Equine Superficial Digital Flexor Tendon Research Articles

Comparison of healing in forelimb and hindlimb surgically induced core lesions of the equine superficial digital flexor tendon.

Even though equine multi-limb tendinopathy models have been reported, it is unknown if fore- and hindlimb tendon healing behave similarly. The aim of this study was to compare the healing process of surgically induced superficial digital flexor tendon (SDFT) core lesions of fore- and hindlimbs in horses. Tendon core lesions were surgically induced in the SDFT of both fore- and hindlimbs in eight horses. One randomly assigned forelimb and one randomly assigned hindlimb were injected with saline one and two weeks post-surgery. The healing process was monitored clinically and ultrasonographically. After 24 weeks, the tendons were harvested and biochemical, biomechanical and histological parameters were evaluated. Twenty-four weeks post-surgery, the forelimb SDFT lesions had a significantly higher colour Doppler ultrasound vascularization score (p = 0.02) and glycosaminoglycan concentration (p = 0.04) and a significantly lower hydroxylysylpyridinoline content (p = 0.03). Our results indicate that fore- and hindlimb SDFT surgically induced lesions exhibit significant differences in several important parameters of tendon healing 24 weeks post-surgery. These differences create significant challenges in using all four limbs and accurately interpreting the results that one might generate. Therefore these findings do not support the use of four-limb models for study of tendon injury until the reasons for these differences are much better understood.

Achilles tendon injuries in elite athletes: lessons in pathophysiology from their equine counterparts.

Superficial digital flexor tendon (SDFT) injury in equine athletes is one of the most well-accepted, scientifically supported companion animal models of human disease (i.e., exercise-induced Achilles tendon [AT] injury). The SDFT and AT are functionally and clinically equivalent (and important) energy-storing structures for which no equally appropriate rodent, rabbit, or other analogues exist. Access to equine tissues has facilitated significant advances in knowledge of tendon maturation and aging, determination of specific exercise effects (including early life), and definition of some of the earliest stages of subclinical pathology. Access to human surgical biopsies has provided complementary information on more advanced phases of disease. Importantly, equine SDFT injuries are only a model for acute ruptures in athletes, not the entire spectrum of human tendonopathy (including chronic tendon pain). In both, pathology begins with a potentially prolonged phase of accumulation of (subclinical) microdamage. Recent work has revealed remarkably similar genetic risk factors, including further evidence that tenocyte dysfunction plays an active role. Mice are convenient but not necessarily accurate models for multiple diseases, particularly at the cellular level. Mechanistic studies, including tendon cell responses to combinations of exercise-associated stresses, require a more thorough investigation of cross-species conservation of key stress pathway auditors. Molecular evidence has provided some context for the poor performance of mouse models; equines may provide better systems at this level. The use of horses may be additionally justifiable based on comparable species longevity, lifestyle factors, and selection pressure by similar infectious agents (e.g., herpesviruses) on general cell stress pathway evolution.

Freeze-Thaw Cycles Enhance Decellularization of Large Tendons

The use of decellularized tendon tissue as a scaffold for tendon tissue engineering provides great opportunities for future clinical and current research applications. The aim of this study was to assess the effect of repetitive freeze-thaw cycles and two different detergents, t-octyl-phenoxypolyethoxyethanol (Triton X-100) and sodium dodecyl sulfate (SDS), on decellularization effectiveness and cytocompatibility in large tendons. Freshly collected equine superficial and deep digital flexor tendons were subjected to decellularization according to four different protocols (1 and 2: freeze-thaw cycles combined with either Triton X-100 or SDS; 3 and 4: Triton X-100 or SDS). Decellularization effectiveness was assessed based on the reduction of vital cell counts, histologically visible nuclei, and DNA content. Transmission electron microscopy was performed to evaluate cellular and extracellular matrix integrity. Further, cytocompatibility of scaffolds that had been decellularized according to the protocols including freeze-thaw cycles (protocols 1 and 2) was assessed by seeding the scaffolds with superparamagnetic iron oxide labeled mesenchymal stromal cells and monitoring the cells histologically and by magnetic resonance imaging for two weeks. Decellularization was significantly more effective when using the protocols including freeze-thaw cycles, leaving only roughly 1% residual nuclei and 20% residual DNA, whereas samples that had not undergone additional freeze-thaw cycles contained roughly 20% residual nuclei and 40% residual DNA. No morphological extracellular matrix alterations due to decellularization could be observed. Scaffolds prepared by both protocols including freeze-thaw cycles were cytocompatible, but the cell distribution into the scaffold tended to be better in scaffolds that had been decellularized using freeze-thaw cycles combined with Triton X-100 instead of SDS.

