Collagen V deficiency during murine tendon healing results in distinct healing outcomes based on knockdown severity.

Tendon function is dependent on proper organization and maintenance of the collagen I tissue matrix. Collagen V is a critical regulator of collagen I fibrils, and while prior studies have shown a negative impact of collagen V deficiency on tendon healing outcomes, these studies are confounded by collagen V deficiency through tendon development. The specific role of collagen V in regulating healing tendon properties is therefore unknown. By using inducible Col5a1 knockdown models and analyzing gene expression, fibril and histological tendon morphology, and tendon mechanical properties, this study defines the isolated role of collagen V through tendon healing. Patellar tendon injury caused large changes in tendon gene expression, and Col5a1 knockdown resulted in dysregulated expression of several genes through tendon healing. Col5a1 knockdown also impacted collagen fibril size and shape without observable changes in scar tissue formation. Surprisingly, heterozygous Col5a1 knockdown resulted in improved stiffness of healing tendons that was not observed with homozygous Col5a1 knockdown. Together, these results present an unexpected and dynamic role of collagen V deficiency on tendon healing outcomes following injury. This work suggests a model of tendon healing in which quasi-static mechanics may be improved through titration of collagen fibril size and shape with modulation of collagen V expression and activity.

Supraspinatus Tendons Have Different Mechanical Properties Across Sex.

Sex differences in the mechanical properties of different musculoskeletal tissues and their impact on tendon function and disease are becoming increasingly recognized. Tendon mechanical properties are influenced by the presence or absence of sex hormones and these effects appear to be tendon- or ligament-specific. The objective of this study was to determine how sex and hormone differences in rats affect supraspinatus tendon and muscle properties. We hypothesized that male supraspinatus tendons would have increased cross-sectional area but no differences in tendon material properties or muscle composition when compared to supraspinatus tendons from female or ovariectomized (OVX) female rats. Uninjured supraspinatus tendons and muscles from male, female, and OVX female rats were collected and mechanical and histological properties were determined. Our analysis demonstrated decreased dynamic modulus and increased hysteresis and cross-sectional area in male tendons. We found that male tendons exhibited decreased dynamic modulus (during low strain frequency sweep and high strain fatigue loading), increased hysteresis, and increased cross-sectional area compared to female and OVX female tendons. Despite robust mechanical differences, tendon cell density and shape, and muscle composition remained unchanged between groups. Interestingly, these differences were unique compared to previously reported sex differences in rat Achilles tendons, which further supports the concept that the effect of sex on tendon varies anatomically. These differences may partially provide a mechanistic explanation for the increased rate of acute supraspinatus tendon ruptures seen in young males.

Poly-N-Acetyl Glucosamine (sNAG) Enhances Early Rotator Cuff Tendon Healing in a Rat Model.

Rotator cuff injuries frequently require surgical repairs which have a high failure rate. Biological augmentation has been utilized in an attempt to improve tendon repair. Poly-N-acetyl glucosamine (sNAG) polymer containing nanofibers has been shown to increase the rate for healing of venous leg ulcers. The purpose of this study was to investigate the healing and analgesic properties of sNAG in a rat rotator cuff injury and repair model. 144 adult male Sprague-Dawley rats underwent a transection and repair of their left supraspinatus tendons. Half of the animals received a sNAG membrane on the tendon-to-bone insertion site. Animals were further subdivided, receiving 1 or 3 days of analgesics. Animals were sacrificed 2, 4, or 8 weeks post-injury. Animals sacrificed at 4 and 8 weeks underwent longitudinal in vivo ambulatory assessment. Histological properties were assessed at 2, 4, and 8 weeks, and mechanical properties at 4 and 8 weeks. In the presence of analgesics, tendons receiving the sNAG polymer had significantly increased max load and max stress at 4 weeks, but not at 8 weeks. Ambulatory improvements were observed at 14 days in stride length and speed. Therefore, sNAG improves tendon-to-bone healing in a rat rotator cuff detachment and repair model.

Aging leads to inferior Achilles tendon mechanics and altered ankle function in rodents.

