Elastic properties of collateral and sesamoid ligaments in the forelimbs of equine cadavers

Kylie A. Legg 1School of Veterinary Science, Massey University, Palmerston North 4410, New Zealand.

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G. Robert Colborne 1School of Veterinary Science, Massey University, Palmerston North 4410, New Zealand.

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Erica K. Gee 1School of Veterinary Science, Massey University, Palmerston North 4410, New Zealand.

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Chris W. Rogers 1School of Veterinary Science, Massey University, Palmerston North 4410, New Zealand.

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Abstract

OBJECTIVE

To evaluate the elastic modulus of various ligaments of the forelimbs of cadaveric horses.

SAMPLE

408 ligaments from 37 forelimbs of 10 Thoroughbred cadavers and cadavers of 9 other horse breeds.

PROCEDURES

Collateral ligaments and straight and oblique sesamoid ligaments were harvested from the proximal interphalangeal, metacarpophalangeal, carpal, and elbow joints of both forelimbs of all 19 horses. Ligament dimensions were measured, and the elastic modulus was determined by tensile testing the ligaments with a strain rate of 1 mm•s−1.

RESULTS

Elastic modulus of the ligaments differed significantly among joints. Highest mean ± SE elastic modulus was for the medial collateral ligament of the metacarpophalangeal joints of Thoroughbreds (68.3 ± 11.0 MPa), and the lowest was for the lateral collateral ligament of the elbow joints of other breeds (2.8 ± 0.3 MPa). Thoroughbreds had a significantly higher elastic modulus for the collateral ligaments of the proximal interphalangeal and metacarpophalangeal joints, compared with values for the other breeds. There was large variation in elastic modulus. Elastic modulus was negatively affected by age. In the ligaments in the distal aspect of the forelimbs, elastic modulus was negatively affected by height at the highest point of the shoulders (ie, withers).

CONCLUSIONS AND CLINICAL RELEVANCE

Cross-sectional area and elastic modulus of collateral ligaments in the forelimbs of equine cadavers differed between breeds and among joints, which may have been reflective of their relative physiologic function under loading during exercise.

Abstract

OBJECTIVE

To evaluate the elastic modulus of various ligaments of the forelimbs of cadaveric horses.

SAMPLE

408 ligaments from 37 forelimbs of 10 Thoroughbred cadavers and cadavers of 9 other horse breeds.

PROCEDURES

Collateral ligaments and straight and oblique sesamoid ligaments were harvested from the proximal interphalangeal, metacarpophalangeal, carpal, and elbow joints of both forelimbs of all 19 horses. Ligament dimensions were measured, and the elastic modulus was determined by tensile testing the ligaments with a strain rate of 1 mm•s−1.

RESULTS

Elastic modulus of the ligaments differed significantly among joints. Highest mean ± SE elastic modulus was for the medial collateral ligament of the metacarpophalangeal joints of Thoroughbreds (68.3 ± 11.0 MPa), and the lowest was for the lateral collateral ligament of the elbow joints of other breeds (2.8 ± 0.3 MPa). Thoroughbreds had a significantly higher elastic modulus for the collateral ligaments of the proximal interphalangeal and metacarpophalangeal joints, compared with values for the other breeds. There was large variation in elastic modulus. Elastic modulus was negatively affected by age. In the ligaments in the distal aspect of the forelimbs, elastic modulus was negatively affected by height at the highest point of the shoulders (ie, withers).

CONCLUSIONS AND CLINICAL RELEVANCE

Cross-sectional area and elastic modulus of collateral ligaments in the forelimbs of equine cadavers differed between breeds and among joints, which may have been reflective of their relative physiologic function under loading during exercise.

Musculoskeletal injury is the most common reason for involuntary loss for the horse racing industry, accounting for 80% of involuntary interruptions to training and 25% of horses exiting from the industry.1,2 The majority (80% to 90%) of these musculoskeletal injuries involve the distal aspect of the forelimbs, with ligaments and tendons as the tissues most commonly affected.1,3–5

Equine limbs primarily move in the sagittal plane, with some capacity for abduction or adduction and limited internal or external rotation.6 Sagittal movement is maintained as a result of the constraint of the collateral ligaments, which support the joints medially and laterally. In the sagittal plane, overextension of the PIP joint is limited by the oblique and straight sesamoid ligaments of the digit, which are attached to the first and second phalanx, respectively. However, to the authors' knowledge, specific mechanical properties of the collateral ligaments and sesamoid ligaments in the distal aspect of the limbs of horses have not been described, and their role in force transmission in the distal aspect of the limbs of horses is relatively unknown.

