Evidence Supporting Intralesional Stem Cell Therapy to Improve Equine Flexor Tendon Healing

a Knowledge Summary by

Sushmitha Durgam BVSc, MS¹

Matthew Stewart BVSc, PhD^1*

¹University of Illinois, Veterinary Clinical Medicine, Champaign, IL 61801, USA
^*Corresponding Author (matt1@illinois.edu)

Question

Does intralesional stem cell therapy improve healing of equine superficial digital flexor tendons?

The evidence

To date, equine experimental and clinical case studies of tendon healing demonstrate improved histological and ultrasonographic features of matrix architecture, respectively with intralesional stem cell therapy. These findings encourage clinical use of stem cell-based therapies to enhance equine tendon healing. A few clinical studies indicate that re-injury rates are lower following stem cell treatment, than without. However, these studies do not provide any firm conclusions regarding the ideal cell source for therapy or optimal treatment protocol. Collaborative research and long-term follow up among the veterinary community is needed to establish the optimal applications for cell-based therapies.

Summary of the evidence

Appraisal, application and reflection

Tendon injuries in horses range from acute tendon strains to chronic tendinopathy, and are both common and challenging problems in equine performance horse practice. The technique and feasibility of cell-based therapy (isolated from bone marrow) for treating equine SDFT injuries was first reported by Smith et al. (2003; 14). Since then, intralesional stem cell therapy, using cells from multiple tissue sources, has become common and commercial services are now available to support cell-based therapies in horses. The purpose of this Knowledge Summary was to evaluate the available evidence from studies evaluating the efficacy of stem cell therapies for equine flexor tendon healing.  

Appraisal

Eleven studies were identified that addressed this issue (note that the outcomes from references 2 and 3 were replicated in reference 10. Reference 10 was used to evaluate the collective outcomes from these manuscripts). These papers segregated into two distinct groups; six studies using experimental models of tendinopathy (references 1, 5, 10, 12, 13 and 17) and six case series of clinical tendinitis patients (references 4, 6, 9-11, and 15; reference 10 included data for both groups). 

Accepting the focused nature of the question under review, there was a remarkable degree of variability in the source of ‘stem cell’ used in these studies (bone marrow [7], blood [1], amnion [1], adipose tissue [2], embryonic [1], and tendon [1]), the protocols used to generate the therapeutic cell populations, donor-recipient matching (three allogeneic sources and 9 autologous sources), the diluents used for the cell injections (saline [5], bone marrow supernatant [4], plasma [1], PRP [1], and fibrin/thrombin [1]) and, in the clinical studies, the specific disciplines the patients were engaged in. Collectively, these sources of variation confounded any coherent meta-analysis.

Of the six experimental studies, five used intratendinous collagenase injections to generate SDFT lesions (5, 10, 12, 13, 17) while Caniglia et al 2012 (1) created a core lesion with a synovial resector. In only two of these studies, were at least some of the outcome assessments conducted in blinded fashion (13, 17). While acknowledging that there are no other accepted alternative models, the extent to which either induced lesion reflects the cumulative tensile strain injury of clinical tendinitis cases is debatable. Self-evidently, neither model is generated by tensile overloading, and our own (5) and other’s (3, 16) experience with the collagenase model has shown that individual horse’s responses to collagenase vary considerably.

Accepting these concerns, in all six experimental studies, cell delivery improved the histological and ‘matrix organisation’ characteristics of the repair tissue, while the ultrasonographic findings were improved in three of the four studies that used ultrasonography for outcome assessment. In all but one experimental study (reference 5), histological outcomes were determined by a score derived from ‘cell and tissue characteristics’ grading schemes. There is clearly an element of subjectivity in these analyses, particularly given that only two were carried out in blinded fashion. Of more general concern, although it makes intuitive sense that a ‘more histologically normal’ repair tissue corresponds to a better functional outcome, the relationship between these outcomes is very poorly defined, particularly since higher level matrix organisation has not been addressed in any study to date.