Spontaneous rupture of the Achilles tendon is increasingly common in the middle aged population. However, the cause for the particularly high incidence of injury in this age group is not well understood. Therefore, the objective of this study was to identify age-specific differences in the Achilles tendon-muscle complex using an animal model. Functional measures were performed in vivo and tissues were harvested following euthanasia for mechanical, structural, and histological analysis from young, middle aged, and old rats. Numerous alterations in tendon properties were detected across age groups, including inferior material properties (maximum stress, modulus) with increasing age. Differences in function were also observed, as older animals exhibited increased ankle joint passive stiffness and decreased propulsion force during locomotion. Macroscale differences in tendon organization were not observed, although cell density and nuclear shape did vary between age groups. Muscle fiber size and type distribution were not notably affected by age, indicating that other factors may be more responsible for age-specific Achilles tendon rupture rates. This study improves our understanding of the role of aging in Achilles tendon biomechanics and ankle function, and helps provide a potential explanation for the disparate incidence of Achilles tendon ruptures in varying age groups.

Males have Inferior Achilles Tendon Material Properties Compared to Females in a Rodent Model.

The Achilles tendon is the most commonly ruptured tendon in the human body. Numerous studies have reported incidence of these injuries to be upwards of five times as common in men than women. Therefore, the objective of this study was to investigate the sex- and hormone-specific differences between Achilles tendon and muscle between female, ovariectomized female (ovarian hormone deficient), and male rats. Uninjured tissues were collected from all groups for mechanical, structural, and histological analysis. Our results showed that while cross-sectional area and failure load were increased in male tendons, female tendons exhibited superior tendon material properties and decreased muscle fiber size. Specifically, linear and dynamic moduli were increased while viscoelastic properties (e.g., hysteresis, percent relaxation) were decreased in female tendons, suggesting greater resistance to deformation under load and more efficient energy transfer, respectively. No differences were identified in tendon organization, cell shape, cellularity, or proteoglycan content. Additionally, no differences in muscle fiber type distribution were observed between groups. In conclusion, inferior tendon mechanical properties and increased muscle fiber size may explain the increased susceptibility for Achilles tendon injury observed clinically in men compared to women.

Ground reaction forces are more sensitive gait measures than temporal parameters in rodents following rotator cuff injury.

Gait analysis is a quantitative, non-invasive technique that can be used to investigate functional changes in animal models of musculoskeletal disease. Changes in ground reaction forces following injury have been observed that coincide with differences in tissue mechanical and histological properties during healing. However, measurement of these kinetic gait parameters can be laborious compared to the simpler and less time-consuming analysis of temporal gait parameters alone. We compared the sensitivity of temporal and kinetic gait parameters in detecting functional changes following rotator cuff injury in rats. Although these parameters were strongly correlated, temporal measures were unable to detect greater than 50% of the functional gait differences between injured and uninjured animals identified simultaneously by ground reaction forces. Regression analysis was used to predict ground reaction forces from temporal parameters. This model improved the ability of temporal parameters to identify known functional changes, but only when these differences were large in magnitude (i.e., between injured vs. uninjured animals, but not between different post-operative treatments). The results of this study suggest that ground reaction forces are more sensitive measures of limb/joint function than temporal parameters following rotator cuff injury in rats. Therefore, although gait analysis systems without force plates are typically efficient and easy to use, they may be most appropriate for use when major functional changes are expected.

Achilles tendons from decorin- and biglycan-null mouse models have inferior mechanical and structural properties predicted by an image-based empirical damage model.

Achilles tendons are a common source of pain and injury, and their pathology may originate from aberrant structure function relationships. Small leucine rich proteoglycans (SLRPs) influence mechanical and structural properties in a tendon-specific manner. However, their roles in the Achilles tendon have not been defined. The objective of this study was to evaluate the mechanical and structural differences observed in mouse Achilles tendons lacking class I SLRPs; either decorin or biglycan. In addition, empirical modeling techniques based on mechanical and image-based measures were employed. Achilles tendons from decorin-null (Dcn(-/-)) and biglycan-null (Bgn(-/-)) C57BL/6 female mice (N=102) were used. Each tendon underwent a dynamic mechanical testing protocol including simultaneous polarized light image capture to evaluate both structural and mechanical properties of each Achilles tendon. An empirical damage model was adapted for application to genetic variation and for use with image based structural properties to predict tendon dynamic mechanical properties. We found that Achilles tendons lacking decorin and biglycan had inferior mechanical and structural properties that were age dependent; and that simple empirical models, based on previously described damage models, were predictive of Achilles tendon dynamic modulus in both decorin- and biglycan-null mice.