Ligaments and tendons are viscoelastic. They have a characteristic load-deformation pattern when stretched, with an elastic linear region representing stiffness of the material. The elastic modulus characterizes the stiffness of a material irrespective of its geometry, as determined by use of the following equation: elastic modulus = (k × l)/A, where k is stiffness, l is length, and A is the CSA. The elastic modulus is dependent on the organization, orientation, and type of collagen fibers and the interaction among tissue constituents such as the ground substance.7 A higher elastic modulus indicates a stiffer material. In an animal, ligaments at various anatomic locations have differences in mechanical properties (stiffness) and healing responses that relate to structural demands, relative magnitudes of load, and biochemical composition.8–10

Across species, variability in the mechanical properties of ligaments among individuals is high and can be influenced by age, genetic components (eg, breed or height), and environmental factors (eg, exercise).11,12 Greater understanding of biological constraints of the soft tissue elements of the distal aspect of the limbs would assist in modification of training programs and could reduce the incidence of injuries to these tissues. There is a lack of information about the mechanical properties of ligaments in the forelimbs of horses. Therefore, the aims of the study reported here were to determine the elastic modulus of the collateral and sesamoid ligaments and to relate these measurements to horse breed and height (highest point at the shoulders [ie, withers]).

Materials and Methods

Sample

The medial and lateral collateral ligaments from the PIP, MCP, carpal, and elbow joints and straight and oblique sesamoid ligaments of both forelimbs were harvested from a convenience sample of horses that were euthanized for nonorthopedic reasons at the Massey University School of Veterinary Science. Height (obtained by use of a standard aluminum stick used to measure horse height) and body weight (obtained by use of an electronic scalea) were measured before horses were euthanized. Horses with obvious pathological conditions of the ligaments on gross examination were excluded from the study. Data on history, breed, and reason for euthanasia were obtained from each horse's clinical record. All data collection and testing were conducted by 1 investigator (KAL).

Ligaments were collected within 6 hours after horses were euthanized, after the limbs had been refrigerated at 3°C for 24 to 72 hours, or, in the case of 1 horse, after the limbs had been frozen at −20°C for > 7 days. Ligaments were resected from their attachment sites on the sides of the joints. The investigator was careful to ensure that the band of parallel ligament fibers remained intact. Discrete boundaries of each ligament integral to the fibrous joint capsule were determined by palpation to assess relative thickness such that the ligament could be isolated from the rest of the fibrous connective tissue. The medial collateral ligament of the elbow joint is a compound ligament, and only the short caudal section was harvested. Immediately after ligaments were harvested, they were double bagged in labeled airtight plastic bags with a small volume of physiologic saline (0.9% NaCl) solution and frozen at −20°C until subsequent testing in a materials laboratory.13

Procedures

Samples were thawed at 20°C for a minimum of 6 hours before testing. The CSAs of the frozen and thawed ligament samples were measured by use of dial calipersb with a precision of ± 0.02 mm. Ligaments were assumed to have a rectangular cross section.9,11,12 Measurements were obtained in the middle of each ligament. The CSA was measured at 5 locations along the length of 6 arbitrarily selected ligaments from 5 horses to investigate consistency of CSA measurements.

After ligaments were thawed, they were blotted on paper towels to remove excess fluid. They then were placed in serrated jaw clamps in a materials testing machinec to elongate the ligaments to failure in a controlled environment at a room temperature of 20°C. Cross-sectional dimensions of the ligaments were measured again with the ligaments placed under physiologically low tension (10 N), which was considered a negligible load of < 1% of the biological failure load.14 Resting length of each ligament was the distance between the crossheads of the clamps at 10 N of tension.

Each sample was exposed to 10 cycles of a preconditioning load from 0 to 50 N at a crosshead speed of 1 mm•s−1 to ensure that a consistent strain history was applied to each sample.10,14 Immediately after completion of the preconditioning cycles, each sample was tested to failure at a strain rate of 1 mm•s−1. Crosshead displacement and applied force were recorded simultaneously at a rate of 200 Hz with commercial software.d Ligament failure (ie, rupture) or slippage of a ligament from the clamps was recorded for each test. Each test was conducted within 3 minutes after the thawed ligament was removed from the plastic bag.