With the possible exception of Lacitignolo et al 2008 (10; 24 weeks), the experimental studies were conducted over time frames far less (7-16 weeks) than the ‘several months’ interval generally required for clinical tendinitis cases to resolve. It is possible, and even perhaps to be expected, that many of the significant differences between cell-treated and control samples detected at early time points in the experimental studies disappear over longer time frames, as healing in the ‘control’ groups catches up.   

Only two of the experimental studies (5,13) included biomechanical testing to provide some functional assessment of outcome. However, it is debatable how well single ‘tensile load to failure’ testing translates to the rapid, submaximal cyclic loading that flexor tendons are subjected to clinically. None of the experimental studies addressed the most clinically relevant issue; namely, do horses with tendinitis perform better after receiving cell-based therapies? The ultrasonographic, histological, biochemical, transcriptional and biomechanical analyses are, at best, indirect indices of ‘healing quality’ and functional return. 

Collectively, outcomes from the experimental studies should be considered as B2 evidence.

Of the six clinical case series, three had very small case numbers and/or no control group and/or marginal outcome assessments (4, 10, 11) and can be considered to be little more than anecdotal (grade E) in value. The study by Godwin et al 2012 (6) is substantive by virtue of the large number of cases (141) enrolled in the analyses. There was no control/placebo group in this study; the authors used previously published outcomes from analogous patient groups for comparison. Further, the great majority of Godwin et al’s patients (105) were National Hunt horses, and so their outcomes should be confined to this discipline. Accepting this, 98% of treated horses returned to racing (albeit after an 8+ month layoff) and the re-injury rate (27%) was approximately half that reported in two studies from similar National Hunt populations. Although the Evidentiary Value of the Godwin study should, at best, be classified as an uncontrolled prospective study (C), the clinical significance of the outcomes is high (grade 1 to 2).

Lange-Consiglio et al 2013 (9) compared the responses of 95 performance horses with tendon or ligament injuries randomly assigned to be treated with allogeneic amnion-derived stem cells (AMSC; 51 cases) or autologous bone marrow-derived stem cells (BMMSC; 44 cases) in a fibrin carrier. This study included show jumpers, dressage horses, eventers, trotters and thoroughbreds, with lesions in the superficial and deep digital flexor tendons and suspensory ligaments and a range of clinical signs and lesion severity, although these variables were distributed fairly evenly across both treatment groups. Horses treated with AMSCs returned to training earlier than horses receiving BMMSCs and the re-injury rates were considerably less in the AMSC group (4%) than the BMMSC group (23%). The overall BMMSC group re-injury rate was reassuringly similar to the rate reported by Godwin et al, above, although Lange-Consiglio’s Thoroughbreds responded substantially better (12.5% recurrence) than Godwin’s thoroughbreds (50%). The study by Lange-Consiglio et al 2013 should be considered B2. 

In the final clinical study, by Smith et al 2013 (15), was a prospective, randomised, controlled clinical trial involving 12 horses with end-stage tendinitis. Further, the histological evaluations of the tendon samples were blinded. Although the horses in the study were not exercised above trotting speeds, there were substantial improvements in ultrasonographic, histological and biomechanical characteristics of healing tissue six months after Intralesional BMMSC treatment. From an experimental design perspective, this study provides the most compelling results supporting stem cell use for flexor tendinitis in horses and should be classified as B2.  

Application

From an evidentiary perspective, the collective data supporting cell-based therapy for equine flexor tendinitis is not strong. In this review, we have raised concerns regarding the clinical applicability of the experimental tendinitis models, the relevance of indirect ‘healing quality’ outcomes to functional success, inadequate or nonexistent control groups and experimental design issues with published clinical trials. The clinical reality is that conventional approaches to managing flexor tendinitis/tendinopathy in performance horses require prolonged and intensive therapy and rehabilitation efforts yet carry a guarded prognosis. Any novel therapeutic option, whether cell-based, biologic or pharmaceutical, will likely be embraced by the equine veterinary community if that option holds some promise of an improved outcome. This perspective makes any attempt to conduct a randomised, controlled, prospective clinical trial challenging, particularly given the funding constraints that veterinary researchers routinely work under.

