Equine Superficial Digital Flexor Tendon Research Articles

Mechanical properties, collagen and glycosaminoglycan content of equine superficial digital flexor tendons are not affected by training.

Physical activity can activate extracellular matrix (ECM) protein synthesis and influence the size and mechanical properties of tendon. In this study, we aimed to investigate whether different training histories of horses would influence the synthesis of collagen and other matrix proteins and alter the mechanical properties of tendon. Samples from superficial digital flexor tendon (SDFT) from horses that were either (a) currently race trained (n = 5), (b) previously race trained (n = 5) or (c) untrained (n = 4) were analysed for matrix protein abundance (mass spectrometry), collagen and glycosaminoglycan (GAG) content, ECM gene expression and mechanical properties. It was found that ECM synthesis by tendon fibroblasts invitro varied depending upon the previous training history. In contrast, fascicle morphology, collagen and GAG content, mechanical properties and ECM gene expression of the tendon did not reveal any significant differences between groups. In conclusion, although we could not identify any direct impact of the physical training history on the mechanical properties or major ECM components of the tendon, it is evident that horse tendon cells are responsive to loading invivo, and the training background may lead to a modification in the composition of newly synthesised matrix.

Blood flow restriction training does not negatively alter the mechanical strength or histomorphology of uninjured equine superficial digital flexor tendons.

Low load exercise training with blood flow restriction (BFR) has become increasingly used by human physical therapists to prescribe controlled exercise following orthopaedic injury; its effects on the equine superficial digital flexor tendon (SDFT), however, are unknown. To investigate outcomes of pressure specific BFR walking exercise on uninjured equine SDFT biomechanics and histomorphology. Controlled in vivo experiment. Four forelimbs of four horses were exposed to 40 BFR-walk sessions (10-min interval walking) on a treadmill over a 56-day study period with their contralateral forelimbs serving as untreated controls. Similarly, four forelimbs of four control horses were exposed to 40 sham cuff walk sessions. On study Day 56, all horses (n = 8) were humanely euthanised and forelimb SDFTs underwent non-destructive biomechanical testing and corresponding histomorphological analysis. Significance in biomechanical parameters between treatment groups was analysed using a mixed-effects ANOVA with Tukey's post-hoc tests. Statistically significant differences in SDFT stiffness for both first (p = 0.02) and last cycles (p = 0.03) were appreciated within the BFR treated group only, with BFR exposed forelimbs being significantly stiffer than the contralateral unexposed forelimbs. When normalised to cross-sectional area, no significant differences were appreciated among treatment groups in elastic modulus for the first (p = 0.5) or last cycles (p = 0.4). No histological differences were appreciated among treatment groups according to Bonar, Movin, or musculotendinous junction evaluation criteria. Short-term comparisons were performed in a small sample population without correlation to performance outcome measures. Optimal occlusion percentages and walk protocols remain unknown. This study demonstrated no negative impact of BFR on mechanical strength of the equine SDFT; however, evidence suggests that BFR results in increased tendon stiffness based on biomechanical testing and subsequent calculations. No consistent detrimental histomorphological changes were seen.

ESTABLISHING NOVEL MARKERS OF TENDON CELL POPULATIONS

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to age-related injury. Tendons have poor healing capacity and a lack of effective treatments can lead to ongoing pain, reduced function and re-injury. It is therefore important to identify the mechanisms underpinning age-related tendinous changes in order to develop more effective treatments. Our recent single cell sequencing data has shown that tendon cell populations have extensive heterogeneity and cells housed in the tendon interfascicular matrix (IFM) are preferentially affected by ageing. There is, however, a lack of established surface markers for cell populations in tendon, limiting the capacity to isolate distinct cell populations and study their contribution to age-related tendon degeneration. Here, we investigate the presence of the cell surface proteins MET proto-oncogene (MET), integrin subunit alpha 10 (ITGA10), fibroblast activation protein alpha (FAP) and platelet derived growth factor receptor alpha (PDGFRA) in the equine SDFT cell populations and their co-localisation with known markers.Using Western blot we validated the specificity of selected antibodies in equine tissue before performing immunohistochemistry to establish the location of the respective proteins in the SDFT. We subsequently used double labelling immunofluorescence with the established mural cell marker desmin (DES) to distinguish between tenocyte and mural cell populations.In situ, MET, ITGA10, and FAP presence was found in cells throughout the tendon whereas PDGFRA was present in cells within the IFM. Double labelling immunofluorescence with the mural cell marker DES showed lack of co-localisation between PDGFRA and DES suggesting PDGFRA is labelling an IFM cell population distinct from those associated with blood vessels.PDGFRA is a promising target for the specific cell sorting of IFM-localised tenocytes, enabling their isolation and subsequent characterisation.Acknowledgments: The authors acknowledge the Biotechnology and Biological Sciences Research Council (BB/W007282/1) for funding this work.

