Equine Suspensory Ligament Research Articles

Ultrasonography of the suspensory ligament branches in yearling and 2-year-old Thoroughbred sales horses: Prevalence, progression of findings and associations with racing performance.

Equine suspensory ligament branch (branch) ultrasonography is becoming increasingly commonplace presale. No ultrasonographical branch reference data exists for Thoroughbred sales horses. To define the prevalence of ultrasonographical findings in the forelimb suspensory branches of yearling and 2-year-old sales Thoroughbreds and to analyse associations with racing performance. To track changes in branch findings between 1 and 2 years of age in horses that present for sale at both ages. Prospective cohort study using an enrolled sample. Horses were enrolled from a 2016 yearling sale and five 2017 2-year-old sales with consignor permission. Ultrasonography was performed immediately prior to the sales. Ultrasonographical findings relating to branch size, fibrillar pattern, the presence of hyperechoic foci, periligamentar tissue thickness and the adjacent proximal sesamoid bone surface were examined. Associations with racing performance from 2 to 4 years of age were investigated using multivariate regression analyses. Clinical follow-up was sought to ascertain why horses that did not race never started. A total of 593 sales yearlings and 367 2-year-olds had forelimb branch ultrasonography performed. Grade ≥2 fibrillar branch change was present in 8.9% of yearlings and 14.4% of 2-year-olds. A 0.25 cm increase in branch width was associated with a 49-day delayed start to racing careers (P < 0.001, 95% confidence interval (95% CI): 21-77 days). The presence of grade 2 hyperechoic foci was associated with significantly lower total earnings (P = 0.01, 95% CI: $2000-$16 022) and lower earnings per start (P = 0.003, 95% CI: $349-$1718) in United States Dollars. Grade 3 fibrillar branch change had clinically important reductions in the probability of racing, the calibre of racing performance and earnings. Grade 1 fibrillar pattern was associated with significantly higher earnings per start (P = 0.004, 95% CI: $2641-$5759). The findings are applicable to horses prepared for public auction and deemed fit to be entered for sale. The results may underestimate the proportion of severe lesions in horses not entered for sale. Reference values specific to young Thoroughbreds have been established. Grade 1 fibrillar branch change should be regarded as an acceptable appearance in sales yearlings and 2-year-olds. Approximately one-third of grade 2 yearling branches progressed to a grade 3 lesion. Evidence of enlarged branch width and grade 2 hyperechoic foci at 2-year-old sales constitute a risk to racing performance.

Evaluation of the Catabolic and Anabolic Gene Expression Effects and Histology Changes induced by Platelet-Rich Gel Supernatants in Equine Suspensory Ligament Explants Challenged with Lipopolysaccharide

Evaluation of the Catabolic and Anabolic Gene Expression Effects and Histology Changes induced by Platelet-Rich Gel Supernatants in Equine Suspensory Ligament Explants Challenged with Lipopolysaccharide

Histological and immunohistochemical evaluation of two cell therapy protocols in equine suspensory ligament repair

ABSTRACT This study aimed to histologically evaluate the quality of tissue repair in equine suspensory ligament treated with two cell therapy protocols. All four limbs of six animals were operated simultaneously to remove a fragment in each ligament using a skin biopsy punch. Two days later, intralesional injections were performed using bone marrow mononuclear fraction (BM group), cultivated cells derived from adipose tissue (AT group), saline (positive control group), or no treatment (negative control group), in such way that each horse received all treatments. After sixty days biopsies were performed for histological analysis (H &amp; E, Masson’s trichrome and picrosirius red) and immunohistochemistry analysis (collagen type III). Histological findings (H &amp; E and Masson’s trichrome), birefringence intensity (through picrosirius) and collagen type III expression (through immunohistochemistry) were analyzed. Samples from treated groups had better birefringence intensity (P=0.007) and fiber alignment scores were superior compared to controls, though not statistically significant (P=0.08). Presence of inflammatory cells and intense staining for collagen type III occurred in all groups demonstrating an active healing process. In conclusion, both protocols resulted in improvement of tissue repair indicating their potential to be used as an adjuvant treatment of equine suspensory ligament disorders.

