amplify numbers. Both the uncertain nature of primary isolates and the possibility of heterogeneity introduced during culture and expansion provides a challenge for regulatory compliance, which risks compromising clinical testing [27] . This is currently less onerous in the veterinary field, although it is increasing. Characterization can also be performed using antibodies specific to cell-surface antigens, either qualitatively through immunohistochemistry, or through fluorescence-activated cell sorting (FACS) to select for specific subpopulations. Such techniques are wellestablished for human MSCs, where a well-defined library is available (including CD105, CD73, CD90, STRO-1 and CD271) but currently, there are much more limited numbers of such identifying markers in the horse, as there is a lack of equine-specific antibodies and cross-reactivity with those available from other species is highly variable. However, as such, CD44 (a non-specific cell surface marker), CD29 and CD90 have found to be positive on equine MSCs, while being negative for CD45, and possibly CD34, CD79alpha and MHC-II [28–31] . Cell phenotype and gene expression profiling have also been used to define the phenotype of a cell population and MSCs may show specific gene expression profiles even whilst in their undifferentiated state [32] . Post-transcriptional gene stabilization may also play a role in the control of differentiation and this too could be used to characterize a population of cells. The lack of clinical markers for equine MSCs makes the construction of hierarchical models of multilineage differentiation difficult [33] and there remains a strong need for a universal, practical, laboratory definition of ‘stemness’, applicable across the range of sources from which MSCs can be obtained.

676

Regen. Med. (2014) 9(5)

Embryo-derived stem cells (ESCs) are self-renewing, pluripotent cells obtained from day 7 preimplantation equine blastocysts, following a similar method to that described in mice [34] . Since no teratoma formation was observed in mice, the terms ‘embryo-derived’ and ‘ES-like’ are used to differentiate these cells from ‘true embryonic stem cells’, although to what extent this discrimination is important is unclear. When co-cultured with embryonic feeder cells, equine ESCs have been successfully expanded and shown to retain the expression of many ESC markers and to differentiate down endodermal, ectodermal and mesodermal cell lines [35] . Whereas autologous and allogeneic MSCs injected into experimentally-induced equine tendon lesions were shown to have very poor survival rates ( 60% throughout the 90 day study period) [36] . In addition, no abnormal growths were detected and both histological and ultrasonographical repair progressed normally [36] . Additionally, fetally-derived embryoniclike cells injected into a collagenase-generated SDFT defect resulted in significant improvements in tissue architecture, tendon size, lesion size and linear fiber pattern, whilst not affecting the expression of specific matrix genes or total proteoglycan, collagen or DNA content of the lesion, suggesting regeneration, rather than repair [37] . Although slightly encumbered by the associated ethical considerations and specialist culture techniques, ESCs are an exciting future prospect in equine regenerative therapy. Induced pluripotent stem (iPS) cells are a recent focus gaining considerable attention for their potential clinical applications as they represent the only pluripotent, autologous type of stem cell not associated with the practical and ethical difficulties of ESCs [38] . Equine iPS cells have been generated [39,40] but their clinical validation is eagerly awaited. Certain strains of iPS cells have been shown to form teratomas in mice [41] but the tumorogenicity of equine iPS cell has yet to be determined, which is an important prerequisite prior to their clinical use. Sources of stem cells for clinical use

The most common source of MSCs is the bone marrow (BM-MSCs) and in the horse, size and anatomy assist in ease of collection and the generation of adequate yields. However, over the last decade, it has become evident that MSCs persist in, and can be isolated from, numerous tissues in the adult horse. In addition to bone marrow, the other common clinically used source is adipose tissue. Adipose tissue is an accessible, reliable, abundant site for MSC isolation, although culture is still needed to produce a cell population similar to that retrieved by culture of bone marrow

future science group

The current ‘state of play’ of regenerative medicine in horses: what the horse can tell the human 

or other sources. The more common use of this source clinically, however, utilizes the ‘stromal vascular fraction’ after digestion and flotation of the adipose cells, yielding a very different and heterogeneous cell composition compared with culture-isolated adipose stem cells (ASCs). ASCs are biologically similar, although possibly not identical, to MSCs [42] and have been shown to mediate repair by the secretion of paracrine factors, inhibiting inflammation and apoptosis and promoting angiogenesis and wound remodeling [43] . In human medicine, umbilical cord blood (UCB) is a well-established source of hematopoietic stem cells. Hematopoietic stem cells from either bone marrow or umbilical cord blood remain the only mainstream clinical use of stem cells in human medicine. MSCs have been derived from human cord blood, but these cells’ clinical utility remains to be determined. In the veterinary field, an increasing number of reports have been published on the isolation and laboratory properties of equine UCB-derived MSCs [44] . Despite the lack of in vivo studies on their therapeutic use, the commercial availability of blood banks in which to store UCB from horses bred for performance is becoming established. UCB offers some advantages over other sources of MSCs, including a pain- and risk-free (for both mare and foal) collection procedure, but yield is reportedly variable. Improved isolation methods have made it feasible to isolate MSC from cord blood [45–47] so that the greatest limitation is blood collection failure at the farm. Differentiation potential appears variable, depending on the protocol selected [48] , and migration activity low [49] . Interestingly, equine UCB-MSC may have higher chondrogenic potency than other equine MSC sources [49,50] . The use of UCB in the horse is in its infancy, but already opinions are divided regarding future benefits. The effect of isolation protocols on the resultant population’s potency requires further investigation. A further source of MSCs includes the amniotic membrane (AMSCs) [52,53] . Again, a noninvasive, lowcost collection procedure and reportedly good differentiation potential combined with rapid proliferation make these potentially attractive source of cells [53] . Allogeneic AMSCs offer the potential advantage of more rapid treatment, which may explain the lower rates of reinjury seen following SDFT  treatment using AMSCs compared with BM-MSCs [54] . MSCs can also be recovered from synovial fluid (SF) and synovium. Mesenchymal progenitors cells have been reported in SF of patients with osteoarthritis and cruciate ligament injury [55,56] . The most likely source of these cells is either the synovium, where MSCs are present [57] , or the subchondral bone marrow [58] . Equine SF-MSCs display similar growth