Mechanical properties of ligaments

The elastic modulus for each ligament was calculated as the slope of the linear region of the stress-strain curve between 2% and 35% of strain.10,15 The elastic modulus was a ratio, calculated as force to normalized CSA divided by strain.

Consistency of measurements

Repeatability of the elastic modulus measurement was determined for 6 ligaments (lateral collateral ligament of the left PIP joint, oblique sesamoid ligament of the left forelimb, straight sesamoid ligament of the left forelimb, medial collateral ligament of the right MCP joint, lateral collateral ligament of the right carpal joint, and lateral collateral ligament of the right elbow joint) obtained from 1 horse. For each of these 6 ligaments, stress-strain measurements were obtained 5 consecutive times by immediately reclamping the ends of the ligament that had failed or slipped from the clamps and retesting.

Data analysis

The authors were not aware of any data published on the elastic modulus of equine collateral ligaments; therefore, sample size estimates were calculated from elastic modulus data of the superficial digital flexor tendon of horses.14 It was estimated that 60 samples (2 ligaments/joint × 2 joints/horse = 15 horses) would be required to provide 85% power to fit a regression line with an effect size of 0.35 measured by use of the Cohen f2 method, as follows: f2 = R2/(1 – R2), where f2 is the effect size and R2 is the squared multiple correlation.

Data were initially used to calculate descriptive statistics and create box plots. Data were tested for normality by use of the Shapiro-Wilk test. All data were nonnormally distributed; thus, differences among groups were tested by use of the nonparametric Kruskal-Wallis test. Comparisons were made by use of pairwise Wilcoxon nonparametric tests.

The elastic modulus and CSA of the ligaments from each joint were regressed against measures of horse height, age, sex, and breed. Data were fitted by use of a multiple linear regression model, with final model fit assessed via backward stepwise analysis by fitting height, age, sex, breed, joint, location (medial vs lateral), and forelimb (right vs left) as predictor variables. Effects for the final model were deemed significant at P < 0.05.

Pearson product-moment correlations were used to examine the association between horse height and body weight with the frozen and thawed (at 10 N of tension) CSA measurements of the ligaments. All statistical analyses were performed with commercial software.e

Results

Sample

A total of 408 ligaments were harvested from 37 forelimbs of 19 horses. The horses were categorized as 10 Thoroughbreds and 9 other horse breeds (6 sport horses, 2 Standardbreds, and 1 Shetland Pony). Thoroughbreds comprised 10 horses (3 mares and 7 geldings) that ranged from 1 to 18 years of age (median, 8 years of age). Reasons for euthanasia included neurologic disorders (n = 3), laminitis (2), colic (1), chronic lameness (1), fracture of the distal aspect of a limb (1), failure to recover after surgery (1), and an untreatable medical condition (1). The other breeds comprised 9 horses (5 mares and 4 geldings) that ranged from 8 to 19 years of age (median, 14 years of age). Reasons for euthanasia included an untreatable medical condition (n = 3), colic (2), failure to recover after surgery (2), laminitis (1), and chronic lameness (1). Horses with both active (eg, race training or eventing) and inactive (eg, paddock mates) histories were included in both groups.

Height of the horses measured at the withers ranged from 102 to 175 cm. Body weight ranged from 180 to 637 kg. Height was not recorded for 2 horses; height for those 2 horses was estimated by extrapolating a value from the linear height-weight correlation graph drawn by use of the height and body weight data recorded for all the horses. The Pearson moment correlation between height and body weight was 0.85, which led to the decision to use only height in the multivariate analysis.

Ligaments were harvested within 6 hours after horses were euthanized (n = 13 horses), after limbs were refrigerated for 24 to 72 hours (5), and after limbs were frozen for > 7 days (1). Ligaments were harvested from both forelimbs of all horses, except they were harvested from only the left forelimb of one of the Thoroughbreds. The majority (372/408) of the ligaments slipped out of the clamps before ligament failure. However, a clear linear region was observed in the stress-strain curve before slippage or failure, which allowed calculation of the elastic modulus.

Consistency of measurements

The CV of the width and thickness measurements for the ligaments was 9% and 12%, respectively. The CV of the width and thickness measurements of the ligaments under 10 N of tension was 9% and 10%, respectively. Pooled CV of the elastic modulus measurements was 32%.