In vivo Evaluation of Acoustoelastography in the Normal Equine Superficial Digital Flexor Tendon

IntroductionSuperficial digital flexor tendon (SDFT) injury is common in athletic horses. Recovery requires balancing rest and controlled exercise, resulting in high risk of re‐injury. It is difficult to determine the level of work the damaged tissue can sustain based on serial ultrasound examination alone. However, as small changes in sonographic appearance are associated with large changes in biomechanical strength, it is difficult to determine the degree of activity the tissue can sustain.AimsAcoustoelastography (AEG) is a new ultrasound‐based model to evaluate tendon function. Acoustoelastography deduces stiffness gradient, the rate of change of normalised stiffness as a function of strain, by analysing the changes in echo intensity observed in cine loops captured from gradually deforming tendon. The goal of this project was to establish a reproducible method for applying AEG in the normal equine SDFT.MethodsFifteen horses with no history of lameness and normal lameness examinations were recruited with informed owner consent and in accordance with the University Research Animal Resources Center. Stiffness gradient index (SGI) and dispersion values (DV) for the palmar SDFT were evaluated at 3 sites (5, 10 and 15 cm distal to the accessory carpal bone [DACB]) by 2 observers. Lifting of the contralateral forelimb during image acquisition resulted in the required SDFT deformation. Interobserver repeatability, intraobserver repeatability, and right‐to‐left limb symmetry were evaluated.ResultsThe SGIs and DVs for the SDFT at different locations, as well as effects of age or gender, showed no statistical difference (P>0.05). Interclass correlations evaluating repeatability within the same observer, between observers, and symmetry between right and left limbs demonstrated excellent agreement.Conclusions and practical significanceThis study shows that AEG is a feasible and repeatable technique for measuring stiffness gradients in normal equine SDFTs. This study will provide the basis for developing a simple, noninvasive evaluation of tendon function that could vastly improve the ability to detect, monitor and treat tendon injuries.Ethical animal researchAll animal protocols were approved by the University of Wisconsin Research Animal Resources Center. Client‐owned horses were used for the study and informed consent was obtained. Sources of funding: Research supported by Companion Animal Grant through University of Wisconsin. Competing interests: Co‐author/collaborator Ray Vanderby has patent on acoustoelastography post processing ultrasound‐based tissue evaluation technique.

STEM CELL THERAPY FOR TENDINOPATHY: LESSONS FROM A LARGE ANIMAL MODEL

IntroductionTendon disorders compromise pain-free activity and often progress to chronic pain with a major impact on quality of life. Achilles tendinopathy (AT) is particularly common with many proposed therapies. However,...

LARGE PROTEOGLYCAN METABOLISM IN TENDINOPATHY

IntroductionOne of the hallmarks of developing tendinopathy after injury is the accumulation of toluidine-blue-positive sulfated glycosaminoglycan, attributed to increased expression and synthesis of the large proteoglycans (PGs) aggrecan and versican....