The Interfascicular Matrix of Energy Storing Tendons Houses Heterogenous Cell Populations Disproportionately Affected by Aging.

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to injury, with incidence increasing with aging, peaking in the 5th decade of life in the human Achilles tendon. The interfascicular matrix (IFM), which binds tendon fascicles, plays a key role in energy storing tendon mechanics, and aging alterations to the IFM negatively impact tendon function. While the mechanical role of the IFM in tendon function is well-established, the biological role of IFM-resident cell populations remains to be elucidated. Therefore, the aim of this study was to identify IFM-resident cell populations and establish how these populations are affected by aging. Cells from young and old SDFTs were subjected to single cell RNA-sequencing, and immunolabelling for markers of each resulting population used to localise cell clusters. Eleven cell clusters were identified, including tenocytes, endothelial cells, mural cells, and immune cells. One tenocyte cluster localised to the fascicular matrix, whereas nine clusters localised to the IFM. Interfascicular tenocytes and mural cells were preferentially affected by aging, with differential expression of genes related to senescence, dysregulated proteostasis and inflammation. This is the first study to establish heterogeneity in IFM cell populations, and to identify age-related alterations specific to IFM-localised cells.

Examining the Potential of Vitamin C Supplementation in Tissue-Engineered Equine Superficial Digital Flexor Tendon Constructs.

Because equine tendinopathies are slow to heal and often recur, therapeutic strategies are being considered that aid tendon repair. Given the success of utilizing vitamin C to promote tenogenesis in other species, we hypothesized that vitamin C supplementation would produce dose-dependent improvements in the tenogenic properties of tendon proper (TP) and peritenon (PERI) cells of the equine superficial digital flexor tendon (SDFT). Equine TP- and PERI-progenitor-cell-seeded fibrin three-dimensional constructs were supplemented with four concentrations of vitamin C. The gene expression profiles of the constructs were assessed with 3'-Tag-Seq and real-time quantitative polymerase chain reaction (RT-qPCR); collagen content and fibril ultrastructure were also analyzed. Moreover, cells were challenged with dexamethasone to determine the levels of cytoprotection afforded by vitamin C. Expression profiling demonstrated that vitamin C had an anti-inflammatory effect on TP and PERI cell constructs. Moreover, vitamin C supplementation mitigated the degenerative pathways seen in tendinopathy and increased collagen content in tendon constructs. When challenged with dexamethasone in two-dimensional culture, vitamin C had a cytoprotective effect for TP cells but not necessarily for PERI cells. Future studies will explore the effects of vitamin C on these cells during inflammation and within the tendon niche in vivo.

Interleukin-6 upregulates extracellular matrix gene expression and transforming growth factor β1 activity of tendon progenitor cells