future science group

Review

characteristics and trilineage capacity to BM-MSCs, but when used to coat tendon explants, were less effective at preventing the loss of extracellular matrix proteins and should not be considered homologous when used in a clinical context [59] . In addition, although MSC numbers are increased in pathological joint fluid samples [56,60] the absolute numbers present in a sample are small, which effectively precludes the routine clinical use of SF-MSCs, certainly without culture, and the synovium may be a more reliable source. The surface zone of articular cartilage represents an alternative source of stem cells from which chondrocyte progenitor cells have been isolated in the horse [61] . Under chondrogenic differentiation, these chondroprogenitors demonstrate phenotypic stability as a hyaline cartilage phenotype after extended expansion in monolayer culture. By contrast, when equine chondroprogenitors were compared with equine bone marrow stromal cells, tissue formed by bone marrow stromal cells in vitro had an endochondral phenotype, suggesting that the chondrocyte progenitors may be a better choice for joint therapy. Tendon-derived progenitor cells have been isolated from equine tendon [62] using a differential adherence pre-plating technique, and these cells appeared to confer some beneficial histological effects on the treatment of collagenase-induced superficial digital tendinitis, but more detailed comparisons with alternative cell therapies are needed [63] . The multiple sources of MSCs for use in equine cell therapies raise additional questions and challenges, as well as opportunities. Investigation has shown that these subpopulations of cells can differ greatly from one another in differentiation ability and gene expression profiles, yet positive results are reported both in vitro and in experimental models of disease. In the light of these variations, it is likely that the defining characteristic of a stem cell, the presence (or absence) of which is critical in conferring therapeutic success to a population, has yet to be determined. Alternatively, in the future, treatment of a certain tissue may become aligned to stem cells obtained from a particular source, if it is established that the subpopulations resident therein impart specific benefits and locally-appropriate promotion of healing under specific circumstances. Stem cell maintenance, differentiation & mechanisms of action

Stem cells residing in vivo occupy conditions to which they are optimally evolved. In vitro expansion, purification and subsequent reimplantation into a foreign tissue must be accompanied by genomic integrity in order to minimize the risks of neoplastic transformation or premature aging.

www.futuremedicine.com

677

Review  Smith, Garvican & Fortier It is no longer thought that MSCs exert their effect through differentiation in a tissue-specific manner, but that they remain viable and initiate a paracrine effect, secreting bioactive molecules and delivering soluble factors to the host cells. Numerous studies have demonstrated that MSCs are potentially immunosuppressive, both in vivo and in vitro [64–66] . Although MSCs display reduced expression of both class I and II MHC antigens [67,68] it would be expected that alloreactive T cells would still mount a response; however, allogeneic equine MSCs have been administered in a variety of delivery methods, including intradermally [69] , intraarticularly [68] and into an injured tendon [70] , with no apparent immune response. Equine MSCs effect a reduction in proliferation of mixed alloreactive lymphocyte populations, including through indirect culture (when cells were separated by a transwell), or via the use of MSC-conditioned media [71] , further supporting the theory proposed from other species that soluble factors secreted by MSCs are responsible for their immune privilege [64,72] . In recent years, MSCs have been linked to the phenomenon known as ‘macrophage switching’. Two phenotypes of macrophage exist; classically activated macrophages are predominantly proinflammatory, whilst alternatively activated macrophages (AAMϕ) have roles in mediation of wound healing, angiogenesis and the deposition of new extracellular matrix [73,74] . The two phenotypes are not formed from distinct populations and a single macrophage can be classically activated in the early stages of healing and thereafter switch to become alternatively activated and participate in tissue resolution. In humans, macrophage switching can be partly induced by co-culture with MSCs [75] , resulting in the production of a novel type of ‘MSC-educated macrophage’ with characteristics similar to AAM. In vitro, MSCs are also capable of undergoing polarization into either a proinflammatory or an immunosuppressive phenotype [76] through stimulation of specific Toll-like receptors (TLRs) on the cell surface. TLR signals are released following tissue damage, recruiting immune cells and, potentially, MSCs, which were shown to express multiple TLRs, the antagonism of which greatly affected their migration, invasion and secretion of immune-modulating factors [77,78] . It is not known specifically whether equine MSCs adopt the same polarization, although the analysis of tissue after MSC treatment in experimental studies would be more consistent with their influence on inflammation rather than by exerting a true regenerative effect [79] . Studies of the effects of stem cell implantation in partially transected infraspinatus tendons in sheep [80] have demonstrated that MSCs had a beneficial effect on induced tendon pathology. Such effects were

678

Regen. Med. (2014) 9(5)

observed not only when injected acutely, but also in the face of established degenerative change, although there was no effect of MSCs on the actual lesion site. This is consistent with the therapeutic effect of MSCs being primarily due to secretion of soluble factors reducing degeneration rather than their differentiation into tenocytes and/or augmenting repair of the injury. The action of stem cells may be enhanced by scaffolds because cells detect the geometry of their surroundings and this in turn can influence cell behavior. However, most utilized clinically are biopolymers (e.g., fibrin glue) and lack sophistication. New scaffold technology involving biodegradable scaffolds that can release anabolic factors may offer significant advantages in the future and help manipulate stem cell mechanisms optimally. Cell tracking & fate after implantation