The role of animal models in tendon research.

Tendinopathy is a debilitating musculoskeletal condition which can cause significant pain and lead to complete rupture of the tendon, which often requires surgical repair. Due in part to the large spectrum of tendon pathologies, these disorders continue to be a clinical challenge. Animal models are often used in this field of research as they offer an attractive framework to examine the cascade of processes that occur throughout both tendon pathology and repair. This review discusses the structural, mechanical, and biological changes that occur throughout tendon pathology in animal models, as well as strategies for the improvement of tendon healing. Cite this article: Bone Joint Res 2014;3:193-202.

Rotator cuff tears: what have we learned from animal models?

Rotator cuff tendon tears are among the most common soft tissue injuries that occur at the shoulder. Despite advancements in surgical repair techniques, rotator cuff repairs experience a high rate of failure. The common occurrence of tears and the frequency of re-tears require a further understanding of the mechanisms associated with injuries, healing, and regeneration of the rotator cuff. This paper reviews in vivo studies using the various animal shoulder models of the rat, rabbit, sheep, canine, and primate. These animal models have been used to study intrinsic and extrinsic factors leading to shoulder degeneration, various suture techniques, effects of post-surgical treatment, numerous biologic and synthetic scaffolds, and an assortment of biologic augmentations used to accelerate healing. These effects can be examined in a controlled manner using animal models without other confounding factors that sometimes limit clinical studies. The clinically impactful results will be explained to highlight the specific knowledge gained from using animal models in rotator cuff research.

Long durations of immobilization in the rat result in enhanced mechanical properties of the healing supraspinatus tendon insertion site.

Rotator cuff tears frequently occur and can lead to pain and decreased shoulder function. Repair of the torn tendon back to bone is often successful in relieving pain, but failure of the repair commonly occurs. Post-operative activity level is an important treatment component that has received minimal attention for the shoulder, but may have the potential to enhance tendon to bone healing. The objective of this study was to investigate the effect of short and long durations of various activity levels on the healing supraspinatus tendon to bone insertion site. Rotator cuff tears were surgically created in Sprague-Dawley rats by detaching the supraspinatus tendon from its insertion on the humerus and these tears were immediately repaired back to the insertion site. The post-operative activity level was controlled through shoulder immobilization (IM), cage activity (CA), or moderate exercise (EX) for durations of 4 or 16 weeks. The healing tissue was evaluated utilizing biomechanical testing and a quantitative polarized light microscopy method. We found that activity level had no effect on the elastic properties (stiffness, modulus) of the insertion site at four weeks post injury and repair, and a decreased activity level had a positive effect on these properties at 16 weeks (IM>CA=EX). Furthermore, a decreased activity level had the greatest positive effect on these properties over time (IM>CA=EX). The angular deviation of the collagen, a measure of disorganization, was decreased with a decrease in activity level at 4 weeks (IM

Rat supraspinatus tendon expresses cartilage markers with overuse.

The goals of this study were to investigate the response of the rat supraspinatus tendon to overuse at the molecular level using transcriptional profiling, and to identify potential markers of tendinopathy. Adult rats were subjected to an overuse protocol that consists of downhill running (10% grade) at 17 m/min for 1 h/day, 5 days/week, for a total of either 1, 2, or 4 weeks. Another group of rats served as nonrunning time 0 controls. Transcriptional profiling was performed on the supraspinatus and patellar tendons using an Affymetrix rat genome array. A gene was considered to be differentially expressed if the p value from an ANOVA test was less than 0.01 and the difference between runners and controls was at least twofold at any time point. The supraspinatus tendon had increased expression of well-known cartilage genes such as col2a1, aggrecan, and sox9. These genes were not regulated in the patellar tendon, an internal comparator. Few genes associated with inflammation, or angiogenesis, were differentially expressed, and no significant change in the regulation of matrix metalloproteinases was detected. The results of this study suggest that by expressing more cartilage genes, the tendon is converting toward a fibrocartilage phenotype as a result of the repetitive loading and repeated compression of the tendon as it passes through the acromial arch.

Development of tendon structure and function: regulation of collagen fibrillogenesis.