BackgroundProlonged inflammation during tendon healing and poor intrinsic healing capacity of tendon are causal factors associated with tendon structural and functional degeneration. Tendon cells, consisting of mature tenocytes and tendon progenitor cells (TPC) function to maintain tendon structure via extracellular matrix (ECM) synthesis. Tendon cells can succumb to tissue cytokine/chemokine alterations during healing and consequently contribute to tendon degeneration. Interleukin-(IL-)1β, IL-6 and TNFα are key cytokines upregulated in injured tendons; the specific effects of IL-6 on flexor tendon-derived TPC have not been discerned.MethodsPassage 3 equine superficial digital flexor tendon (SDFT)-derived TPC were isolated from 6 horses. IL-6 impact on the viability (MMT assay with 0, 1, 5 and 10 ng/mL concentrations), migration (scratch motility assay at 0, 10ng/mL concentration) of TPC in monolayer culture were assessed. IL-6 effect on tendon ECM and chondrogenic gene expression (qRT-PCR), TGFβ1 gene expression and activity (ELISA), and MMP-1, -3 and − 13 gene expression of TPC was evaluated.ResultsIL-6 decreased TPC viability and migration. IL-6 treatment at 10 ng/mL significantly up-regulated TGFβ1 gene expression (6.3-fold; p = 0.01) in TPC, and significantly increased the TGFβ1 concentration in cell culture supernates. IL-6 (at 10 ng/mL) significantly up-regulated both tendon ECM (COL1A1:5.3-fold, COL3A1:5.4-fold, COMP 5.5-fold) and chondrogenic (COL2A1:3.9-fold, ACAN:6.2-fold, SOX9:4.8-fold) mRNA expression in TPC. Addition of SB431542, a TGFβ1 receptor inhibitor, to TPC in the presence of IL-6, attenuated the up-regulated tendon ECM and chondrogenic genes.ConclusionIL-6 alters TPC phenotype during in vitro monolayer culture. Pro- and anti-inflammatory roles of IL-6 have been implicated on tendon healing. Our findings demonstrate that IL-6 induces TGFβ1 activity in TPC and affects the basal TPC phenotype (as evidenced via increased tendon ECM and chondrogenic gene expressions). Further investigation of this biological link may serve as a foundation for therapeutic strategies that modulate IL-6 to enhance tendon healing.

Decoding the transcriptomic expression and genomic methylation patterns in the tendon proper and its peritenon region in the aging horse

ObjectivesEquine tendinopathies are challenging because of the poor healing capacity of tendons commonly resulting in high re-injury rates. Within the tendon, different regions – tendon proper (TP) and peritenon (PERI) – contribute to the tendon matrix in differing capacities during injury and aging. Aged tendons have decreased repair potential; the underlying transcriptional and epigenetic changes that occur in the TP and PERI regions are not well understood. The objective of this study was to assess TP and PERI regional differences in adolescent, midlife, and geriatric horses using RNA sequencing and DNA methylation techniques.ResultsDifferences existed between TP and PERI regions of equine superficial digital flexor tendons by age as evidenced by RNASeq and DNA methylation. Cluster analysis indicated that regional distinctions existed regardless of age. Genes such as DCN, COMP, FN1, and LOX maintained elevated TP expression while genes such as GSN and AHNAK were abundant in PERI. Increased gene activity was present in adolescent and geriatric populations but decreased during midlife. Regional differences in DNA methylation were also noted. Notably, when evaluating all ages of TP against PERI, five genes (HAND2, CHD9, RASL11B, ADGRD1, and COL14A1) had regions of differential methylation as well as differential gene expression.

Use of mesenchymal stem cells for tendon healing in veterinary and human medicine: getting to the "core" of the problem through a one health approach.

The purpose of this manuscript, which is part of the Currents in One Health series, is to take a comparative approach to stem cell treatment for tendon injury and consider how the horse might inform treatment in other veterinary species and humans. There is increasing experimental and clinical evidence for the use of bone marrow-derived mesenchymal stem cells to treat tendon injuries in the horse. The same evidence does not currently exist for other species. This manuscript will review why the equine superficial digital flexor tendon core lesion might be considered optimal for stem cell delivery and stem cell interaction with the injury environment and will also introduce the concept of stem cell licensing for future evaluation. The companion Currents in One Health by Koch and Schnabel, AJVR, October 2023, addresses in detail what is known about stem cell licensing for the treatment of other diseases using rodent models and how this information can potentially be applied to tendon healing.

The tendon interfascicular basement membrane provides a vascular niche for CD146+ cell subpopulations.

Introduction: The interfascicular matrix (IFM; also known as the endotenon) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Methods: Immunolabelling of equine superficial digital flexor tendon was used to identify the interfascicular matrix niche, localising expression patterns of CD31 (endothelial cells), Desmin (smooth muscle cells and pericytes), CD146 (interfascicular matrix cells) and LAMA4 (interfascicular matrix basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. Results: Labelling for CD146 using standard histological and 3D imaging of large intact 3D segments revealed an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms extensive, interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralisation (osteogenesis) and lipid production (adipogenesis). Discussion: This study demonstrates that the interfascicular matrix is a unique tendon cell niche, containing a vascular-rich network of basement membrane, CD31+ endothelial cells, Desmin+ mural cells, and CD146+ cell populations that are likely essential to tendon structure and/or function. Contrary to our hypothesis, interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes. Instead, our results indicate CD146 as a pan-vascular marker within the tendon interfascicular matrix. Together with previous work demonstrating that endogenous tendon CD146+ cells migrate to sites of injury, our data suggest that their mobilisation to promote intrinsic repair involves changes in their relationships with local interfascicular matrix vascular and basement membrane constituents.