Elastic modulus

The elastic modulus of the collateral and sesamoid ligaments had some variability within groups and between medial and lateral sides of the same joint (Table 1). The highest mean ± SE elastic modulus was 68.3 ± 11.0 MPa for the medial collateral ligament of the MCP joint of Thoroughbreds, and the lateral collateral ligament of the elbow joint for the other breeds had the lowest elastic modulus (2.8 ± 0.3 MPa). Results for the Kruskal-Wallis test indicated significant differences between the elastic modulus of the lateral and medial collateral ligaments of the carpal joint for the other breeds and lateral and medial collateral ligaments of the elbow joint for all horses. Thoroughbreds had a significantly higher elastic modulus in the collateral ligaments for the PIP and MCP joints, compared with results for the other breeds.

Table 1—

Mean ± SE value for the elastic modulus of ligaments from 37 forelimbs of 19 horses (10 Thoroughbreds and 9 other horse breeds comprising 6 sport horses, 2 Standardbreds, and 1 Shetland Pony).

 Elastic modulus (MPa)
LigamentThoroughbredOther
PIP joint
 Medial collateral26.3 ± 3.912.5 ± 1.9*
 Lateral collateral28.1 ± 3.811.1 ± 1.6
Oblique sesamoid
 Medial part35.4 ± 5.536.0 ± 5.1
 Lateral part37.6 ± 4.332.9 ± 5.4
 Straight sesamoid50.1 ± 6.639.1 ± 4.2
MCP joint
 Medial collateral68.3 ± 11.034.4 ± 3.1
 Lateral collateral57.4 ± 9.835.9 ± 4.1
Carpal joint
 Medial collateral41.8 ± 8.026.4 ± 2.9
 Lateral collateral47.2 ± 4.939.9 ± 3.5
Elbow joint
 Medial collateral64.1 ± 20.132.5 ± 13.3
 Lateral collateral4.1 ± 0.6§2.8 ± 0.3§

Within a ligament, value differs significantly (*P = 0.005 and †P < 0.001) from the value for Thoroughbreds

Within a joint, value differs significantly (‡P = 0.01 and §P < 0.001) from the value for the medial collateral ligament.

Multivariate linear regression revealed a significant relationship between horse height and elastic modulus for only the PIP joint (Table 2). There was a significant effect of medial-lateral location in the joint for the carpal and elbow joints. Age had a weak negative correlation with the elastic modulus for all collateral ligaments, and breed had a significant effect for the PIP and MCP joints. Sex and forelimb (with and without horse as an independent factor) did not significantly affect the elastic modulus of collateral ligaments. All the screening variables (height, age, breed, sex, forelimb, and location) did not have a significant effect, as determined on the basis of results of the regression equation, on the elastic modulus of the sesamoid ligaments.

Table 2—

Results of multilinear regression analysis of the elastic modulus of the collateral ligaments from the forelimbs of 19 horses.

JointPredictorβ coefficientModel R2P value
PIP  0.50< 0.001
 Intercept60.60  
 Height–0.25  
 Age–0.78  
 Breed12.37  
 LocationNS  
 ForelimbNS  
MCP  0.20< 0.001
 Intercept61.10  
 HeightNS  
 Age–1.90  
 Breed19.23  
 LocationNS  
 ForelimbNS  
Carpal  0.29< 0.001
 Intercept67.60  
 HeightNS  
 Age–2.11  
 BreedNS  
 Location–9.30  
 ForelimbNS  
Elbow  0.18< 0.001
 Intercept36.39  
 HeightNS  
 Age–2.09  
 BreedNS  
 Location45.28  
 ForelimbNS  

Height represented height of a horse measured at the highest point of the shoulders (ie, withers). Breed represented Thoroughbreds versus other breeds. Location represented medial versus lateral. Forelimb represented right versus left.

NS = Value for the β coefficient did not differ significantly (P ≥ 0.05) from 0.

CSA of the collateral ligaments

The CSA of the collateral ligaments differed for each joint and on the basis of medial-lateral location for the carpal and elbow joints (Table 3). Thoroughbreds had a smaller CSA of the collateral ligaments for the PIP and MCP joints than did the other breeds. The CSAs of the collateral ligaments of the PIP joint, oblique sesamoid ligaments, and lateral collateral ligaments of the elbow joint all had a significant weak positive relationship with horse height (R2 = 0.20, R2 = 0.16, and R2 = 0.16, respectively). The CSAs of the straight sesamoid ligaments, collateral ligaments of the MCP joint, collateral ligaments of the carpal joint, and medial collateral ligaments of the elbow joint were not associated with horse height.

