Adult stem cells in equine tendon therapy
Equine tendon or ligament injuries most commonly occur in the superficial digital flexor tendon (SDFT), a large, energy-storing tendon similar to the human Achilles tendon, or in the suspensory ligament (SL), a ligamentous muscle containing only small amounts of myofibers (54). Typical equine tendon lesions are located in the center of the SDFT and ultrasonographically appear as hypoechogenic areas, surrounded with more or less intact tendon tissue with similarities to human Achilles tendinopathy. The existence of these intratendinous lesions, filled with tissue that is less dense than the surrounding tendon tissue, facilitates a direct intralesional application of therapeuticals. Therefore, ultrasound-guided intralesional injection of a cell suspension is the common approach to introduce stem cells to the injury site, which has been used in all studies mentioned below (2–4, 6–8, 55–60).
In 2001, promising results were reported after application of autologous bone marrow into damaged SLs (61). In 2003, a more developed approach for the treatment of strain-induced SDFT injury was published which utilized autologous, culture expanded, bone marrow derived MSCs that were suspended in blood plasma for application (2). Re-examinations 10 days and 6 weeks after treatment showed no adverse effects such as lameness or changes in tendon substance, while the cross-sectional area of the tendon at maximum injury zone had decreased by 10% (2). Since then, this approach has been evaluated and improved upon in several clinical and experimental in vivo studies, but the basic therapeutic concept has remained unchanged.
Multiple MSC populations, ranging from culture-expanded bone marrow derived MSCs to culture-expanded adipose tissue or tendon derived MSCs (59, 60) have been used for cell therapy applications. So far, the clinical outcome after using these alternative MSC sources in tendon therapy seems to be comparable. Furthermore, with possible relevance for potential applications in human medicine, the use of the mononuclear cell (MNC) fractions without subsequent selection of MSCs by culture expansion is also being evaluated (7, 58). Moreover, the effects of allogeneic MSC application have been investigated, and no enhanced immunological response was found compared to autologous cell applications (4, 62, 63). However, due to species specific differences of the immune system, this approach may not be easily transferrable to human medicine.
Clinical results following treatment with autologous bone marrow derived MSCs are promising and re-injury rates are reduced compared to those observed after conventional therapy (8, 55, 56, 64). In the earliest of these studies, 9 out of 10 race horses that had received MSC treatment of SDFT core lesions could return to racing and did not reinjure within the observed period of 2 years. Ultrasonographic evaluation showed dense filling of the tendon lesions with parallel fibers (55) whereas horses in the control group (n = 15) showed ultrasonographic evidence of fibrosis within the tendon lesions, and all of those horses reinjured after a median time of 7 months (55). A clinical study including a larger number of cases was published in 2011 (8): 113 race horses had received an intratendinous injection of autologous MSCs suspended in bone marrow supernatant for treatment of SDFT lesions and could be followed up for a minimum of 3 years. One hundred eleven of these horses could return back to racing; however, horses that had been used otherwise (breeding, other sports, etc.) after the injury had already been excluded from the study. 27.4% of these 111 horses suffered a reinjury, which was significantly less than observed after conventional treatments (8). Analysis of our own clinical data revealed similar results as in the studies described above; only 15.5% of 58 horses with SDFT or SL lesions treated with MSCs had suffered a reinjury or had retired from sports within the follow-up period, while the other 84.5% had returned back to their previous level of performance (n = 43) or were in full training (n = 6) (56).
Besides the overall clinical outcome, these studies also showed that re-injuries tend to appear less frequently when a larger number of MSCs (at least ∼10 × 106 MSCs) is applied (8, 55), the patients are younger (8, 56) or when the time between the incidence of injury and cell application is shorter (8). Further, the athletic discipline for which the horse is used seems to have a major influence on treatment success (8, 56). However, it should be noted that MSC treatment does not replace or shorten the standard rehabilitation but is rather an additional treatment that improves the outcome.
Experimental in vivo studies lead to the assumption that this improvement might mainly be due to the fact that MSCs enhance the structural organization of the repaired tissue. Autologous adipose derived nucleated cell fractions, used in a collagenase-induced tendinopathy horse model, significantly improved tendon fiber architecture compared to the control group, as revealed by histology 6 weeks after cell injection (58). However, biochemical and molecular analyses of tendon compositions revealed no significant differences except for a higher cartilage oligomeric protein (COMP) expression in treated tendons (58). A comparably designed study conducted by the same group, using bone marrow derived MSCs and insulin-like growth factor-I gene-enhanced bone marrow derived MSCs, arrived at similar results. While histology scores were significantly better in both MSC treated groups compared to the control group, no changes in tendon composition could be found. Additionally, MSC treated tendons showed greater stiffness at mechanical testing than control group tendons, although this difference was not significant (57). In a study which directly compared the use of bone marrow derived MNCs or MSCs, it was found that both treatments equally enhanced tendon healing compared to the controls (7). Improvements were evident at ultrasonographic measurements as well as at the histological and immunocytochemical evaluations 21 weeks after cell application, revealing a better structural organization of the healing tissue, marked by dense filling of the defect and parallel alignment of collagen fibers (7). Interestingly, in this study, changes in tendon compositions could also be detected, with higher collagen I and lower collagen III contents in the treatment groups (7), which might be due to the fact that horses were euthanized after a longer period of time compared to the other studies described here (57, 58). Similarly, a recent study revealed that MSC treatment had no effect on collagen fibril size within the tendon lesions at 12 weeks posttreatment (65), although the light microscopical appearance of tendinopathy was shown to be improved as early as 6 weeks after cell application (58). Summarizing the results obtained in the horse so far, MSC application seems to improve tendon healing, as demonstrated by the decreased re-injury rates and the better structural organization of the healing tissue. However, it still remains unclear how the cells influence the healing process.
Embryonic and fetal stem cells in equine tendon therapy.
The current approach for embryonic stem cell therapy is to directly apply allogeneic, pluripotent embryonic or fetal derived cells (3, 6). It was shown that ESCs distribute more widely in the tissue and display significantly higher survival rates compared to MSCs after 90 days. Yet the histology and ultrasonographic assessment revealed no improvement of tendon architecture compared to the serum injected controls (3). In contrast, in a study using fetal derived embryonic-like cells, it was found that after 8 weeks, tendon architecture was significantly better than in controls, although again, no differences in tendon composition could be demonstrated (6). In neither of those studies, teratoma formation was observed. However, this might be unique to equine ESCs, as a previous study reported that these equine cells did not induce teratomas in SCID mice, either (66).