Short-Term Exposure to Ciprofloxacin Reduces Proteoglycan Loss in Tendon Explants

Fluoroquinolone antibiotics are associated with increased risk of tendinopathy and tendon rupture, which can occur well after cessation of treatment. We have previously reported that the fluoroquinolone ciprofloxacin (CPX) reduced proteoglycan synthesis in equine tendon explants. This study aimed to determine the effects of CPX on proteoglycan catabolism and whether any observed effects are reversible. Equine superficial digital flexor tendon explant cultures were treated for 4 days with 1, 10, 100 or 300 µg/mL CPX followed by 8 days without CPX. The loss of [35S]-labelled proteoglycans and chemical pool of aggrecan and versican was studied as well as the gene expression levels of matrix-degrading enzymes responsible for proteoglycan catabolism. CPX suppressed [35S]-labelled proteoglycan and total aggrecan loss from the explants, although not in a dose-dependent manner, which coincided with downregulation of mRNA expression of MMP-9, -13, ADAMTS-4, -5. The suppressed loss of proteoglycans was reversed upon removal of the fluoroquinolone with concurrent recovery of MMP and ADAMTS mRNA expression, and downregulated TIMP-2 and upregulated TIMP-1 expression. No changes in MMP-3 expression by CPX was observed at any stage. These findings suggest that CPX suppresses proteoglycan catabolism in tendon, and this is partially attributable to downregulation of matrix-degrading enzymes.

Assessing the functional properties of tenogenic primed mesenchymal stem cells in ex vivo equine tendon and ligament explants: A preliminary study

Injuries to equine tendons and ligaments are career-compromising, causing reduced performance and premature retirement. Promising treatment alternatives have been investigated in the field of mesenchymal stem cells (MSCs). In this study, the tissue adherence and protein expression of tenogenic primed mesenchymal stem cells (tpMSCs) after administration to ex vivo tendon and ligament explants is investigated. First, collagen type I (COL I) and smooth muscle actin (SMA) expression was assessed in cytospins prepared from native MSCs and tpMSCs. Second, equine superficial digital flexor tendon and suspensory ligament explants were cultivated, and a lesion was treated with both cell types. Subsequently, cell adhesion to the explants and the amount of COL I and SMA positive cells was evaluated. The cytospins revealed a significantly higher COL I and lower SMA expression in tpMSCs compared to native MSCs. In the explants, tpMSCs showed a significantly higher tendon and ligament adherence. Furthermore, a significantly higher percentage of COL I positive and a lower percentage of SMA positive cells were observed in the lesions treated with tpMSCs. The results of these explant co-cultures may demonstrate at least a part of the mechanism of action and functional properties of tpMSCs in restoring function to tendons and ligaments.

Applicability of ARFI elastography in detecting elasticity changes of the equine superficial digital flexor tendon with induced injury.

The objectives of this prospective, experimental study were to describe changes in the stiffness of the equine superficial digital flexor tendon (SDFT) after induced injury, deep digital flexor tendon (DDFT), accessory ligament (AL-DDFT), and suspensory ligament (SL) during 90 days of healing using acoustic radiation force impulse (ARFI) elastography. Eight healthy horses were selected. Preinjury B mode and ARFI evaluations were performed bilaterally in the palmar metacarpal region. Injury was induced only on the left forelimb (G2) by a single injection of collagenase in SDFT, 15 cm distal to the accessory carpal bone. The right forelimb was used as a control (G1). Evaluations were performed at eight timepoints: one before injury (T0) and seven (T1-T7) after injury (3, 15, 30, 40, 60, 75, and 90 days post-induction). Tendinopathies were visualized as hypoechoic areas with loss of parallel tendon fiber pattern. Injured SDFTs presented mainly cool colors (soft) from T1 to T3, and from T4, there was an increase in warm colors (hard), close to the appearance of tendons of G1. In the first four timepoints, there was a decrease in stiffness compared to G1 (P < 0.001). On T1 and T2, a cutoff value <6.21 m/s to determine tendinopathy of the SDFT was established (75.8% sensitivity and 92.03% specificity). Stiffness changes in the DDFT, AL-DDFT, and SL of injured limbs occurred at different timepoints. Tendinopathy significantly altered the stiffness of the injured tendon and the adjacent tissues. ARFI made it possible to detect these changes, helping to monitor the reparation of this injury.