Active Hyaluronidase 2 Expression in the Granulation Tissue Formed in the Healing Process of Equine Superficial Digital Flexor Tendonitis

Upregulation of hyaluronidase 2 (HYAL2), one of somatic hyaluronidase (HAase), was demonstrated in granulation tissue during the healing of equine superficial digital flexor tendon injuries. The activity of HAase was assessed by hyaluronan (HA)-containing gel zymography and in situ zymography using frozen sections obtained from normal and injured tendon tissues. Elevated HAase activity was identified in the extract from the tendinopathic tissues, with lower levels of the activity in normal tendons. In situ zymography using fluorescently-labeled HA demonstrated HAase activity in the granulation tissue formed in the injured region. In addition, in situ hybridization analysis indicated that fibroblastic cells in the granulation tissue of the injured tendon actively expressed HYAL2 but not HYAL1. Quantitative RT-PCR further confirmed a higher level of amplicons corresponding to HYAL2 in tendonitis-derived samples. These results suggest that elevated HYAL2 activity in the granulation tissue could participate in controlling the healing process in equine tendonitis.

Immunolocalization of Collagens (I and III) and Cartilage Oligomeric Matrix Protein in the Normal and Injured Equine Superficial Digital Flexor Tendon

This is a descriptive study of tendon pathology with different structural appearances of repair tissue correlated to immunolocalization of cartilage oligomeric matrix protein (COMP) and type I and III collagens and expression of COMP mRNA. The material consists of nine tendons from seven horses (5–25 years old; mean age of 10 years) with clinical tendinopathy and three normal tendons from horses (3, 3, and 13 years old) euthanized for non-orthopedic reasons.The injured tendons displayed different repair-tissue appearances with organized and disorganized fibroblastic regions as well as areas of necrosis. The normal tendons presented distinct immunoreactivity for COMP and expression of COMP mRNA and type I collagen in the normal aligned fiber structures, but no immunolabeling of type III collagen. However, immunoreactivity for type III collagen was present in the endotenon surrounding the fiber bundles, where no expression of COMP could be seen. Immunostaining for type I and III collagens was present in all of the pathologic regions indicating repair tissue. Interestingly, the granulation tissues showed immunostaining for COMP and expression of COMP mRNA, indicating a role for COMP in repair and remodeling of the tendon after fiber degeneration and rupture.The present results suggest that not only type III collagen but also COMP is involved in the repair and remodeling processes of the tendon.

Evaluation of equine superficial digital flexor tendon lesions

Tendon injuries are commonplace in the equine athlete and are reported to be one of the most prevalent forms of musculoskeletal injuries occurring in horses competing in all disciplines. The superficial digital flexor tendon (SDFT) is the more commonly injured of the flexor tendons, accounting for 75–93% of clinical cases; research suggests that lesions typically occur at the mid-metacarpal level in the ‘core’ or central region of the SDFT. Aims and objectives: The aim of this research was to increase knowledge regarding the susceptibility to injury of specific topographical regions within the SDFT, and to identify any relationship between topographical location and lesion severity to laterality and the equestrian discipline the subjects competed in. Method: Secondary data were ultrasound scans from the forelimb SDFTs of horses (n =100) presenting with primary ‘core’ lesions. Topographical location, percentage distribution of the lesion entirety and the echogenicity of lesions were ascertained and graded. Statistical analysis identified if any significant differences were present between discipline competed in and right and left forelimb lesion severity within the population. Results: No significant differences were exposed between competitive discipline or right and left forelimb lesions severity (p >0.05). Lesions that present in the mid-metacarpal region of the SDFT comprised the greater percentage of the total clinical lesion and exhibited higher echogenicity scores, suggesting they were more severe in nature. Conclusions: The study confirmed that the mid-metacarpal region of the SDFT exhibited more severe legions in comparison with the proximal and distal regions; however, no differences between disciplines or with reference to laterality were observed. Although these results can only be applied to this population, they suggest that ultrasonographic evaluation of topography and echogenicity translated to severity scores can aid in the diagnosis of tendon pathology and subjects’ subsequent rehabilitation regimen.