Table 3—

Mean ± SE value for the CSA of ligaments from 37 forelimbs of 19 horses (10 Thoroughbreds and 9 other horse breeds comprising 6 sport horses, 2 Standardbreds, and 1 Shetland Pony).

 CSA (mm2)
LigamentThoroughbredOther
PIP joint
 Medial collateral67.3 ± 9.2120.6 ± 15.9*
 Lateral collateral63.8 ± 8.7125.0 ± 12.4
Oblique sesamoid
 Medial part39.1 ± 2.638.1 ± 3.6
 Lateral part36.1 ± 3.439.5 ± 3.6
 Straight sesamoid58.9 ± 3.248.7 ± 2.9
MCP joint
 Medial collateral31.6 ± 2.943.8 ± 3.9*
 Lateral collateral38.2 ± 3.151.7 ± 5.2
Carpal joint
 Medial collateral80.5 ± 7.380.1 ± 3.2
 Lateral collateral64.3 ± 10.5§57.9 ± 4.2
Elbow joint
 Medial collateral68.7 ± 8.394.1 ± 15.5
 Lateral collateral303.7 ± 19.9375.5 ± 20.3

Within a ligament, value differs significantly (*P = 0.01, †P < 0.001, and ‡P < 0.05) from the value for Thoroughbreds

Within a joint, value differs significantly (§P < 0.05 and ‖ P < 0.001) from the value for the medial collateral ligament.

Discussion

In the study reported here, the elastic modulus of the collateral ligaments from the PIP, MCP, carpal, and elbow joints and the straight and oblique sesamoid ligaments of the equine forelimb was assessed. Elastic modulus of ligaments differed among the joints of the forelimb. The differences in elastic modulus of these ligaments were consistent with the proposed function of each ligament.8–10,16 This variation in elastic behavior reflected the predicted variation of the forces across the joints of the equine forelimb and complemented previous results in that the mechanical properties of ligaments differ according to their location and functional demands.

Medial-lateral similarities in the elastic modulus of ligaments of the PIP and MCP joints were detected. This could have reflected the function these ligaments play in supporting the suspensory apparatus of the equine forelimb. They form part of the network of soft tissues that tightly wrap the distal aspect of the limbs and are constantly loaded with the force of body weight. Minimal extrasagittal motion has been measured in the PIP and MCP joints during trotting in a straight line on a level surface (mean ± SD, 3 ± 1° and 1.8 ± 0.9° respectively),17,18 as opposed to motion in the carpal and elbow joints (13 ± 6° and 22 ± 6°, respectively),19,20 which are exposed to a greater degree of frontal plane (abduction and adduction) motion.

The elastic modulus of the medial collateral ligament of the elbow joint and lateral collateral ligament of the carpal joint was higher than the elastic modulus of the corresponding lateral or medial ligament on the opposite side of the joint. This was in agreement with findings for collateral ligaments of the carpal joint in dogs, whereby the elastic modulus of the lateral collateral ligaments was greater than that of the medial collateral ligaments.9 The disparity in body size and conformational differences between horses and dogs could result in an exaggeration of these differences, which are consistent with their function to constrain extrasagittal movement caused by a perturbed hoof or foot landing. If this were the case, it appears that the elastic modulus of ligaments may not be directly comparable in allometric equations among species, as has previously been supposed for tendons.21

The strain on collateral ligaments during locomotion depends on the frontal position and angle of the ground reaction force vector, and these will depend on the position of the foot under the trunk and the angle of the limb in the stance phase with increasing velocity.22 It could be hypothesized that increasing adduction of the limb during the stance phase with increasing velocity would cause differential compressive loading of the lateral aspect of the proximally located joints and abduction of those joints. This appeared to be the hypothesized effect at the elbow joint, which was constrained against abduction by the stiffer medial collateral ligaments of that joint.

In addition to differences in the elastic modulus among anatomic sites, wide variation in mechanical properties among individual horses was found in the present study, as has been observed in other studies7,11,23–26 of mammalian ligaments. Breed, height, and age influenced the elastic modulus, notably the collateral ligaments from the more distally located PIP and MCP joints.