The ability to track cells implanted as treatment into an animal is important in furthering our understanding of the processes by which they have an effect. Given the lack of monoclonal antibodies available to identify or track the fate of equine MSCs, cell labeling using dyes, such as CM-DiL, offers a quick, easy alternative that requires no genetic manipulation, in contrast to genetic labeling (such as with green fluorescent protein) commonly used in small animal studies [81] . Equine MSCs labeled with CM-DiL remained viable, retained their differentiation capacity and were identified within both naturally occurring and surgically created SDF tendon lesions up to 170 days postimplantation (Figure 2) . No evidence of migration to adjacent areas of tendon was observed [82] . An alternative tracking method is to label MSCs with hexamethylpropyleneamine oxime (HMPAO) and Technetium99m (Tc99m), which allows in vivo tracking using a gamma camera. MSCs labeled with Tc99m have been used in both normal and experimentally created lesions [83,84] and in horses with naturally occurring tendinopathy [85] . In spite of the different experiments there was remarkable similarity in cell retention 24 hours after intralesional injection, where only 24% of implanted cells remained. Cell administration through intraarterial, and intravenous regional limb perfusion were also evaluated. Intra-arterial administration was more reliable than intravenous regional perfusion for diffuse distribution throughout the distal limb. However, intra-arterial regional perfusion carried some risks of vascular thrombosis and ischemic necrosis unless the tourniquet was omitted proximal to the injection site [86] . In studies of naturally occurring tendinopathy using intravenous injection, no labeling of the injured tendon was observed, although labeling efficiencies were low [85] . Redistribution of cells was observed

future science group

The current ‘state of play’ of regenerative medicine in horses: what the horse can tell the human 

A

C

E

G

B

D

F

H

Review

Figure 2. The effect on intralesional treatment of equine tendinopathy with mesenchymal stem cells. Transverse and longitudinal ultrasonographs taken before treatment (A & B) and 1 month after implantation (C & D) showing rapid in-filling of the hypoechoic lesion in the superficial digital flexor tendon. Evaluation of the tendon matrix 6 months after treatment shows better organization (E & F) with crimp pattern (G & H) in the mesenchymal stem cell-treated tendons (E & G) compared with saline-injected controls (F & H).

to the lung field, primarily following intravascular administration. By contrast, retention and survival of ESCs in an experimentally induced lesion were significantly higher than BM-MSCs. ESCs also migrated to adjacent areas of damage within the affected tendon, which was not observed with BM-MSCs [70] . Experimental & clinical effects of stem cell treatment in horses

Execution of blinded, placebo-controlled clinical trials is difficult in veterinary species, in particular the horse, because of the reluctance of owners and trainers to allow randomization. Until individual horse owners’ willingness to accept placebo treatment improves and/or the horse industry adapts policies similar to other major livestock industries compensating the individual animal owner for participation in such studies, the scientific value of these studies will be greatly underutilized. Furthermore, the purchase and upkeep of experimental animals for ultimate euthanasia at the conclusion of the study period is often prohibitively expensive and ethically complicated. In many aspects, the horse represents an effective, relevant model for human musculoskeletal disease. Commonly damaged equine tendons display physiological, mechanical and environmental similarities to those human tendons that are frequently injured, as well as suffering age-related degeneration and demonstrating failed healing, manifesting in the horse predominantly as a high reinjury rate for the most common injury (superficial digital flexor tendinopathy) (Figure 1) . For just over a decade, BM-MSCs have been used to treat naturally occurring superficial flexor tendon injury, following the development of a protocol for isolation and implantation [87] . During this

future science group

time, minimal numbers of adverse reactions have been reported for many thousands of horses treated. Histological evaluation of both induced [88,89] and naturally occurring injuries [90] in equine tendons show consistently improved organization in the reparative tissues [90,91] , compared with controls, although differences in composition and ultrastructure have been less convincing [92] . MSC-treated tendons have shown crimped organized collagen fibers, minimal inflammatory cells and no evidence of abnormal or neoplastic tissue [91] . An audit of an adequately powered clinical data set showed statistically lower rates of reinjury compared with historical controls treated with a variety of conventional therapies [91] . The use of naturally occurring tendon injury in this trial yields not only directly relevant clinical data for the equine industry, but also supportive data for the translation of this technology into the human field. Superficially similar results have been reported following the use of allogeneic ASCs (in combination with PRP) [93] and umbilical cord MSCs [94] for the treatment of over-strain injury, although in both cases, sample sizes were small and follow-up of limited length. Similar disease states of cartilage also exist in the horse and human. Articular cartilage is of a similar thickness in both species, enabling the creation and evaluation of multiple, large defects, assessment of which is aided by adequate arthroscopic access. In addition, re-evaluation of lesions and postoperative progression can be monitored clinically, alongside controlled exercise. An effective cartilage repair must generate a tissue structure capable of sustaining functional load and integrating with adjacent articular cartilage and the underlying subchondral bone. Treatment of cartilage defects using bone marrow concentrate [95] and MSCs [96] has been

