Tendon injuries in both the human and horse represent a challenge due to persistent inflammation combined with inadequate reparative cells and a poorly organised extracellular matrix. The potential of mesenchymal stem cells (MSCs) in regenerating tendon injuries remains to be fully realised. The main mechanism of action by MSCs is considered to be primarily mediated via paracrine mechanisms. This may involve the production and release of extracellular vesicles (EVs) by stem cells with a sub-fraction of these EVs (<100 nm diameter) called exosomes that appear to be the main paracrine effectors. EVs can be readily prepared from MSCs and offer a clinically relevant therapy. However, EVs for tendon repair need to be fully characterised. The horse represents a highly relevant model of tendon and ligament injuries as it shares many features of mechanical loading, function and aetiopathology with the human. We have isolated and characterised EVs from equine MSCs for modulating tendon cell phenotype in an in vitro tendon injury model using IL-1ß. EVs can be isolated from IL-1ß stimulated MSCs although their levels are not significantly increased over controls suggesting that the nature of the stimulated EV cargo may be more important than absolute levels of released EVs.

Summary Statement

Transportation media and injection protocol have implications for the viability of MSCs used for intra-lesional treatment of tendon injuries. Every effort should be made to implant cells within 24h of laboratory re-suspension, using a needle bore larger than 21G.

Summary

Treatment of equine naturally occurring over-strain tendinopathy with mesenchymal stem cells suspended in bone marrow supernatant resulted in significant improvements compared to saline treated tendons in the normalisation of biomechanical, morphological, and compositional parameters with no adverse effects.

Hyaline cartilage defects are a significant clinical problem for which a plethora of cartilage repair techniques are used. One such technique is cartilage replacement therapy using autologous chondrocyte or mesenchymal stem cell (MSC) implantation (ACI). Mesenchymal stem cells are increasingly being used for these types of repair technique because they are relatively easy to obtain and can be expanded to generate millions of cells. However, implanted MSCs can terminally differentiate and produce osteogenic tissue which is highly undesirable, also, MSCs generally only produce fibrocartilage which does not make biomechanically resilient repair tissue, an attribute that is crucial in high weight-bearing areas. Tissue-specific adult stem cells would be ideal candidates to fill the void, and as we have shown previously in animal model systems [Dowthwaite et al, 2004, J Cell Sci 117;889], they can be expanded to generate hundreds of millions of cells, produce hyaline cartilage and they have a restricted differential potential. Articular chondroprogenitors do not readily terminally differentiate down the osteogenic lineage.

At present, research focused on isolating tissue-specific stem cells from articular cartilage has met with modest success. Our results demonstrate that using differential adhesion it is possible to easily isolate articular cartilage progenitor populations from human hyaline cartilage and that these cells can be subsequently expanded in vitro to a high population doubling whilst maintaining a normal karyotype. Articular cartilage progenitors maintain telomerase activity and telomere length that are a characteristic of progenitor/stem cells and differentiate to produce hyaline cartilage.

In conclusion, we propose the identification and characterisation of a novel articular cartilage progenitor population, resident in human cartilage, which will greatly benefit future cell-based cartilage repair therapies.

Introduction: Tendon injury is an important cause of injury in racehorses, with flexor tendon and suspensory ligament injuries accounting for 46% of all musculoskeletal injuries at British racecourses (1). In the galloping horse the superficial digital flexor tendon (SDFT) undergoes strains that are close to the functional limit of the tendon (2) and it is hypothesised that exercise induces cumulative microdamage in the SDFT of skeletally mature horses which may predispose to clinical disease. We hypothesised that matrix metalloproteinases (MMPs) play a role in the process of tendon degeneration induced by cyclical loading and investigated this using an in vitro model.

Methods: Mid-metacarpal SDFTs were harvested from Thoroughbred horses that were euthanased for non-orthopaedic reasons. Tendon explants (2mm x 2mm x 60mm) were maintained in DMEM and placed in custom designed loading cassettes which were cyclically loaded in an incubator using a Dartec materials testing device for 24 hours with 5% strain and at a frequency of 1Hz. Control explants were placed in similar cassettes but were not loaded. The ultimate tensile strength (UTS) of the tendon was assessed using a destructive test at the end of the 24 hour loading period. The experiments were repeated using non-viable tendon explants, or in the presence of a pan-MMP specific inhibitor (Illomastat, 25 μM).

Results: Cyclical loading induced a 30% decrease in the UTS of tendons of immature and young mature (< 10 years of age) horses but this increased to a 50% reduction in older (10–30 years of age) horses compared to controls. This loss of UTS was prevented in tendon explants with non-viable cells or with a pan-MMP inhibitor applied to the live explants prior to cyclical loading.

Conclusions: The results suggest that an MMP mediated mechanism plays a pivotal role in tendon degeneration following cyclical loading in vitro. Current work including analysis of gene expression and quantification of MMPs within the tendon tissue aims to identify the key MMPs responsible for the loss of tendon UTS following cyclical loading. This will hopefully enable therapeutic strategies to be developed to slow or stop the age-associated tendon degeneration that predisposes to overstrain injury, and thereby help prevent this common orthopaedic disease in horses.

Objective: To challenge the validity of using biomarker concentrations in synovial fluid for the assessment of joint pathology.

Hypothesis: Synovial fluid biomarker concentrations are influenced by both cartilage and synovial fluid volumes.

Methods: Synovial fluid volumes were determined from the equine metacarpophalangeal (MCP), proximal inter-phalangeal (PIP) and distal interphalangeal (DIP) joints, which have different disease prevalences.

Chondrocyte density was calculated from a defined site in each joint.

Cartilage volume was measured by novel application of Peripheral Quantitative Computed Tomography (pQCT).

Cartilage oligomeric matrix protein (COMP), glycos-aminoglycans (GAG) and total protein (TP) concentrations were measured and then adjusted for cartilage and synovial fluid volume and compared between joints.

Results: Mean synovial fluid volume was significantly greater in the MCP than the distal joints (p< 0.0001) (3.2 ±0.5ml, 0.5 ±0.1ml and 0.6 ±0.1ml respectively). In contrast, the DIP had the greatest cartilage volume compared to the proximal joints (5360 ±667mm3 2640mm3, 1940 ±331mm3 respectively). There was no significant difference in the cartilage cellularity between all joints.

The DIP had higher TP, COMP and GAG concentrations, however, when values were expressed per unit cartilage volume the opposite was found, with the MCP then exhibiting significantly higher concentrations.

Conclusions: These data show the joint with the highest prevalence to osteoarthritis has the lowest biomarker synovial fluid concentrations but the highest biomarker levels per unit cartilage, suggesting a higher release. These results indicate that meaningful interpretation of biomarkers in synovial fluid require consideration of both fluid and cartilage volume.