Stephanie Georgina Dakin

Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy.

Tendon injuries are a common age-related degenerative condition where current treatment strategies fail to restore functionality and normal quality of life. This disease also occurs naturally in horses, with many similarities to human tendinopathy making it an ideal large animal model for human disease. Regenerative approaches are increasingly used to improve outcome involving mesenchymal stem cells (MSCs), supported by clinical data where injection of autologous bone marrow derived MSCs (BM-MSCs) suspended in marrow supernatant into injured tendons has halved the re-injury rate in racehorses. We hypothesized that stem cell therapy induces a matrix more closely resembling normal tendon than the fibrous scar tissue formed by natural repair. Twelve horses with career-ending naturally-occurring superficial digital flexor tendon injury were allocated randomly to treatment and control groups. 1X107 autologous BM-MSCs suspended in 2 ml of marrow supernatant were implanted into the damaged tendon of the treated group. The control group received the same volume of saline. Following a 6 month exercise programme horses were euthanized and tendons assessed for structural stiffness by non-destructive mechanical testing and for morphological and molecular composition. BM-MSC treated tendons exhibited statistically significant improvements in key parameters compared to saline-injected control tendons towards that of normal tendons and those in the contralateral limbs. Specifically, treated tendons had lower structural stiffness (p<0.05) although no significant difference in calculated modulus of elasticity, lower (improved) histological scoring of organisation (p<0.003) and crimp pattern (p<0.05), lower cellularity (p<0.007), DNA content (p<0.05), vascularity (p<0.03), water content (p<0.05), GAG content (p<0.05), and MMP-13 activity (p<0.02). Treatment with autologous MSCs in marrow supernatant therefore provides significant benefits compared to untreated tendon repair in enhancing normalisation of biomechanical, morphological, and compositional parameters. These data in natural disease, with no adverse findings, support the use of this treatment for human tendon injuries.

Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair.

Macrophages (Mϕ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mϕ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3–6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mϕ sub-populations were quantified based on surface antigen expression of CD172a (pan Mϕ), CD14highCD206low (pro-inflammatory M1Mϕ), and CD206high (anti-inflammatory M2Mϕ) to assess potential polarised phenotypes. In addition, the Lipoxin A4 receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mϕ and FPR2/ALX. In contrast, M1Mϕ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mϕ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mϕ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A4 (LXA4) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E2. Stimulation with either mediator induced LXA4 release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mϕ.

Inflamm-Aging and Arachadonic Acid Metabolite Differences with Stage of Tendon Disease

The contribution of inflammation to the pathogenesis of tendinopathy and high prevalence of re-injury is not well established, although recent evidence suggests involvement of prostaglandins. We investigated the roles of prostaglandins and inflammation-resolving mediators in naturally occurring equine tendon injury with disease stage and age. Levels of prostaglandins E2 (PGE2), F2α (PGF2α), lipoxin A4 (LXA4) and its receptor FPR2/ALX were analysed in extracts of normal, sub-acute and chronic injured tendons. To assess whether potential changes were associated with altered PGE2 metabolism, microsomal prostaglandin E synthase-1 (mPGES-1), prostaglandin dehydrogenase (PGDH), COX-2 and EP4 receptor expression were investigated. The ability of tendons to resolve inflammation was determined by assessing FPR2/ALX expression in natural injury and IL-1β stimulated tendon explants. Alterations in the profile of lipid mediators during sub-acute injury included low PGE2 and elevated LXA4 levels compared to normal and chronic injuries. In contrast, PGF2α levels remained unchanged and were three-fold lower than PGE2. The synthetic capacity of PGE2 as measured by the ratio of mPGES-1:PGDH was elevated in sub-acute injury, suggesting aberrations in tendon prostaglandin metabolism, whilst COX-2 and EP4 receptor were unchanged. Paradoxically low tendon PGE2 levels in early injury may be attributed to increased local clearance via PGDH or the class switching of lipid mediators from the prostaglandin to the lipoxin axis. PGE2 is therefore implicated in the development of tendon inflammation and its ensuing resolution. Whilst there was no relationship between age and tendon LXA4 levels, there was an age-associated decline in FPR2/ALX receptor expression with concurrent increased PGE2 levels in injury. Furthermore, uninjured tendon explants from younger (<10 years) but not older horses (≥10 years) treated with IL-1β responded by increasing FPR2/ALX suggesting aged individuals exhibit a reduced capacity to resolve inflammation via FPR2/ALX, which may present a potential mechanism for development of chronic tendinopathy and re-injury.

Resolving an inflammatory concept: the importance of inflammation and resolution in tendinopathy.

Injuries to the superficial digital flexor tendon (SDFT) are an important cause of morbidity and mortality in equine athletes, but the healing response is poorly understood. One important drive for the healing of connective tissues is the inflammatory cascade, but the role of inflammation in tendinopathy has been contentious in the literature. This article reviews the processes involved in the healing of tendon injuries in natural disease and experimental models. The importance of inflammatory processes known to be active in tendon disease is discussed with particular focus on recent findings related specifically to the horse. Whilst inflammation is necessary for debridement after injury, persistent inflammation is thought to drive fibrosis, a perceived adverse consequence of tendon healing. Therefore the ability to resolve inflammation by the resident cell populations in tendons at an appropriate time would be crucial for successful outcome. This review summarises new evidence for the importance of resolution of inflammation after tendon injury. Given that many anti-inflammatory drugs suppress both inflammatory and resolving components of the inflammatory response, prolonged use of these drugs may be contraindicated as a therapeutic approach. We propose that these findings have profound implications not only for current treatment strategies but also for the possibility of developing novel therapeutic approaches involving modulation of the inflammatory process.

Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage- specific Fragmentation and Differential Cleavage of COMP ( Cartilage Oligomeric Matrix Protein)*

Background: Tendon disease is characterized by extensive remodeling of the extracellular matrix. Results: Novel COMP cleavage fragments were identified in both an in vitro inflammatory model and natural disease. Conclusion: Inflammatory mediators drive distinct COMP fragmentation at different stages of tendon disease. Significance: Novel COMP neo-terminal fragments provide opportunities for developing markers for tendon injury. During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.

The relationship between in vivo limb and in vitro tendon mechanics after injury: a potential novel clinical tool for monitoring tendon repair.

REASONS FOR PERFORMING THE STUDYnHighly prevalent superficial digital flexor tendon (SDFT) injury results in compromised tendon function through fibrosis and high frequency of re-injury due to altered biomechanical function. This study investigated the consequences of SDF tendinopathy on limb mechanics in relation to the mechanical properties of injured tendon.nnnOBJECTIVESnTo develop and validate a noninvasive in vivo assessment of tendon mechanics to investigate the effect of recent SDFT injury on limb stiffness index, providing an objective method to assess quality of healing.nnnHYPOTHESESnLimb stiffness index would reduce as a consequence of SDFT injury and progressively increase during tendon healing and correlate with in vitro mechanical properties of the respective SDFTs.nnnMETHODSnKinematic analysis was performed at walk in 10 horses that had sustained career-ending SDFT injury. Stiffness index was derived from limb force recorded via a series of force plates and measurement of change in metacarpophalangeal joint angle using 3D motion analysis software. Horses were subjected to euthanasia 7 months after injury, the SDFTs removed and subjected to nondestructive in vitro mechanical testing.nnnRESULTSnLimb stiffness index was reduced following SDFT injury in comparison with the contralateral limb and increased during the convalescent period, approximating that of the contralateral limb by 7 months post injury. There was a significant positive correlation between in vivo limb stiffness index and in vitro SDFT stiffness.nnnCLINICAL RELEVANCEnThe ability to assess and monitor SDFT mechanical competence through limb stiffness measurement techniques in horses recovering from SDFT injury and the possibility of corroborating this with functional tendon healing may permit a more objective and accurate assessment of optimal tendon repair in the horse. This technique may be a useful method for assessing the efficacy of treatment regimens for tendinopathy and could be utilised to predict time to safe return to performance or re-injury.

Advances in the understanding of tendinopathies: A report on the Second Havemeyer Workshop on equine tendon disease

Tendons are formed during the second half of embryonic developmentwhen tendon precursor cells deposit narrow-diameter (∼30 nm) collagenfibrils that are parallel to the long axis of the tissue. During post nataldevelopment, the narrow fibrils are replaced by large-diameter (up to500 nm)fibrils.Theabilityoftendontotransmitforcefrommuscletobone,and to dissipate forces during locomotion, is directly attributable to thecollagen fibrils. How the fibrils are synthesised, how they are alignedparallel to the tendon long axis, and how this arrangement can bereinstated during tendon healing are poorly understood. Ultrastructuralstudies of tendon lesions show the reappearance of narrow-diametercollagen fibrils and cells with slender cytoplasmic protrusions (calledfibripositors) that normally only occur in tendon during embryonicdevelopment. Recapitulation of development is a hypothesis that isgaining increasing support from researchers of tendon disease. A betterunderstanding of the genetic, molecular and environmental cues duringembryonicdevelopmentisexpectedtoprovidebetterinsightsintohowtoimprove the rate and fidelity of tendon repair in mature horses. Tendondevelopment can conveniently be considered to have an early ‘cellular’phaseandasubsequent‘matrix’phase.Inthematrix-dominatedphaseoftendon development 3D scanning electron microscopy of mouse tendonsuggests that fibripositors of the cells are the site of new fibril formationandthemechanicalinterfacebetweenthecellandtheextracellularmatrix.It is hypothesised that fibripositors exert pulling forces on collagen fibrils,and their cellular forces require functional myosin II, which is anintracellular molecular motor that is part of the actinomyosin system. Adetailed understanding of how cells set the tensional homeostasis oftendon is expected to lead to new strategies for regulating collagen fibrilassemblyinhealthandintendinopathy.

Relationship between the shape of the central and third tarsal bones and the presence of tarsal osteoarthritis.

Osteoarthritis (OA) of the low motion joints of the tarsus, commonly termed bone spavin, is common in horses. Whilst the aetiology of this condition is multifactorial, it has been suggested that dorsal wedging of the central tarsal bone (CTB) and third tarsal bone (TTB) may predispose to the development of this disease. The aim of this study was to investigate the relationship between tarsal bone conformation and osteoarthritis of the proximal intertarsal (PIT), distal intertarsal (DIT) and tarsometatarsal (TMT) joints. It was hypothesised that wedging of the CTB and TTB would be associated with OA in these joints. Multiplanar reconstructions based on computed tomographic (CT) images were used to measure the height of the central and third tarsal bones at their dorsal and plantar aspects in three parasagittal planes in cadaver specimens. A wedging index was calculated as the ratio between the dorsal and plantar measurements. All tarsal bones were graded for OA on CT images. There was a significant moderate negative correlation between the wedging index of the CTB and OA of the DIT (ρu2009=u2009-0.45, Pu2009<0.01), TMT (ρu2009=u2009-0.49, Pu2009<0.01) and PIT joints (ρu2009=u2009-0.43, Pu2009<0.01). Dorsal wedging of the TTB was seen in mild and moderate grades of OA, but severe cases of OA were associated with plantar wedging. Our study suggests that wedging of the small tarsal bones is associated with OA in the associated joints and hence care should be taken in foals to prevent the development of wedging.