www.futuremedicine.com

679

Review  Smith, Garvican & Fortier trialed in the horse and, although some clinical success has been reported following cell-scaffold implantation, this was not associated with any long-term beneficial effect [96] . No synergy was observed when stem cells and PRP were combined in an experimental study on tendon injuries in sheep [97] , an observation consistent with the hypothesis that while MSCs are immunosuppressive PRP is proinflammatory thereby negating the positive effects of stem cell implantation. An alternative approach utilized a direct injection of MSCs into the joint space, which reportedly carries long-term advantages, with repair tissue remaining firm following a year of strenuous exercise [98] . However, MSCs do not appear to be effective at stimulating repair in all types of osteoarthritis and so further study is necessary before drawing definitive conclusions on efficacy [99] . In conclusion, regenerative medicine is regarded as a ‘state-of-the-art’ technique and one which will continue to attract considerable attention, excitement and funding. However, the current use of stem cells and other biological products in the horse has not been conclusively shown to induce a truly regenerative effect and its benefits most probably lie in the ability of the implanted cells to modulate the natural repair mecha-

nisms in the different tissues. Improved cell retention and survival remain important goals for the improvement of this technology. Equine research is likely to be at the forefront of many breakthroughs in clinical application because of the ease of translation and, in reciprocation, advances in the understanding of stem cell biology will continue to benefit the equine athlete. Future perspective Considerable advances have been made in the last 10 years in translating this technology from the laboratory to the successful treatment of naturally occurring disease in large animals. The most important immediate challenge relates to improving retention after implantation, which we believe will be best offered by ‘smart’ scaffolds that mimic cell matrix interactions. We foresee that MSC treatment will continue to offer significant clinical advantages, although the most important indications are likely to be more limited than the current ‘scatter-gun’ approach and most likely related to clinical situations where their immunomodulatory properties carry the most benefit. MSC therapy will therefore extend into treatment of immunemediated and chronic inflammatory diseases and the

Executive summary Background • The horse is an excellent large animal model in which to test new treatments for degenerative musculoskeletal diseases, due to the presence of naturally occurring disease in a species that ages and exercises. • Treatment of veterinary diseases with stem cells is at a more advanced stage than that of human therapy for a comparative disease because of a permissive regulatory framework.

Type & characterization of stem cells • Trilineage differentiation is the most commonly used definition of ‘stemness’ in veterinary research, but it is frustratingly inadequate, expensive, time consuming and does not address concerns such as heterogeneity and population maintenance. • The defining characteristic of a stem cell, the presence (or absence) of which is critical in conferring therapeutic success to a population, has yet to be determined.

Sources of stem cells for clinical use • In the future, treatment of a certain tissue may become aligned to stem cells obtained from a particular source.

Stem cell maintenance, differentiation & mechanisms of action • Mesenchymal stem cells (MSCs) produce soluble immunomodulatory trophic factors that are likely to play a major role in the observed clinical beneficial effects on injured tendon.

Cell tracking & fate after implantation • Bone marrow MSCs are retained within both naturally occurring and surgically created tendon lesions for up to 170 days, but in small numbers. • Following implantation, bone marrow MSCs do not migrate to adjacent areas of tendon, in contrast to embryo-derived stem cells, in which migration is observed.

Experimental & clinical effects of stem cell treatment in horses • Auditing of clinical data sets has indicated efficacy of stem cell treatment in reducing tendon reinjury rates in athletic horses. • The efficacy of MSCs for the treatment of naturally occurring joint disease is less convincing, although is potentially related to specific types and stages of cartilage lesions. • Naturally occurring tendon and joint disease in the horse has yielded not only directly relevant clinical data for the equine industry, but also supportive data for the translation of this technology into the human field.

680

Regen. Med. (2014) 9(5)

future science group

The current ‘state of play’ of regenerative medicine in horses: what the horse can tell the human 

cell’s innate characteristics that best address these diseases will be augmented. The necessity of a two-stage procedure for autologous treatment will be replaced by the simpler, cheaper option of allogeneic cells. This will dictate better characterization of preimplantation phenotype and open up the potential to tailor cell selection for specific injuries and stages. True regenerative strategies are likely to rely on a combination of pluripotent stem cells and the appropriate environment to allow the recapitulation of tissue development. Acknowledgements This review is based on presentations from a recent Havemeyer symposium on equine regenerative medicine (see [99] ). The authors would like to thank the conference delegates, selected from leading authorities in both equine and human regenerative medicine fields from whose presentations much of this review has been based; Ana-Liz Alves (São Paulo State University, Brazil), Jennifer Barrett (Marion duPont Scott Equine Medical Center, USA), Frank Barry ( National University of Ireland, Ireland), Gordon Blunn (University College London, UK), Walter Brehm (University of Leipzig, Germany), Bruce Bunnell (Tulane University Health Sciences Center, USA), Peter Clegg (University of Liverpool, UK), Antonio Crovace (Università degli Studi di Bari Aldo Moro, Italy), Jay Dudhia (Royal Vet-

References

erinary College, UK), David Frisbie (Colorado State University, USA), Laurie Goodrich (Colorado State University, USA), Allen Goodship (University College London, UK), Debbie Guest (Animal Health Trust, UK), Brian Johnstone (Indiana University, USA), Yoshinori Kasashima (Equine Research Institute, Japan), Thomas Koch (Ontario Veterinary College, Canada), Chris Little (University of Sydney, Australia), Helen McCarthy (Cardiff University, UK), Laurie McDuffee (University of Prince Edward Island, Canada), Wayne McIlwraith (Colorado State University, USA), Alan Nixon (Cornell University, USA), Gene Pranzo (Havemeyer Foundation, USA), Iris Ribitsch (University of Veterinary Medicine in Vienna, Austria), John Schimenti (Cornell University, USA), Michael Schramme (Ecole Nationale Veterinaire de Lyon, France), Allison Stewart (University of Illinois, USA), and Martin Vidal (University College Davis, USA).