While acknowledging the questionable evidentiary value of some studies, there is a consistent collective body of evidence supporting the findings that cell-based therapies improve tendon matrix repair and reorganisation, both in experimental and clinical subjects, and significantly reduce recurrence rates following return to work. In light of these conclusions, and the lack of any more promising alternatives, cell-based therapies should be considered for treating equine flexor tendinitis cases and, by extension, for ligament injuries also. As the applications of these therapies evolve, there are many variables that need to be investigated and optimised. Some of the major issues are as follow: 

1. Standardisation/optimisation of cell source(s), cell processing, and diluents
2. Optimisation of timing and dose(s) of cell delivery following injury
3. Agreement on effective post-therapeutic case management and rehabilitation protocols
4. Standardisation of meaningful outcome measures for specific equine disciplines

Conclusions and Reflection

Cell-based therapy, in both the human and veterinary fields, is a highly dynamic, but poorly defined field of discovery. It is clear that clinical applications of cell-based therapies have far outstripped our understanding of the biological mechanisms by which stem cells influence healing. As noted above, there is little or no rationale for deciding on many of the variables that influence cell-based therapy efficacy. Somewhat contentiously, the actual need for a ‘stem cell’ population, as opposed to any population of cells isolated from a tissue or fluid, has not been demonstrated through properly controlled experiments. Further, data from recent cell-tracking studies (5,7,16) clearly show that exogenous stem cells are cleared from the injection site within a few weeks and do not contribute to the pool of tenocytes and/or progenitor cell engaged in tendon repair/regeneration beyond this time. It is probable that secreted stem cell cytokines and/or trophic factors contribute substantially to stem cells’ impact on tissue repair (8). Identifying these factors could simplify and standardise ‘biologic therapy’ considerably.

Regardless of the specific biologic therapy in question, randomised, appropriately controlled studies using a consistent and translatable experimental model or clinical caseload, standardised treatment and rehabilitation protocols and credible outcome assessments, will be required to make meaningful headway on clarifying efficacy. Given the costs and case numbers required for these clinical trials, multi-center/practice collaboration and significant financial support from private industry and foundations will be necessary. Depending on regulatory developments in human medicine, the veterinary community might also stand to benefit from biomedical research efforts focused on validating cell-based therapies for people.

Methodology Section

Search Strategy
Databases searched and dates covered:	Search terms were applied in PubMed Central accessed on NCBI website (1910-2016), CAB abstracts database accessed on OVID platform (1973-2015) and Scopus
Search terms:	horse AND tendonitis OR tendinitis OR stem cell OR flexor tendon
Dates searches performed:	August 2016

Exclusion / Inclusion Criteria
Exclusion:	Non-English language, review articles, case reports, conference proceedings/abstracts
Inclusion:	Experimental studies and clinical case series evaluating the effect of intralesional stem cell therapy on equine superficial digital flexor tendon healing.

Search Outcome
Database	Number of results	Excluded – did not answer PICO question	Excluded – review articles	Excluded – not in English	Total relevant papers
NCBI PubMed	24	11	2	0	11
Scopus	18	8	0	1	9
CAB Direct	26	10	0	6	10
Total relevant papers when duplicates removed					11

The authors declare no conflicts of interest.