Thoroughbreds had a significantly smaller CSA of the collateral ligaments in the PIP, MCP, and elbow joints than did the other breeds. Also, they had a significantly higher elastic modulus (stiffer) for the collateral ligaments of the distally located joints. A higher elastic modulus in the collateral ligaments could result in a tighter, better-performing joint with less deflection, which would result in a better-performing racehorse. Differences in elastic modulus on the basis of breed have been reported for 2 breeds of pigs, whereby the mean ± SD elastic modulus of the collateral ligaments of the carpal joints of the lighter-weight breed was significantly higher than that of the heavier breed (400.0 ± 47.5 MPa vs 327 ± 54.4 MPa).11

The higher elastic modulus for ligaments of Thoroughbreds could have been influenced by their adaptation to higher forces that are imposed by early race training.27 Adaptive response of ligaments to exercise has not been clearly defined, but evidence suggests that musculoskeletal tissues can be conditioned during the early growth phase of animals.28–30 High outlying values for the elastic modulus were detected for the collateral ligaments of the MCP joint of 2 Thoroughbred yearlings. Training data were unavailable, so it was unknown whether the yearlings had been subjected to training before they were euthanized. However, if the horses had been in training, the high elastic modulus could have represented an early adaptive response of the ligaments to that training. No data were available for skeletally immature horses that were breeds other than Thoroughbred, so the discrete effect of training could not be investigated. For the analysis in the present study, we assumed that exercise status would be approximately similar between Thoroughbreds and the other breeds.

The decline in mechanical properties of ligaments with age is a well-described phenomenon that is attributable to cumulative microdamage and degeneration of the ligament substance,7 and this decline was evident in the elastic modulus of the PIP, MCP, and carpal joints and the medial collateral ligaments of the elbow joint in the study reported here. Absence of a relationship of the elastic modulus between the sesamoid ligaments and lateral collateral ligaments of the elbow joint could have been attributable to their inherent properties, or it may have been affected by procedural errors during the study or difficulties of in vitro measurement of ligament properties. This lower mechanical stiffness was evident for the collateral ligaments in the mares of the present study and is consistent with more compliant ligaments in women and female pigs31; however, this effect was not evident after breed was included in the statistical model.

The negative relationship between horse height and elastic modulus in the PIP and MCP joints is in contrast with the finding of independence of elastic properties between tendons and body mass,21 although in the larger collateral ligaments of the carpal and elbow joints, this relationship with body mass was not found. The smaller, more distally located ligaments could have been more greatly affected by a more rapid growth rate associated with greater height at the withers in horses than were the larger proximally located collateral ligaments, which would have led to the detected compromise in ligament properties. Larger horses are likely to have greater bending moments in the distal aspect of their limbs.32 Larger bending moments lead to higher stresses in ligaments; thus, the lack of increase in elastic modulus of ligaments may account for the higher rates of injury observed in taller horses because these horses perform closer to the safety margins of their ligaments.32–34

Data were limited by the opportunistic manner for collection of the 37 forelimbs from 19 horses of various breeds. However, despite high variation among individual horses, there was sufficient information to ascertain the aforementioned significant differences in elastic modulus of ligaments among joints of the distal aspect of the forelimb, lateral or medial location, breed, height, and age of the horse. The data set provided a good cross section of horses and was representative of horses with both active (eg, race training or eventing) and inactive (eg, paddock mates or teaching horses) histories.

Accurate and reproducible laboratory measurement of the tensile properties of ligaments is difficult. In vitro measurement of elastic modulus is influenced by a number of variables, including geometry of the tissue (CSA, length, and shape), strain rate or rate of elongation, temperature, and hydration status. Equine ligaments were assumed to have a rectangular CSA, similar to results for studies of the collateral ligaments of dogs,9 pigs,11 and rabbits.12 Because of the proportionally long limbs of horses, deviations from a rectangular CSA may be greater in horses than in other species, and the CSA was found to differ by a mean of 14% along the length of a sample. This variation likely accounted for some of the large variability in elastic modulus.

Resting length of a ligament was defined as the length of the ligament under 10 N of tension, which represented a physiologically low load (1% to 20% of the maximum load) and was in accordance with tension loads and protocols used in other studies.12,14 Sample elongation at a low strain rate was determined by use of the displacement value of the crosshead of the testing machine. The pooled CV for repeated measurements of elastic modulus was 32%, which indicated that slippage and possible machine deformation may have contributed to a portion of the final variation in elastic modulus.