Efficacy of a commercial dry sleeve cryotherapy system for cooling the equine metacarpus.

To determine the ability of a commercial cryotherapy system (Game Ready Equine) to cool the metacarpal subcutaneous tissue and the superficial digital flexor tendon (SDFT) in horses. Experimental study. Six healthy adult horses. Thermocouples were implanted into the metacarpal subcutaneous tissues and the SDFT of six horses. Two treatments (cryotherapy or cryotherapy with 5-50 mmHg intermittent compression) were randomly assigned to forelimbs and performed for 20 minutes. Temperatures were compared to the target range of 10-19°C and between groups. Only one limb in the cryotherapy/compression group reached the target range after cryotherapy. Temperatures did not differ between treatment groups at time 0. Lowest temperatures achieved in the subcutaneous tissue (p=.0043) and SDFT (p=.005) were 4.9 and 7.6°C lower when intermittent compression was applied. Similarly, applying compression induced a maximum change in temperature of approximately 7.0°C in the subcutaneous tissue (p=.014) and 10.2°C in the SDFT (p=.0001). The cryotherapy system did not cool equine subcutaneous tissue or SDFT to the target temperature range, except in one limb. Combining cryotherapy with intermittent compression did result in lower temperatures and a greater change in temperature of the subcutaneous tissue and SDFT. When using this cryotherapy system, the addition of intermittent compression should be considered to achieve lower temperatures and potentially greater reduction in inflammation. Further studies are warranted to determine the effect of longer treatment times, higher compression settings, and the optimal temperature for benefits in normal and diseased equine tissues.

Ciprofloxacin reduces tenocyte viability and proteoglycan synthesis in short-term explant cultures of equine tendon.

Fluoroquinolones are an effective, broad-spectrum antibiotic used to treat an array of bacterial infections. However, they are associated with an increased risk of tendinopathy and tendon rupture even after discontinuation of treatment. This condition is known as fluoroquinolone-associated tendinopathy, the underlying mechanisms of which are poorly understood. While many factors may be involved in the pathophysiology of tendinopathies in general, changes in tenocyte metabolism and viability, as well as alteration of proteoglycan metabolism are prominent findings in the scientific literature. This study investigated the effects of ciprofloxacin, a common fluoroquinolone, on cell viability, proteoglycan synthesis, and proteoglycan mRNA expression in equine superficial digital flexor tendon explants after 96 h treatment with between 1–300 µg/mL ciprofloxacin, and again after 8 days discontinuation of treatment. Ciprofloxacin caused significant reductions in cell viability by between 25–33% at all dosages except 10 µg/mL, and viability decreased further after 8 days discontinuation of treatment. Proteoglycan synthesis significantly decreased by approximately 50% in explants treated with 100 µg/mL and 300 µg/mL, however this effect reversed after 8 days in the absence of treatment. No significant mRNA expression changes were observed after the treatment period with the exception of versican which was down-regulated at the highest concentration of ciprofloxacin. After the recovery period, aggrecan, biglycan and versican genes were all significantly downregulated in explants initially treated with 1–100 µg/mL. Results from this study corroborate previously reported findings of reduced cell viability and proteoglycan synthesis in a whole tissue explant model and provide further insight into the mechanisms underlying fluoroquinolone-associated tendinopathy and rupture. This study further demonstrates that certain ciprofloxacin induced cellular changes are not rapidly reversed upon cessation of treatment which is a novel finding in the literature.