Equine suspensory ligament and tendon explants cultured with platelet-rich gel supernatants release different anti-inflammatory and anabolic mediators.

The aim of this study was to evaluate the release of pro- and anti-inflammatory as well as anabolic mediators stimulated by a leukocyte-reduced platelet-rich gel supernatant (Lr-PRGS) and a leukocyte-reduced plasma supernatant (Lr-PL) at two concentrations (25 and 50%) on normal equine suspensory ligament explants (SLEs) and tendon explants (TEs). SLEs and TEs from six horses were independently incubated for 48 h with Lr-PRGS and Lr-PL at concentrations of 25 and 50%, respectively. Samples were collected from the incubated tissues at 1 h and 48 h, which were employed for ELISA determination of interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), IL-4, IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor isoform BB (PDGF-BB), transforming growth factor beta-1 (TGF-β1, and hyaluronic acid (HA). Overall, 50% Lr-PRGS induced significantly less IL-1β release than the other hemoderivatives in both tissues. At 48 h, both Lr-PRGS and 25% Lr-PL induced significantly higher TNF-α concentrations in SLEs when compared to TEs, whereas both Lr-PRGS concentrations induced significantly higher IL-4 concentrations in SLEs in comparison to TEs. IL-1ra release was not different between tissues. However, this cytokine was significantly higher in tissue explants cultured with both Lr-PRGS concentrations. HA concentration was lower in tissue explants cultured with all hemoderivatives at two concentrations when compared to the control group. The positive effects observed for ligaments and tendons treated with Lr-PRGS may be mediated by the inhibition of IL-1β release of and increased release of IL-4 and IL-1ra. Furthermore, PDGF-BB could be a polypeptide responsible for mediating the release of anti-inflammatory cytokines in SLEs and TEs incubated with Lr-PRGS.

B-mode and power Doppler ultrasonography of the equine suspensory ligament branches: A descriptive study on 13 horses.

Ultrasonography is routinely used to achieve the diagnosis of equine suspensory ligament desmopathy. In human medicine, power Doppler ultrasonography has also been found to be useful for the diagnosis of tendon/ligament injuries. The aim of this prospective, pilot study was to assess the presence or absence of power Doppler signal in suspensory ligament branches and compare B-mode findings with power Doppler findings in suspensory ligament branches of lame and non-lame limbs. Thirteen horses were used (eight lame horses, with lameness related to pain in the suspensory ligament branches, and five non-lame horses). Ten lame limbs and 24 sound limbs were assessed by B-mode and power Doppler ultrasonography. The severity of power Doppler signal was scored by two independent readers. The B-mode ultrasonographic examination revealed abnormalities in branches of lame limbs and in branches of sound limbs. Suspensory ligament branches that were considered normal in B-mode showed no power Doppler signal. However, power Doppler signal was detected in suspensory ligament branches that were abnormal in B-mode, both in lame and sound limbs. Power Doppler scores were subjectively higher in suspensory ligament branches of lame limbs and in branches with more severe B-mode changes. Findings supported the use of power Doppler as an adjunctive diagnostic test for lame horses with suspected suspensory desmopathy.

Characterization of collagen fibrils after equine suspensory ligament injury: An ultrastructural and biochemical approach

Suspensory ligament (SL) injuries are an important cause of lameness in horses. The mechanical properties of connective tissue in normal and pathological ligaments are mainly related to fibril morphology, as well as collagen content and types. The purpose of this study was to evaluate, using biochemical and ultrastructural approaches, the alterations in collagen fibrils after injury. Eight Warmblood horses with visible signs of injury in only one forelimb SL were selected and specimens were examined by transmission electron microscope (TEM). Collagen types I, III and V were purified by differential salt precipitation after collagen extraction with acetic acid containing pepsin. TEM revealed abnormal organization as well as alterations in the diameter and shape of fibrils after SL injury. The bands corresponding to types I, III and V collagen were assessed by densitometry after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis indicated that the proportions of type III and type V collagen were higher (P < 0.001) in damaged tissues compared with normal tissues with a mean increase of 20.9% and 17.3%, respectively. Concurrently, a decrease (P < 0.001) in type I collagen within damaged tissues was recorded with a mean decrease of 15.2%. These alterations could be the hallmark of a decrease in the tissue quality and mechanical properties of the ligament. The findings provide new insight for subsequent research on tissue regeneration that may lead to the development of future treatment strategies for SL injury.