The low elastic modulus of the lateral collateral ligament of the elbow joint was likely affected by the large CSA and orientation of the collagen fibrils. Collagen fibrils were oriented in a diagonal direction, with the tensile force applied longitudinally. A lower tensile strain and smaller linear region of the stress-strain curve were obtained during tensile testing of this ligament because it slipped out of the clamps more easily than did the other ligaments. Collagen fibrils are strongest along their axes; thus, this diagonal orientation may have resulted in an abnormally low elastic modulus. In addition, the elastic modulus was calculated by use of the CSA for the entire sample and with the assumption that the fibers were longitudinally oriented, whereas in reality the collagen fibrils under direct load in the lateral collateral ligament of the elbow joint represented only a portion of the total CSA. However, direction of the applied force approximated the ground reaction force during the stance phase of locomotion.

Both temperature and hydration status affect the mechanical behavior of ligamentous tissue. The testing environment of 20°C and short duration of the tests ensured that the ligaments remained as close to an in vivo environment as possible within the constraints of the testing apparatus. However, there is an inversely proportional relationship of tensile load and temperature for a given strain,35 which may have resulted in higher than normal values for elastic modulus.

Ligaments were not tested until structural failure. Most of the ligaments slipped from the clamps instead of failing; however, most of the ligaments were too short to cryofreeze in the clamps without affecting the ligament properties. Before ligaments slipped from the serrated jaw clamps, a linear region was observed in the stress-strain curve, which allowed us to calculate the elastic modulus. Failure or slippage occurred at a strain of 7% to 35%. This was greater than the accepted values for the toe region of the curve of 1.5% to 3%, which indicated that the linear region of the stress-strain curve was reached for each ligament. Ligaments with high elastin content can be strained from 30% to 60% without damage.10 Therefore, results for the study reported here were within the predicted range of elastic behavior for ligaments. The elastic modulus of collateral ligaments of the equine carpal joint were comparable to that of adult humans (50 to 60 MPa36) and dogs (approx 50 to 80 MPa9) but substantially less than that of the collateral ligaments of the carpal joint in pigs (327.6 ± 54.4 MPa to 477.8 ± 47.5 MPa11) and goats (516 ± 158 MPa37). These differences may reflect the evolution of horses as a cursorial species, with collateral ligaments that are more flexible and elastic than those of more sedentary species. This allows for greater movement in the distal aspects of the limbs and highlights the importance of accurate breed-specific elastic modulus values for individual ligaments.

Inaccuracies in methods attributable to the aforementioned limitations were considered to be less than the high individual variability for mechanical properties of ligaments in other studies9,14,38,39 in which the CV ranged from 13% to 52%. In addition, data on ligament properties for the present study agree with similar data reported elsewhere.9,38,40

Accurate representations of changes in limb loading patterns by use of computer models with accurate data will help to determine factors that are involved with injury to the limbs of horses. This could lead to development and maintenance of racetrack surfaces or horseshoes that could reduce the incidence of injuries in Thoroughbred racehorses. In addition, rehabilitation techniques for injuries to the distal aspects of limbs could be improved as knowledge about force transmission in the limbs increases. For the Thoroughbred racing industry, this information could substantially reduce wastage and rehabilitation costs attributable to ligament and tendon injuries.

Elastic modulus of collateral ligaments differed in joints from distally to proximally along the forelimbs of equine cadavers; significant differences were consistent with joint location and function of each ligament. Thoroughbreds had a higher elastic modulus in the PIP and MCP joints than did horses of other breeds. There was a negative relationship between horse height and elastic modulus for only the PIP joint. Further studies on the properties of ligaments and their function in the distal aspect of the forelimbs of horses will increase the understanding of factors that lead to musculoskeletal injuries.

Acknowledgments

Supported by the School of Veterinary Science and Institute of Food Science and Technology at Massey University.

The authors declare that there were no conflicts of interest.

ABBREVIATIONS

CSA

Cross-sectional area

CV

Coefficient of variation

MCP

Metacarpophalangeal

PIP

Proximal interphalangeal

Footnotes

a.

Yaohua XK 3190-A12, AVIC Shanghai Yaohua Weighing System Co Ltd, Shanghai, People's Republic of China.

b.

Mitutoyo series 505-633-50, Hobeca Trading Co Ltd, Auckland, New Zealand.

c.

Stable Microsystems TA.XT plus texture analyzer, Stable Micro Systems Ltd, Godalming, England.

d.

Exponent TEE32, version 6, Stable Micro Systems Ltd, Godalming, England.

e.

R Studio, version 1.0.143, R Studio Inc, Boston, Mass.

References

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