1. Caniglia, C.J. et al (2012) 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 Veterinary Journal, 44 (4), pp. 587-593. http://dx.doi.org/10.1111/j.2042-3306.2011.00514.x
2. Crovace, A. et al (2007) Cell Therapy for Tendon Repair in Horses: An Experimental Study. Veterinary Research Communications, 31 (S1), pp. 281-283. http://dx.doi.org/10.1007/s11259-007-0047-y
3. Crovace, A. et al (2010) 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. Veterinary Medicine International, 2010, Article ID 250978. http://dx.doi.org/10.4061/2010/250978
4. Del Bue, M. et al (2008) Equine Adipose-Tissue Derived Mesenchymal Stem Cells and Platelet Concentrates: Their Association in Vitro and in Vivo. Veterinary Research Communications, 32 (S2), pp. 51-55. http://dx.doi.org/10.1007/s11259-008-9093-3
5. Durgam, S.S. et al (2016) Tendon-Derived Progenitor Cells Improve Healing of Collagenase-Induced Flexor Tendinitis. Journal of Orthopaedic Research, doi: 10.1002/jor.23251, [Epub ahead of print]. http://dx.doi.org/10.1002/jor.23251
6. Godwin, E.E. et al (2012) Implantation of Bone Marrow-Derived Mesenchymal Stem Cells Demonstrates Improved Outcome in Horses with Overstrain Injury of the Superficial Digital Flexor Tendon. Equine Veterinary Journal, 44 (1), pp. 25-32. http://dx.doi.org/10.1111/j.2042-3306.2011.00363.x
7. Guest, D. et al (2010) Equine embryonic stem-like cells and mesenchymal stromal cells have different migration patterns following their injection into damaged superficial digital flexor tendon. Equine Veterinary Journal, 42 (6), pp. 636-642. http://dx.doi.org/10.1111/j.2042-3306.2010.00112.x
8. Lange-Consiglio, A. et al A (2013) 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 Cells and Development, 22 (22), pp. 3015-3024. http://dx.doi.org/10.1089/scd.2013.0214
9. Lange-Consiglio, A. et al B (2013) Investigating the Efficacy of Amnion-Derived Compared with Bone Marrow-Derived Mesenchymal Stromal Cells in Equine Tendon and Ligament Injuries. Cytotherapy, 15 (8), pp. 1011-1020. http://dx.doi.org/10.1016/j.jcyt.2013.03.002
10. Lacitignola, L. et al (2008) Cell Therapy for Tendinitis, Experimental and Clinical Report. Veterinary Research Communications, 32 (Suppl 1), pp. S33-S38. http://dx.doi.org/10.1007/s11259-008-9085-3
11. Marfe, G. et al. (2012) A New Clinical Approach: Use of Blood-Derived Stem Cells (BDSCs) for Superficial Digital Flexor Tendon Injuries in Horses. Life Sciences, 90, pp. 825-830. http://dx.doi.org/10.1016/j.lfs.2012.03.004
12. Nixon, A.J. et al (2008) Effect of Adipose-Derived Nucleated Cell Fraction on Tendon Repair in Horses with Collagenase-Induced Tendinitis. American Journal of Veterinary Research, 69 (7), pp.928-937. http://dx.doi.org/10.2460/ajvr.69.7.928
13. Schnabel, L.V. et al (2009) 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. Journal of Orthopaedic Research, 27 (8), pp. 1392-1398. http://dx.doi.org/10.1002/jor.20887
14. Smith, R.K. et al (2003) Isolation and Implantation of Autologous Equine Mesenchymal Stem Cells from Bone Marrow into the Superficial Digital Flexor Tendon as a Potential Novel Treatment. Equine Veterinary Journal, 35 (1), pp. 99-102. http://dx.doi.org/10.2746/042516403775467388
15. Smith, R.K.W. et al (2013) Beneficial Effects of Autologous Bone Marrow-Derived Mesenchymal Stem Cells in Naturally Occurring Tendinopathy. PLosOne, 8 (9), e75697. http://dx.doi.org/10.1371/journal.pone.0075697
16. Sole, A. et al (2013) 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 Veterinary Journal, 45 (6), pp. 726-731. http://dx.doi.org/10.1111/evj.12063
17. Watts, A.E. et al (2011) Fetal Derived Embryonic-Like Stem Cells Improve Healing in a Large Animal Flexor Tendonitis Model. Stem Cell Research and Therapy, 2 (4), pp. 1-12.