Financial & competing interest disclosures The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. 7

Heinemeier KM, Schjerling P, Heinemeier J, Magnusson SP, Kjaer M. Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C. FASEB J. 27(5), 2074–2049 (2013).

••

Incorporation of radiation into the cells of people exposed to atomic radiation conclusively demonstrates the lack of tissue renewal in the tendon.

8

Heinemeier KM, Bjerrum SS, Schjerling P, Kjaer M. Expression of extracellular matrix components and related growth factors in human tendon and muscle after acute exercise. Scand. J. Med. Sci. Sports 23, e150–e161 (2013).

9

Mokone GG, Gajjar M, September AV et al. The guaninethymine dinucleotide repeat polymorphism within the tenascin-C gene is associated with achilles tendon injuries. Am. J. Sports Med. 33, 1016–1021 (2005).

10

Mokone GG, Schwellnus MP, Noakes TD, Collins M. The COL5A1 gene and Achilles tendon pathology. Scand. J. Med. Sci. Sports 16, 19–26 (2006).

11

Tully LJ, Murphy A, Smith RKW et al. Polymorphisms in TNC and COL5A1 genes are associated with risk of superficial digital flexor tendinopathy in National Hunt Thoroughbred racehorses. Equine Vet. J. 46(3), 289–293 (2014).

Papers of special interest have been highlighted as: • of interest; •• of considerable interest 1

Smith RK, Birch HL, Goodman S, Heinegard D, Goodship AE. The influence of ageing and exercise on tendon growth and degeneration – hypotheses for the initiation and prevention of strain-induced tendinopathies. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 133, 1039–1050 (2002).

2

Lui PP, Maffulli N, Rolf C, Smith RK. What are the validated animal models for tendinopathy? Scand. J. Med. Sci. Sports 21, 3–17 (2011).

••

A discussion of the suitability and validity of various animal models for studying human tendinopathies.

3

Dudhia J, Scott CM, Draper ER et al. Aging enhances a mechanically-induced reduction in tendon strength by an active process involving matrix metalloproteinase activity. Aging Cell 6, 547–556 (2007).

4

Lavagnino M, Arnoczky SP, Egerbacher M, Gardner KL, Burns ME. Isolated fibrillar damage in tendons stimulates local collagenase mRNA expression and protein synthesis. J. Biomech. 39, 2355–2362 (2006).

5

Smith RK, Goodship AE. The effect of early training and the adaptation and conditioning of skeletal tissues. Vet. Clin. North Am. Equine Pract. 24, 37–51 (2008).

12

Thorpe CT, Streeter I, Pinchbeck GL et al. Aspartic acid racemization and collagen degradation markers reveal an accumulation of damage in tendon collagen that is enhanced with aging. J. Biol. Chem. 285, 15674–15681 (2010).

Goodship AE, Brown PN, Silver IA, Jenkins D, Kirby M. Use of carbon fibre for tendon repair. Vet. Rec. 102 (14), 322 (1978).

13

Goodship AE, Brown PN, Yeats JJ, Jenkins DH, Silver IA. An assessment of filamentous carbon fibre for the treatment of tendon injury in the horse. Vet. Rec. 106, 217–221 (1980).

6

future science group

Review

www.futuremedicine.com

681

Review  Smith, Garvican & Fortier Eliashar E, Schramme MC, Schumacher J, Ikada Y, Smith RK. Use of a bioabsorbable implant for the repair of severed digital flexor tendons in four horses. Vet. Rec. 148, 506–509 (2001).

28

15

Gibson KT, Burbidge HM, Robertson ID. The effects of polyester (terylene) fibre implants on normal equine superficial digital flexor tendon. NZ Vet. J. 50, 186–194 (2002).

de Mattos Carvalho A, Alves AL, Golim MA et al. Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue. Vet. Immunol. Immunopathol. 132, 303–306 (2009).

29

16

Dowling BA, Dart AJ, Hodgson DR, Rose RJ, Walsh WR. The effect of recombinant equine growth hormone on the biomechanical properties of healing superficial digital flexor tendons in horses. Vet. Surg. 31, 320–324 (2002).

Ranera B, Lyahyai J, Romero A et al. Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue. Vet. Immunol. Immunopathol. 144, 147–154 (2011).

17

Dahlgren LA, van der Meulen MC, Bertram JE, Starrak GS, Nixon AJ. Insulin-like growth factor-I improves cellular and molecular aspects of healing in a collagenase-induced model of flexor tendinitis. J. Orthop. Res. 20, 910–919 (2002).

30

De Schauwer C, Piepers S, Van de Walle GR et al. In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry. Cytometry 81, 312–323 (2012).

18

Ortved KF, Nixon AJ, Mohammed HO, Fortier LA. Treatment of subchondral cystic lesions of the medial femoral condyle of mature horses with growth factor enhanced chondrocyte grafts: a retrospective study of 49 cases. Equine Vet. J. 44, 606–613 (2012).

31

Radcliffe CH, Flaminio MJ, Fortier LA. Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations. Stem Cells Dev. 19, 269–282 (2010).

32

19

Smith JJ, Ross MW, Smith RK. Anabolic effects of acellular bone marrow, platelet rich plasma, and serum on equine suspensory ligament fibroblasts in vitro. Vet. Comp. Orthop. Traumatol. 19, 43–47 (2006).