Ovine amniotic epithelial cells: In vitro characterization and transplantation into equine superficial digital flexor tendon spontaneous defects

In vitro expanded and frosted ovine amniotic epithelial cells (oAECs) were evaluated for their phenotype, stemness and attitude to differentiate into tenocytes. Fifteen horses with acute tendon lesions were treated with one intralesional injection of oAECs. Tendon recovery under controlled training was monitored. In vitro expanded oAECs showed a constant proliferative ability, a conserved phenotype and stable expression profile of stemness markers. Differentiation into tenocytes was also regularly documented. US controls showed the infilling of the defect and early good alignment of the fibers and 12 horses resumed their previous activity. Histological and immunohistochemical examinations in an explanted tendon demonstrated the low immunogenicity of oAECs that were able to survive in the healing site. In addition, oAECs supported the regenerative process producing ovine collagen type I amongst the equine collagen fibers. Considering our results, oAECs can be proposed as a new approach for the treatment of spontaneous equine tendon injuries.

Equine superficial digital flexor tendon force and axial speed of sound: a calibration method under clinical conditions

Equine superficial digital flexor tendon force and axial speed of sound: a calibration method under clinical conditions

Effects of ground surface on the equine superficial digital flexor tendon loading at the walk and trot

Effects of ground surface on the equine superficial digital flexor tendon loading at the walk and trot

A Preliminary Study of Diagnostic Color Doppler Ultrasonography in Equine Superficial Digital Flexor Tendonitis

We hypothesized that semi-quantitative determinations of changeable blood flows in granulation and scar tissues during tendon healing could be helpful for differentiation between the acute phase rich in blood vessels and the remodeling phase with fewer vessels. Ten Thoroughbreds with injured superficial digital flexor tendon (SDFT) in a forelimb were used for evaluation of blood flows in the injured tendons of racehorses using color Doppler (CD) ultrasonography. Using longitudinal CD images, features of maximum color activities were defined. These were rhythmically blinking, tiny to small signals (grade 1), pulsatile expanded dots (grade 2), and dynamic streams (grade 3). Grade of color activity in CD ultrasonography could be useful for quantitative assessment of equine SDFT repair.

Short‐term cast immobilisation is effective in reducing lesion propagation in a surgical model of equine superficial digital flexor tendon injury

Larger superficial digital flexor tendon (SDFT) injuries have a poorer prognosis than smaller lesions. During the inflammatory phase enlargement of the initial lesion is frequently noted, with biomechanical loading being recently proposed to play an important role. To evaluate the effect of lower limb cast immobilisation on tendon lesion propagation in an equine model of surgically induced SDFT injury. Core lesions were surgically induced in both front SDFTs of 6 young mature horses. At the end of surgery, one leg was randomly placed in a lower limb cast and the other leg (control) was bandaged for 10 days. Computerised ultrasonographic tissue characterisation performed at Days 10, 15, 21, 28, 35 and 42 allowed measurement of lesion length (cm) and width (expressed as a percentage of whole tendon cross-section). On Day 42 horses were subjected to euthanasia and both SDFTs were sectioned every centimetre to assess the lesion length macroscopically. Statistics were performed to compare cast vs. control legs with significance set at P<0.05. When all time points were combined, lesion length was 19% shorter (P<0.0001) and lesion width 57% smaller (P = 0.0002) in the cast legs (6.13 ± 0.12 cm; 6.90 ± 0.64%) than in the control legs (7.30 ± 0.21 cm; 10.85 ± 1.22%). On Day 42 the lesion length on macroscopic evaluation was 19% shorter (P = 0.04) in the cast (7.00 ± 0.36 cm) than in the control legs (8.33 ± 0.33 cm). Cast immobilisation for 10 days effectively reduced lesion propagation (length and width) compared to bandaging in an in vivo model of artificially-induced tendon lesions. A short period of cast immobilisation during the early phase of tendon healing may be an easy and cost-effective way to reduce the initial enlargement of lesion size and hence to improve prognosis.