Cyclic tensile tests of Shetland pony superficial digital flexor tendons (SDFTs) with an optimized cryo-clamp combined with biplanar high-speed fluoroscopy

BackgroundLong-term cyclic tensile testing with equine palmar/plantar tendons have not yet been performed due to problems in fixing equine tendons securely and loading them cyclically. It is well established that the biomechanical response of tendons varies during cyclic loading over time. The aim of this study was to develop a clamping device that enables repetitive cyclic tensile testing of equine superficial digital flexor tendon for at least 60 loading cycles and for 5 min.ResultsA novel cryo-clamp was developed and built. Healthy and collagenase-treated pony SDFTs were mounted in the custom-made cryo-clamp for the proximal tendon end and a special clamping device for the short pastern bone (os coronale). Simultaneously with tensile testing, we used a biplanar high-speed fluoroscopy system (FluoKin) to track tendon movement. The FluoKin system was additionally validated in precision measurements. During the cyclic tensile tests of the SDFTs, the average maximal force measured was 325 N and 953 N for a length variation of 2 and 4 % respectively. The resulting stress averaged 16 MPa and 48 MPa respectively, while the modulus of elasticity was 828 MPa and 1212 MPa respectively. Length variation of the metacarpal region was, on average, 4.87 % higher after incubation with collagenase. The precision of the FluoKin tracking was 0.0377 mm, defined as the standard deviation of pairwise intermarker distances embedded in rigid bodies. The systems accuracy was 0.0287 mm, which is the difference between the machined and mean measured distance.ConclusionIn this study, a good performing clamping technique for equine tendons under repetitive cyclic loading conditions is described. The presented cryo-clamps were tested up to 50 min duration and up to the machine maximal capacity of 10 kN. With the possibility of repetitive loading a stabilization of the time-force-curve and changes of hysteresis and creep became obvious after a dozen cycles, which underlines the necessity of repetitive cyclical testing. Furthermore, biplanar high-speed fluoroscopy seems an appropriate and highly precise measurement tool for analysis of tendon behaviour under repetitive load in equine SDFTs.

An ex vivo biomechanical comparison of two suture materials and two pattern combinations for equine superficial digital flexor tendon tenorrhaphy

To compare biomechanical characteristics of three-loop pulley (3LP) pattern versus Bunnell technique (BT) using polydioxanone (PDS) suture; to determine the influence of polyester tape (PT) versus PDS on the BT for equine superficial digital flexor tendon (SDFT) tenorrhaphy; to compare BT with PT versus 3LP with PDS. Ex vivo biomechanical study. Forty equine forelimb SDFT. Two experiments were performed: (1) 10 SDFT pairs were repaired with 3LP or BT using PDS; (2) 10 SDFT pairs were repaired with PDS or PT using BT. Load at failure, mode of failure, load at 2 mm gap, and gap at failure were obtained using a material testing machine. In experiment 1, 3LP + PDS1 had higher loads at failure (p < .001) and at 2 mm gap (p < .001), and smaller gap at failure than BT + PDS1 (p=.024). In experiment 2, BT + PT2 had higher loads at failure (p < .001) and at 2 mm gap (p=.001), and larger gap at failure (p=.004) than the BT + PDS2 . 3LP + PDS1 and BT + PT2 mostly failed by suture/implant pull-through while BT + PDS failed by suture breakage. BT + PT2 had greater load (p=.035) and gap at failure (p < .001) than 3LP + PDS1, with no difference in load at 2 mm gap (p=.14). The use of BT may be justified over 3LP if combined with PT. However, the larger size of the PT required stab incisions in the tendon for placement and was subjectively more difficult to place than PDS. The BT + PT, although the strongest among the tested repairs, would only be able to withstand 12%-24% of the load encountered by the SDFT at walk.

Strain Elastography of Injured Equine Superficial Digital Flexor Tendons: A Reliability Study of Manual Measurements.

Simple SummaryStrain elastography is an ultrasound-based technique that assesses the mechanical properties of tissues and gives a relative representation of elasticity. Early diagnosis of tendon injuries and long-term monitoring of the healing process are key to equine practice; thus, an accurate method is needed for analyzing and interpreting the images obtained with strain elastography. The first aim of the study was to demonstrate the intraoperator repeatability and interoperator reproducibility of manual measurements of elastograms obtained from injured superficial digital flexor tendons of horses; the second aim was to perform a standardization of the manual measurement method by comparing it with external software. Despite their subjectivity, manual measurements proved to be repeatable and reproducible. In addition, the results obtained with the manual method matched those obtained with the external software.Early diagnosis of tendon injuries and accurate long-term monitoring of the healing process are key for equine veterinarians that use conventional ultrasonography. The development of strain elastography could improve the management of clinical cases. The aim of the study was to assess the intraobserver repeatability and interobserver reproducibility of manual measurements of the colored areas of the tendons within elastograms and to standardize this manual modality by comparing the analysis of the images with ImageJ. Twenty elastograms of the injured superficial digital flexor tendons (SDFTs) of horses were analyzed by two different operators after an acute injury was diagnosed with ultrasonography. Statistical analysis demonstrated excellent intraobserver repeatability (intraclass correlation coefficient, ICC = 0.949) and good interobserver reproducibility (ICC = 0.855) for manual measurements performed with tools available on the ultrasound unit. A good agreement between manual measurements and measurements performed with ImageJ (ICC = 0.849) was then demonstrated. Despite its subjectivity, the manual modality proved to be a valid method for analyzing images obtained with strain elastography.