Clegg PD. Characterisation of mesenchymal stem cells. Presented at: Conference on Regenerative Medicine. Saguaro Lake Ranch, Mesa, AZ, USA, 1–5 May 2011.

33

Barry F, Dwyer R, O’Brien T, Kavanagh C, Duffy G, Murphy M. Differentiation of mesenchymal stem cells. Presented at: Conference on Regenerative Medicine. Saguaro Lake Ranch, Mesa, AZ, USA, 1–5 May 2011.

34

Li F, Lu SJ, Honig GR. Hematopoietic cells from primate embryonic stem cells. Methods Enzymol. 418, 243–251 (2006).

35

Guest DJ, Allen WR. Expression of cell-surface antigens and embryonic stem cell pluripotency genes in equine blastocysts. Stem Cells Dev. 16, 789–796 (2007).

36

Guest DJ, Smith MR, Allen WR. Equine embryonic stemlike cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon. Equine Vet. J. 42, 636–642 (2010).

37

Watts AE, Yeager AE, Kopyov OV, Nixon AJ. Fetal derived embryonic-like stem cells improve healing in a large animal flexor tendonitis model. Stem Cell Res. Ther. 2(1), 4 (2011).

38

Hackett CH, Fortier LA. Embryonic stem cells and iPS cells: sources and characteristics. Vet. Clin. North Am. Equine Pract. 27, 233–242 (2011).

39

Nagy K, Sung HK, Zhang P et al. Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev. 7, 693–702 (2011).

40

Stemcca GM, Bressan F, Maziero R et al. 197 induced pluripotent stem cells (iPS) derived from equine umbilical cord cells using lentivirus vector. Reprod. Fertil. Dev. 26(1), 213 (2013).

41

Nishimori M, Yakushiji H, Mori M et al. Tumorigenesis in cells derived from induced pluripotent stem cells. Hum. Cell. 27(1), 29–35 (2014).

42

Vidal MA, Kilroy GE, Lopez MJ, Johnson JR, Moore RM, Gimble JM. Characterization of equine adipose tissuederived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells. Vet. Surg. 36, 613–622 (2007).

20

21

Schnabel LV, Mohammed HO, Miller BJ et al. Platelet rich plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons. J. Orthop. Res. 25, 230–240 (2007). Schnabel LV, Sonea HO, Jacobson MS, Fortier LA. Effects of platelet rich plasma and acellular bone marrow on gene expression patterns and DNA content of equine suspensory ligament explant cultures. Equine Vet. J. 40, 260–265 (2008).

22

Castelijns G, Crawford A, Schaffer J et al. Evaluation of a filter-prepared platelet concentrate for the treatment of suspensory branch injuries in horses. Vet. Comp. Orthop. Traumatol. 24, 363–369 (2011).

23

Bosch G, van Schie HT, de Groot MW et al. Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: a placebo-controlled experimental study. J. Orthop. Res. 28, 211–217 (2010).

24

Shah M, Foreman, DM, Ferguson MWJ. Neutralisation of TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous rat wounds reduces scarring. J. Cell Sci. 10, 985–1002 (1995).

25

Bosch G, Moleman M, Barneveld A, van Weeren PR, van Schie HT. The effect of platelet-rich plasma on the neovascularization of surgically created equine superficial digital flexor tendon lesions. Scand. J. Med. Sci. Sports 21, 554–561 (2011).

26

27

682

The International Society for Cellular Therapy position statement. Cytotherapy 8, 315–317 (2006).

14

Waselau M, Sutter WW, Genovese RL, Bertone AL. Intralesional injection of platelet-rich plasma followed by controlled exercise for treatment of midbody suspensory ligament desmitis in Standardbred racehorses. J. Am. Vet. Med. Assoc. 232, 1515–1520 (2008). Dominici M, Le Blanc K, Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells.

Regen. Med. (2014) 9(5)

future science group

The current ‘state of play’ of regenerative medicine in horses: what the horse can tell the human 

43

Gimble JM, Bunnell BA, Chiu ES, Guilak F. Concise review: Adipose-derived stromal vascular fraction cells and stem cells: let’s not get lost in translation. Stem Cells 29, 749–754 (2011).

58

Garvican ER, Alves AG, Smith RKW, Dudhia J. Mesenchymal stem cells modulate release of matrix proteins from tendon surfaces in vitro: a potential beneficial therapeutic effect. Regen. Med. 9(3), 295–308 (2014).

44

Koch TG, Heerkens T, Thomsen PD, Betts DH. Isolation of mesenchymal stem cells from equine umbilical cord blood. BMC Biotechnol. 30(7), 26 (2007).

59

45

Koch TG, Thomsen PD, Betts DH. Improved isolation protocol for equine cord blood-derived mesenchymal stromal cells. Cytotherapy 11(4), 443–447 (2009).

Jones EA, English A, Henshaw K et al. Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis. Arthritis Rheum. 50, 817–827 (2004).

60

De Schauwer C, Meyer E, Cornillie P et al. Optimization of the isolation, culture, and characterization of equine umbilical cord blood mesenchymal stromal cells. Tissue Eng. Part C Methods. 17(11), 1061–1070 (2011).

McCarthy HE, Bara JJ, Brakspear K, Singhrao SK, Archer CW. The comparison of equine articular cartilage progenitor cells and bone marrow-derived stromal cells as potential cell sources for cartilage repair in the horse. Vet. J. 192, 345–351 (2012).

61

Cremonesi F, Violini S, Lange-Consiglio A et al. Isolation, in vitro culture and characterization of foal umbilical cord stem cells at birth. Vet. Res. Commun. Suppl. 1, S139–S142 (2008).