Concerted and adaptive alignment of decorin dermatan sulfate filaments in the graded organization of collagen fibrils in the equine superficial digital flexor tendon

The equine superficial digital flexor tendon (SDFT) has a graded distribution of collagen fibril diameters, with predominantly small-diameter fibrils in the region of the myotendinous junction (MTJ), a gradual increase in large-diameter fibrils toward the osteotendinous junction (OTJ), and a mixture of small- and large-diameter fibrils in the middle metacarpal (MM) region. In this study, we investigated the ultrastructure of the SDFT, to correlate the spatial relationship of the collagen fibrils with the graded distribution. The surface-to-surface distances of pairs of fibrils were found to be almost constant over the entire tendon. However, the center-to-center distances varied according to fibril diameter. Decorin is the predominant proteoglycan in normal mature tendons, and has one dermatan sulfate (DS) or chondroitin sulfate (CS) filament as a side chain which is associated with the surfaces of the collagen fibrils via its core protein. We identified a coordinated arrangement of decorin DS filaments in the equine SDFT. The sizes of the decorin DS filaments detected by Cupromeronic blue staining showed a unique regional variation; they were shortest in the MM region and longer in the MTJ and OTJ regions, and a considerable number of filaments were arranged obliquely to adjacent collagen fibrils in the MTJ region. This regional variation of the filaments may be an adaptation to lubricate the interfibrillar space in response to local mechanical requirements. The results of this study suggest that the MTJ region, which receives the muscular contractile force first, acts as a buffer for mechanical forces in the equine SDFT.

First Application of Axial Speed of Sound to Follow Up Injured Equine Tendons

Ultrasonography is an established technique to follow up injured tendons, although the lesions’ echogenicity tends to become normal before the tendon is ready to sustain the stresses imposed by exercise. Normalized axial speed of sound (SOS) has been found to correlate with an injured tendon’s stiffness; therefore, the purpose of this study was to establish whether SOS would be a useful tool in tendon injury follow-up. Axial SOS was measured in 11 equine superficial digital flexor tendons during a 15-week follow-up period and compared with an ultrasonographic grading system. SOS significantly decreased 2 weeks after the surgical induction of a core lesion, showing a minimum between 7 and 10 weeks; ultrasonographic grade showed a minimum at 3 weeks and increased thereafter. The ultrasonographic grading at 15 weeks was correlated to normalized SOS. These results suggest that axial SOS provides complementary information to ultrasonography that could be of clinical interest.

Exogenous collagen cross‐linking recovers tendon functional integrity in an experimental model of partial tear

We investigated the hypothesis that exogenous collagen cross-linking can augment intact regions of tendon to mitigate mechanical propagation of partial tears. We first screened the low toxicity collagen cross-linkers genipin, methylglyoxal and ultra-violet (UV) light for their ability to augment tendon stiffness and failure load in rat tail tendon fascicles (RTTF). We then investigated cross-linking effects in load bearing equine superficial digital flexor tendons (SDFT). Data indicated that all three cross-linking agents augmented RTTF mechanical properties but reduced native viscoelasticity. In contrast to effects observed in fascicles, methylglyoxal treatment of SDFT detrimentally affected tendon mechanical integrity, and in the case of UV did not alter tendon mechanics. As in the RTTF experiments, genipin cross-linking of SDFT resulted in increased stiffness, higher failure loads and reduced viscoelasticity. Based on this result we assessed the efficacy of genipin in arresting tendon tear propagation in cyclic loading to failure. Genipin cross-linking secondary to a mid-substance biopsy-punch significantly reduced tissue strains, increased elastic modulus and increased resistance to fatigue failure. We conclude that genipin cross-linking of injured tendons holds potential for arresting tendon tear progression, and that implications of the treatment on matrix remodeling in living tendons should now be investigated.