The equine forelimb suspensory ligament exhibits a heterogeneous strain pattern under tensile load.

To determine if regional variations in strain patterns occur within the suspensory ligament under tensile load. Local increases in strain may put certain regions of the suspensory ligament at risk and may explain the poor healing and high recurrence rates associated with suspensory branch injuries. The suspensory ligament and its bone attachments were isolated from each of 10 adult equine cadaveric forelimbs and radiodense reference beads were inserted throughout the length of the ligament. Specimens were attached to a custom fixture secured to a materials testing system. Radiographs were acquired at 50, 445, 1112, and 2224 N of applied tensile load. Changes in distances between the beads in each region of the suspensory ligament were measured and the regional strain was calculated. Significant differences were determined using a repeated-measures analysis of variance. The suspensory ligament exhibited significant differences in regional strain (p <0.001). The distal branches of the suspensory ligament had significantly greater strains than the proximal (p = 0.025) and mid-body (p = 0.002) regions. The mid-body of the suspensory ligament also exhibited local strain variation, with the distal mid-body having significantly higher strains than the proximal mid-body (p = 0.038). The equine suspensory ligament demonstrates a heterogeneous strain pattern during tensile loading, with the distal regions exhibiting significantly more strain than the proximal region. The non-homogenous strain pattern could explain the regional difference in injury and re-injury rates.

COMPARISON BETWEEN STANDARD ULTRASONOGRAPHY, ANGLE CONTRAST ULTRASONOGRAPHY, AND MAGNETIC RESONANCE IMAGING CHARACTERISTICS OF THE NORMAL EQUINE PROXIMAL SUSPENSORY LIGAMENT

Previous studies have proposed that standard ultrasonography may not adequately represent the pertinent anatomic characteristics of the equine proximal suspensory ligament. The purpose of the study was to compare the use of standard ultrasonography, angle contrast ultrasonography, MRI, and histology for identification of the anatomic characteristics of the normal equine suspensory ligament in the forelimb. Horses free from forelimb lameness with no palpable abnormalities in the region of the suspensory ligament were included in the study. The proximal suspensory ligaments in 20 forelimbs were examined using the standard ultrasound technique, angle contrast ultrasound technique, and MRI, followed by histologic evaluation. Total transverse (cross-sectional) area of the proximal suspensory ligament was estimated using the standard ultrasound and the angle contrast ultrasound techniques, MRI, and histologic sections for the following parameters: total area of the ligament, ligament fibers, muscle, and fat. The proximal suspensory ligament lobe size and tissue distribution were compared and subjectively graded (0-4) for asymmetry. Subjectively, angle contrast ultrasound technique improved differentiation of fibers from the remaining tissue types and allowed identification of the peripheral ligament margin. There was no significant difference in asymmetry scores between modalities. The asymmetry scores of the right and left forelimbs were significantly different with both ultrasound and MRI, based on the level of measurement. The angle contrast ultrasound technique has limitations compared to MRI. However, it provides additional diagnostic information that is not available with the standard ultrasound technique.

Regional differences in biochemical, biomechanical and histomorphological characteristics of the equine suspensory ligament