Stewart AA, Barrett JG, Byron CR et al. Comparison of equine tendon-, muscle- and bone marrow-derived cells cultured on tendon matrix. Am. J. Vet. Res. 70, 750–757 (2009).

62

48

Gittel CB, Ribitsch J, Brehm W. Efficiency of adipogenic differentiation methods in mesenchymal stromal cells from diverse sources. Regen. Med. 6, 203 (2011).

Alves AG, Stewart AA, Dudhia J et al. Cell-based therapies for tendon and ligament injuries. Vet. Clin. North Am. Equine Pract. 27, 315–333 (2011).

63

49

Burk J, Ribitsch, I, Gittel C et al. Growth and differentiation characteristics of equine mesenchymal stromal cells derived from different sources. Vet. J. 195, 98–106 (2013).

50

Berg L, Koch T, Heerkens T et al. Chondrogenic potential of mesenchymal stromal cells derived from equine bone marrow and umbilical cord blood. Vet. Comp. Orthop. Traumatol. 22, 363–370 (2009).

Lange-Consiglio A, Rossi D, Tassan S et al. Conditioned medium from horse amniotic membrane-derived multipotent progenitor cells: immunomodulatory activity in vitro and first clinical application in tendon and ligament injuries in vivo. Stem Cell Dev. 22(22), 3015–3024 (2013).

64

51

Lange-Consiglio, A, Corradetti B, Bizzaro D et al. Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane. J. Tiss. Eng. Regen. Med. 6(8), 622–635 (2012).

Koch M, Lehnhardt A, Hu X et al. Isogeneic MSC application in a rat model of acute renal allograft rejection modulates immune response but does not prolong allograft survival. Transpl. Immunol. 29(1–4), 43–50 (2013).

65

52

Lange-Consiglio A, Corradetti B, Meucci A, Bizzaro D, Cremonesi F. Characteristics of equine mesenchymal stem cells derived from amnion and bone marrow: in vitro proliferative and multilineage potential assessment. Equine Vet. J. 45(6), 737–744 (2013).

Yoo KH, Jang IK, Lee MW et al. Comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues. Cell Immunol. 259(2), 150–156 (2009).

66

Guest DJ, Ousey JC, Smith MRW. Defining the expression of marker genes in equine mesenchymal stromal cells. Stem Cells Cloning 1, 1–9 (2008).

67

53

Lange-Consiglio A, Tassan S, Corradetti B et al. Investigating the efficacy of amnion-derived compared with bone marrow-derived mesenchymal stromal cells in equine tendon and ligament injuries. Cytotherapy 15(8), 1011–1012 (2013).

Carrade DD, Owens SD, Galuppo LD et al. Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses. Cytotherapy 13(4), 419–430 (2011).

68

54

Jones EA, Crawford A, English A et al. Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single-cell level. Arthritis Rheum. 58, 1731–1740 (2008).

Carrade, DD, Affolter VK, Outerbridge CA et al. Intradermal injections of equine allogeneic umbilical cordderived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions. Cytotherapy 13(10), 1180–1192 (2011).

55

Morito T, Muneta T, Hara K et al. Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology 47, 1137–1143 (2008).

69

56

Nishimura K, Solchaga LA, Caplan AI, Yoo JU, Goldberg VM, Johnstone B. Chondroprogenitor cells of synovial tissue. Arthritis Rheum. 42, 2631–2637 (1999).

Guest DJ, Smith MR, Allen WR. Equine embryonic stemlike cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon. Equine Vet. J. 42(7), 636–642 (2010).

70

57

Endres M, Neumann K, Häupl T et al. Synovial fluid recruits human mesenchymal progenitors from subchondral spongious bone marrow. J. Orthop. Res. 25, 1299–1307 (2007).

Carrade DD, Lame MW, Kent MS et al. Comparative analysis of the immunomodulatory properties of equine adult-derived mesenchymal stem cells. Cell. Med. 4, 1–11 (2012).

71

Kang JW, Kang KS, Koo HC et al. Soluble factors-mediated immunomodulatory effects of canine adipose tissue-derived mesenchymal stem cells. Stem Cells Dev. 17(4), 681–693 (2008).

46

47

future science group

www.futuremedicine.com

Review

683

Review  Smith, Garvican & Fortier 72

85

Trela JM, Spriet M, Padgett KA et al. Scintigraphic comparison of intra-arterial injection and distal intra-venous regional limb perfusion for administration of mesenchymal stem cells to the equine foot. Equine Vet. J. 45(6), 726–731 (2013).

86

Smith RK, Korda M, Blunn GW, Goodship AE. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment. Equine Vet. J. 35, 99–102 (2003).



A novel method for harvesting mesenchymal stem cells and reimplanting them into lesions of the superficial digital flexor tendon, in horses, is described.

87

Schnabel LV, Lynch ME, van der Meulen MC et al. Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons. J. Orthop. Res. 27(10), 1392–1398 (2009).

88

Crovace A, Lacitignola L, Rossi G, Francioso E. Histological and immunohistochemical evaluation of autologous cultured bone marrow mesenchymal stem cells and bone marrow mononucleated cells in collagenase-induced tendinitis of equine superficial digital flexor tendon. Vet. Med. Int. 250978 (2010).

73

Schebesch C, Kodelja V, Müller C et al. Alternatively activated macrophages actively inhibit proliferation of peripheral blood lymphocytes and CD4+ T cells in vitro. Immunology 92(4), 478–486 (1997).