Intrafascicular chondroid-like bodies in the ageing equine superficial digital flexor tendon comprise glycosaminoglycans and type II collagen.

The superficial digital flexor tendon (SDFT) is considered functionally equivalent to the human Achilles tendon. Circular chondroid depositions scattered amongst the fascicles of the equine SDFT are rarely reported. The purpose of this study was the detailed characterization of intrafascicular chondroid-like bodies (ICBs) in the equine SDFT, and the assessment of the effect of ageing on the presence and distribution of these structures. Ultrahigh field magnetic resonance imaging (9.4T) series of SDFT samples of young (1-9 years) and aged (17-25 years) horses were obtained, and three-dimensional reconstruction of ICBs was performed. Morphological evaluation of the ICBs included histology, immunohistochemistry and transmission electron microscopy. The number, size, and position of ICBs was determined and compared between age groups. There was a significant difference (p = .008) in the ICB count between young and old horses with ICBs present in varying number (13-467; median = 47, mean = 132.6), size and distribution in the SDFT of aged horses only. There were significantly more ICBs in the tendon periphery when compared with the tendon core region (p = .010). Histological characterization identified distinctive cells associated with increased glycosaminoglycan and type II collagen extracellular matrix content. Ageing and repetitive strain frequently cause tendon micro-damage before the development of clinical tendinopathy. Documentation of the presence and distribution of ICBs is a first step towards improving our understanding of the impact of these structures on the viscoelastic properties, and ultimately their effect on the risk of age-related tendinopathy in energy-storing tendons.

Ultrastructural evidence for telocytes in equine tendon.

The three-dimensional ultrastructure of the tendon is complex. Two main cell types are classically supported: elongated tenocytes and ovoid tenoblasts. The existence of resident stem/progenitor cells in human and equine tendons has been demonstrated, but their location and relationship to tenoblasts and tenocytes remain unclear. Hence, in this work, we carried out an ultrastructural study of the equine superficial digital flexor tendon. Although the fine structure of tendons has been previously studied using electron microscopy, the presence of telocytes, a specific type of interstitial cell, has not been described thus far. We show the presence of telocytes in the equine inter-fascicular tendon matrix near blood vessels. These telocytes have characteristic telopodes, which are composed of alternating dilated portions (podoms) and thin segments (podomers). Additionally, we demonstrate the presence of the primary cilium in telocytes and its ability to release exosomes. The location of telocytes is similar to that of tendon stem cells. The telocyte-blood vessel proximity, the presence of primary immotile cilia and the release of exosomes could have special significance for tendon homeostasis.

Comparison of Tenocyte Populations from the Core and Periphery of Equine Tendons.

Tendon is a highly organized, dense connective tissue that has been demonstrated to have very little turnover. In spite of the low turnover, tendon can grow in response to loading, which may take place primarily at the periphery. Tendon injuries and recurrence of injuries are common in both humans and animals in sports. It is unclear why some areas of the tendon are more susceptible to such injuries and whether this is due to intrinsic regional differences in extracellular matrix (ECM) production or tissue turnover. This study aimed to compare populations of tenocytes derived from the tendon core and periphery. Tenocytes were isolated from equine superficial digital flexor tendons (SDFTs), and the proliferation capacity was determined. ECM production was characterized by immuno- and histological staining and by liquid chromatography-mass spectrometry-based proteomics. Core and periphery SDFT cultures exhibited comparable proliferation rates and had very similar proteome profiles, but showed biological variation in collagen type I deposition. In conclusion, the intrinsic properties of tenocytes from different regions of the tendon are very similar, and other factors in the tissue may contribute to how specific areas respond to loading or injury.