Desmopathies of the suspensory ligament are relatively frequent and vary in prevalence over different anatomical regions. This variation may be because of regional differences in tissue characteristics. To characterise different regions of healthy forelimb suspensory ligaments (SLs) by means of biochemical, biomechanical and histomorphological techniques. There is substantial topographical heterogeneity in the SL with respect to structural, compositional and functional characteristics. SLs were harvested from 38 limbs and used for biochemical (n = 20), biomechanical (n = 14) and histomorphological (n = 4) evaluation. Sulphated glycosaminoglycan (S-GAG), DNA and collagen content, degree of lysyl hydroxylation and numbers of enzymatic and nonenzymatic cross-links were determined in 7 regions of the SL: lateral and medial part of the origin (OM, OL), mid-body (MB), axial and abaxial parts of the lateral and medial branches (ILAX, ILAB, IMAX and IMAB, respectively). Passive resistance to tensile loading was measured in 5 regions of the SL (all except OL and OM). DNA content was lower in OL and OM than in all other parts. GAG content was also lower in OL and OM and highest in ILAB and IMAB. Collagen content was lower in OL/OM and highest in ILAX/IMAX. Pentosidine levels were highest in OL and significantly lower in the lateral insertion (ILAX/ILAB). There were no differences in hydroxylysylpyridinoline (HP) cross-links or lysyl hydroxylation. Stiffness (P<0.01) and modulus of elasticity (P<0.01) were substantially higher in the MB region than in all other regions except for IMAB. Strain at failure was lower in the MB region (P<0.0001), resulting in a comparable force at rupture as in the other regions. Matrix composition differs to a relatively limited extent between different regions of the SL. The mid-body of the ligament is stiffer than the branches, which have similar properties and relevance and mechanical differences between mid-body and branches/origin may explain some use-related differences in the prevalence of lesions.

Effects of platelet rich plasma and acellular bone marrow on gene expression patterns and DNA content of equine suspensory ligament explant cultures

Suspensory ligament (SL) desmitis is a common source of lameness. The results of this study will determine if blood-derived products stimulate SL matrix synthesis and have potential as regenerative therapies for SL desmitis To determine if various blood-based biological products including plasma, blood, PRP, platelet poor plasma (PPP) and ABM aspirate stimulates anabolic and/or catabolic pathways in suspensory ligaments (SL). The body of the SL was harvested from 6 horses and used to establish explant cultures. Explants were cultured in plasma, blood, PRP, PPP or ABM at concentrations of 10, 50 or 100%. Anabolic responses were assessed by use of quantitative PCR for collagens type I and III, cartilage oligomeric matrix protein (COMP) and decorin. Total DNA and collagen protein content were also measured. Catabolic reactions were measured by quantitative PCR for matrix metalloproteinases 3 and 13 (MMP-3, MMP-13). Acellular bone marrow aspirate at 100% stimulated decorin and COMP mRNA synthesis more than all other treatments at all concentrations. No treatment at any concentration stimulated the catabolic gene MMP-13; only 50% ABM stimulated MMP-13 mRNA expression. Acellular bone marrow is indicated, and might be preferred to plasma, blood or PPP, as a blood-based biological source for SL tissue regenerative therapy. Long-term, placebo controlled case studies are indicated to determine if ABM aids in recovery from SL desmitis. Bone marrow aspirate is an autogenous, readily available biological source for SL regenerative therapy where the aim is to stimulate matrix synthesis.

Anabolic effects of acellular bone marrow, platelet rich plasma, and serum on equine suspensory ligament fibroblasts in vitro

The purpose of this study was to investigate the response of suspensory ligament fibroblasts (SLF) to in vitro stimulation using acellular bone marrow (ABM), platelet rich plasma (in vitro PRP), and serum as potential treatment modalities for suspensory desmitis. Blood, bone marrow, and suspensory ligaments were collected from five horses. SLF were harvested, grown until confluent, and stimulated with various concentrations of ABM, PRP, equine serum, foetal bovine serum, and medium (control). The responses to the treatments were assessed using a combination of radio-labeling for total protein synthesis and an ELISA for quantification of Cartilage Oligomeric Matrix Protein (COMP) production. Addition of all of the samples resulted in significant increases in COMP and total protein synthesis over controls (P<0.001). ABM caused the greatest increase in both COMP and total protein synthesis by the SLF. Equine ABM, PRP, and serum contain anabolic factors that promote matrix synthesis by SLF in vitro, with ABM having the greatest effect. Application of bone marrow to injured ligaments may enhance healing by providing anabolic factors, other than or in addition to mesenchymal stem cells, which stimulate matrix production.