74

Kim J, Hematti P. Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp. Hematol. 37(12), 1445–1453 (2009).

75

Betancourt AM. New cell-based therapy paradigm: induction of bone marrow-derived multipotent mesenchymal stromal cells into pro-inflammatory MSC1 and antiinflammatory MSC2 phenotypes. Adv. Biochem. Eng. Biotechnol. 130, 163–197 (2013).

76

Romieu-Mourez R, François M, Boivin MN, Bouchentouf M, Spaner DE, Galipeau J. Cytokine modulation of TLR expression and activation in mesenchymal stromal cells leads to a proinflammatory phenotype. J. Immunol. 182, 7963–7973 (2009).

77

Betancourt A, Bunnell BA. The anti-inflammatory/proinflammatory switch of MSCs. Presented at: Conference on Regenerative Medicine. Saguaro Lake Ranch, Mesa, AZ, USA, 1–5 May 2011.

89

Smith RK, Werling NJ, Dakin SG et al. Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy. PLoS ONE 8(9), e75697 (2013).

78

Smith RKW, Werling N, Dakin SG et al. Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally-occurring tendinopathy. PLoS ONE 8(9), e75697 (2013).

90

79

Smith MM, Sakurai G, Smith SM et al. Modulation of aggrecan and ADAMTS expression in ovine tendinopathy induced by altered strain. Arthritis Rheum. 58, 1055–1066 (2008).

Godwin EE, Young NJ, Dudhia J, Beamish IC, Smith RK. Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon. Equine Vet. J. 44, 25–32 (2012).

••

Intralesional implantation of mesenchymal stem cells as a treatment for superficial digital flexor tendonopathy was evaluated in National Hunt horses and found to carry no adverse effect, with no aberrant tissue on histological examination and a reinjury rate significantly less than that published for horses treated in other ways, providing evidence for the long-term efficacy of mesenchymal stem cell treatment.

91

Caniglia CJ, Schramme MC, Smith RK. The effect of intralesional injection of bone marrow derived mesenchymal stem cells and bone marrow supernatant on collagen fibril size in a surgical model of equine superficial digital flexor tendonitis. Equine Vet. J. 44(5), 587–593 (2012).

92

Ricco S, Renzi S, Del Bue M et al. Allogeneic adipose tissuederived mesenchymal stem cells in combination with platelet rich plasma are safe and effective in the therapy of superficial digital flexor tendonitis in the horse. Int. J. Immunopathol. Pharmacol. 26(Suppl. 1), 61–68 (2013).

93

Kang JG, Park SB, Seo MS, Kim HS, Chae JS, Kang KS. Characterization and clinical application of mesenchymal stem cells from equine umbilical cord blood. J. Vet. Sci. 14(3), 367–371 (2013).

94

Fortier LA, Potter HG, Rickey EJ et al. Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model. J. Bone. Joint Surg. Am. 92, 1927–1937 (2010).

80

Guo Y, Su L, Wu J et al. Assessment of the green florescence protein labeling method for tracking implanted mesenchymal stem cells. Cytotechnology 64, 391–401 (2012).

81

Kasashima Y, Tomita A, Kuwano A, Goodship AE, Smith RKW. In vivo tracking of implanted bone marrow-derived mesenchymal stem cells labelled by CM-DiL. Presented at: Conference on Regenerative Medicine. Saguaro Lake Ranch, Mesa, AZ, USA, 1–5 May 2011.

82

83

84

684

Fadok VA, Bratton DL, Konowal A, Freed, PW, Westcott JY, Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGFbeta, PGE2, and PAF. J. Clin. Invest. 101(4), 890–898 (1998).

Sole A, Spriet M, Galuppo LD et al. Scintigraphic evaluation of intra-arterial and intravenous regional limb perfusion of allogeneic bone marrow-derived mesenchymal stem cells in the normal equine distal limb using (99m) Tc-HMPAO. Equine Vet. J. 44, 594–599 (2012). Sole A, Spriet M, Padgett KA et al. Distribution and persistence of technetium-99 hexamethyl propylene amine oxime-labelled bone marrow-derived mesenchymal stem cells in experimentally induced tendon lesions after intratendinous injection and regional perfusion of the equine distal limb. Equine Vet. J. 45(6), 726–723 (2013). Becerra P, Valdes Vazquez MA, Dudhia J et al. Distribution of injected technetium(99m)-labeled mesenchymal stem cells in horses with naturally occurring tendinopathy. J. Orthop. Res. 31, 1096–1102 (2013).

Regen. Med. (2014) 9(5)

future science group

The current ‘state of play’ of regenerative medicine in horses: what the horse can tell the human 

95

Wilke MM, Nydam DV, Nixon AJ. Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model. J. Orthop. Res. 25, 913–925 (2007).

97

McIlwraith CW, Frisbie DD, Rodkey WG et al. Evaluation of intra-articular mesenchymal stem cells to augment healing of microfractured chondral defects. Arthroscopy 27, 1552–1561 (2011).

96

Martinello T, Bronzini I, Perazzi A et al. Effects of in vivo applications of peripheral blood-derived mesenchymal stromal cells (PB-MSCs) and platlet-rich plasma (PRP) on experimentally injured deep digital flexor tendons of sheep. J. Orthop. Res. 31, 306–314 (2013).

98

Frisbie DD, Stewart MC. Cell-based therapies for equine joint disease. Vet. Clin. North Am. Equine Pract. 27, 335–349 (2011).

99

Havemeyer Foundation. www.havemeyerfoundation.org

future science group

www.futuremedicine.com

Review

685