In the tendon, the development of mature mechanical properties is dependent on the assembly of a tendon-specific extracellular matrix. This matrix is synthesized by the tendon fibroblasts and composed of collagen fibrils organized as fibers, as well as fibril-associated collagenous and non-collagenous proteins. All of these components are integrated, during development and growth, to form a functional tissue. During tendon development, collagen fibrillogenesis and matrix assembly progress through multiple steps where each step is regulated independently, culminating in a structurally and functionally mature tissue. Collagen fibrillogenesis occurs in a series of extracellular compartments where fibril intermediates are assembled and mature fibrils grow through a process of post-depositional fusion of the intermediates. Linear and lateral fibril growth occurs after the immature fibril intermediates are incorporated into fibers. The processes are regulated by interactions of extracellular macromolecules with the fibrils. Interactions with quantitatively minor fibrillar collagens, fibril-associated collagens and proteoglycans influence different steps in fibrillogenesis and the extracellular microdomains provide a mechanism for the tendon fibroblasts to regulate these extracellular interactions.

Biomechanics of tendon injury and repair.

Many clinical and experimental studies have investigated how tendons repair in response to an injury. This body of work has led to a greater understanding of tendon healing mechanisms and subsequently to an improvement in their treatment. In this review paper, characterization of normal and healing tendons is first covered. In addition, the debate between intrinsic and extrinsic healing is examined, and the cellular and extracellular matrix response following a tendon injury is detailed. Next, clinical and experimental injury and repair methods utilizing animal models are discussed. Animal models have been utilized to study the effect of various activity levels, motions, injury methods, and injury locations on tendon injury and repair. Finally, current and future treatment modalities for improving tendon healing, such as tissue engineering, cell therapy, and gene therapy, are reviewed.

Tendon to bone healing: differences in biomechanical, structural, and compositional properties due to a range of activity levels.

Little knowledge exists about the healing process of the tendon to bone insertion, and hence little can be done to improve tissue healing. The goal of this study is to describe the healing of the supraspinatus tendon to its bony insertion under a variety of loading conditions. Tendons were surgically detached and repaired in rats. Rat shoulders were then immobilized, allowed cage activity, or exercised. Shoulders that were immobilized demonstrated superior structural (significantly higher collagen orientation), compositional (expression of extracellular matrix genes similar to the uninjured insertion), and quasilinear viscoelastic properties (A = 0.30 +/- 0.10 MPa vs. 0.16 +/- 0.08 MPa, B = 17.4 +/- 2.9 vs. 15.1 +/- 0.9, and tau 2 = 344 +/- 161 s vs. 233 +/- 40 s) compared to those that were exercised, contrary to expectations. With this knowledge of the healing response, treatment modalities for rotator cuff tears can be developed.

The localized expression of extracellular matrix components in healing tendon insertion sites: an in situ hybridization study.

The localized expression of a number of extracellular matrix genes was evaluated over time in a novel rat rotator cuff injury model. The supraspinatus tendons of rats were severed at the bony insertion and repaired surgically. The healing response was evaluated at 1, 2, 4, and 8 weeks post-injury using histologic and in situ hybridization techniques. Expression patterns of collagens (I, II, III, IX, X, XII), proteoglycans (decorin, aggrecan, versican, biglycan, fibromodulin), and other extracellular matrix proteins (elastin, osteocalcin, alkaline phosphatase) were evaluated at the healing tendon to bone insertion site. Histologic results indicate a poor healing response to the injury, with only partial recreation of the insertion site by 8 weeks. In situ hybridization results indicate a specific pattern of genes expressed in each zone of the insertion site (i.e., tendon, fibrocartilage, mineralized cartilage, bone). Overall, expression of collagen types I and XII, aggrecan, and biglycan was increased, while expression of collagen type X and decorin was decreased. Expression of collagen type I, collagen type XII, and biglycan decreased over time, but remained above normal at 8 weeks. Results indicate that the rat supraspinatus tendon is ineffective in recreating the original insertion site, even at 8 weeks post-injury, in the absence of biological or biomechanical enhancements.

A noncontact, nondestructive method for quantifying intratissue deformations and strains.

The function of soft connective tissues is frequently characterized by quantifying tissue strain (e.g., during joint motion). Conventional techniques for quantifying tendon and ligament strain typically provide surface measures, using markers, stain lines or instrumentation that may influence the tissue. An alternative approach is to quantify intratendinous strain by applying texture correlation analysis to magnetic resonance (MR) images. This paper reports the accuracy and reproducibility of this approach by (1) assessing the reproducibility of MR images, (2) assessing texture correlation accuracy using simulated displacements, and (3) comparing texture correlation measures of displacement and strain from MR images to conventional techniques.