Biomechanical Testing of a Novel Suture Pattern for Repair of Equine Tendon Lacerations

To compare in vitro biomechanical properties of a novel suture pattern to a current standard for primary repair of equine superficial digital flexor tendon (SDFT) laceration. In vitro randomized, paired design. Cadaveric equine forelimb SDFT (n = 24). The 3-loop pulley (3LP) and 6-strand Savage (SSS) suture patterns were applied to transected equine SDFT. Ultimate failure load, stiffness, mode of failure, and load required to form a 3-mm gap were obtained using a materials testing system and synchronized high-speed video analysis. Statistical comparisons were made using Student's t-test, with significance set at P < .05. The SSS repair failed at a higher ultimate load (421.1 N ± 47.6) than the 3LP repair (193.7 N ± 43.0; P < .001). There was no significant difference in stiffness (P = .99). Failure mode was suture breakage for all SSS repair and suture pull through for all 3LP repair. The maximum load to create a 3-mm gap in the SSS repair (102.0 N ± 22.4) was not significantly different from the 3LP repair (109.9 N ± 16.0; P = .27). SSS tenorrhaphy has improved strength and resistance to pull through compared with 3LP for equine SDFT in a single load-to-failure test. Load required to form a 3-mm gap was not significantly different between SSS and 3LP.

Axial speed of sound for the monitoring of injured equine tendons: A preliminary study

Equine superficial digital flexor tendons (SDFT) are often injured, and they represent an excellent model for human sport tendinopathies. While lesions can be precisely diagnosed by clinical evaluation and ultrasonography, a prognosis is often difficult to establish; the knowledge of the injured tendon's mechanical properties would help in anticipating the outcome. The objectives of the present study were to compare the axial speed of sound (SOS) measured in vivo in normal and injured tendons and to investigate their relationship with the tendons' mechanical parameters, in order to assess the potential of quantitative axial ultrasound to monitor the healing of the injured tendons. SOS was measured in vivo in the right fore SDFTs of 12 horses during walk, before and 3.5 months after the surgical induction of a bilateral core lesion. The 12 horses were then euthanized, their SDFTs isolated and tested in tension to measure their elastic modulus and maximal load (and corresponding stress). SOS significantly decreased from 2179.4±31.4m/s in normal tendons to 2065.8±67.1m/s 3.5 months after the surgical induction, and the tendons' elastic modulus (0.90±0.17GPa) was found lower than what has been reported in normal tendons. While SOS was not correlated to tendon maximal load and corresponding stress, the SOS normalized on its value in normal tendons was correlated to the tendons' elastic modulus. These preliminary results confirm the potential of axial SOS in helping the functional assessment of injured tendon.

The effect of platelet‐rich plasma on the neovascularization of surgically created equine superficial digital flexor tendon lesions

The repair process of tendon injuries, which are common in both human and equine athletes, is slow and the quality of the repair tissue is often inferior to the original tendon tissue, which frequently leads to re-injury. The relatively poor vascularization of tendons is considered to be one of the reasons for their limited healing potential. Recently, platelet-rich plasma (PRP), an autologous concentrate of platelets, rich in growth factors, has been shown to enhance the repair process of injured tendons. This effect has been ascribed to the high levels of growth factors in PRP, several of which are known to be involved in tendon repair. Among many other growth factors, the vascular endothelial growth factor, a powerful stimulator of angiogenesis, is abundantly present in PRP, suggesting that enhancement of neovascularization might be one of the working mechanisms. In this study, the effect of PRP on neovascularization was studied in experimentally induced tendon injuries using color Doppler ultrasonography and immunological staining of Factor VIII. PRP induced significantly more neovascularization than the placebo treatment until at least 23 weeks after treatment, as detected by both Doppler ultrasonography and Factor VIII staining. Neovascularization might be one of the explanations for the long-lasting effect of a single intratendinous treatment with PRP.