Total strain fields of the antero-inferior shoulder capsule under subluxation: a stereoradiogrammetric study.

The antero-inferior capsule (AIC) is the primary restraint to antero-inferior glenohumeral dislocation. This study utilizes a biomechanical model to determine the total strain field of the AIC in a subluxed shoulder. Strains were calculated from two capsule states: a nominal strain state set by inflation and a strained state set by subluxation. Marker coordinates on the AIC were reconstructed from stereoradiographs and strain fields calculated. Peak strain on the glenoid side of the AIC was significantly greater than the humeral side and strain fields were highly variable. This study reports an accurate method for measuring planar strains in a three-dimensional membrane.

Chondrocyte differentiation is modulated by frequency and duration of cyclic compressive loading.

As part of a program of research aimed at determining the role of mechanical forces in connective tissue differentiation, we have developed a model for investigating the effects of dynamic compressive loading on chondrocyte differentiation in vitro. In the current study, we examined the influence of cyclic compressive loading of chick limb bud mesenchymal cells to a constant peak stress of 9.25 kPa during each of the first 3 days in culture. Cells embedded in agarose gel were subjected to uniaxial, cyclic compression at 0.03, 0.15, or 0.33 Hz for 2 h. In addition, load durations of 12, 54, or 120 min were evaluated while holding frequency constant at 0.33 Hz. For a 2 h duration, there was no response to loading at 0.03 Hz. A significant increase in chondrocyte differentiation was associated with loading at 0.15 Hz, and an even greater increase with loading at 0.33 Hz. Holding frequency constant at 0.33 Hz, a loading duration of 12 min elicited no response, whereas chondrocyte differentiation was enhanced by loading for either 54 or 120 min. Although not statistically significant from the 120 min response, average cartilage nodule density and glycosaminoglycan synthesis rate were highest in the 54 min duration group. This result suggests that cells may be sensitive to the level of cumulative (nonrecoverable) compressive strain, as well as to the dynamic strain history.

Proteoglycans and glycosaminoglycan fine structure in the mouse tail tendon fascicle.

The isolated mouse tail tendon fascicle, a functional and homogenous volume of tendon extracellular matrix, was utilized as an experimental system to examine the structure function relationships in tendon. Our previous work using this model system demonstrated relationships between mean collagen fibril diameter and fascicle mechanical properties in isolated tail tendon fascicles from three different groups of mice (3-week and 8-week control and 8-week Mov13 transgenic) K.A. Derwin, L.J. Soslowsky, J. Biomech. Eng. 121 (1999) 598-604. These groups of mice were chosen to obtain tendon tissues with varying collagen fibril structure and/or biochemistry, such that relationships with material properties could be investigated. To further investigate the molecular details of matrix composition and organization underlying tendon function, we report now on the preparation, characterization, and quantitation of fascicle PGs (proteoglycans) from these three groups. The chondroitin sulfate/dermatan sulfate (CS/DS)-substituted PGs, biglycan and decorin, which are the abundant proteoglycans of whole tendons, were also shown to be the predominant PGs in isolated fascicles. Furthermore, similar to the postnatal maturation changes in matrix composition previously reported for whole tendons, isolated fascicles from 8-week mice had lower CS/DS PG contents (both decorin and biglycan) and a higher collagen content than 3-week mice. In addition, CS/DS chains substituted on PGs from 8-week fascicles were shorter (based on a number average) and richer in disulfated disaccharide residues than chains from 3-week mice. Fascicles from 8-week Mov13 transgenic mice were found to contain similar amounts of total collagen and total CS/DS PG as age-matched controls, and CS/DS chain lengths and sulfation also appeared normal. However, both decorin and biglycan in Mov13 tissue migrated slightly faster on sodium dodecyl sulfate polyacrylamide gel electorphoresis (SDS-PAGE) than the corresponding species from 8-week control, and biglycan from the 8-week Mov 13 fascicles appeared to migrate as a more polydisperse band, suggesting the presence of a unique PG population in the transgenic tissue. These observations, together with our biomechanical data [Derwin and Soslowsky, 1999] suggest that compensatory pathways of extracellular matrix assembly and maturation may exist, and that tissue mechanical properties may not be simply determined by the contents of individual matrix components or collagen fibril size.