The dynamics of collagen uncrimping and lateral contraction in tendon and the effect of ionic concentration

Under tensile loading, tendon undergoes a number of unique structural changes that govern its mechanical response. For example, stretching a tendon is known to induce both the progressive “uncrimping” of wavy collagen fibrils and extensive lateral contraction mediated by fluid flow out of the tissue. However, it is not known whether these processes are interdependent. Moreover, the rate-dependence of collagen uncrimping and its contribution to tendon's viscoelastic mechanical properties are unknown. Therefore, the objective of this study was to (a) develop a methodology allowing for simultaneous measurement of crimp, stress, axial strain and lateral contraction in tendon under dynamic loading; (b) determine the interdependence of collagen uncrimping and lateral contraction by testing tendons in different swelling conditions; and (c) assess how the process of collagen uncrimping depends on loading rate. Murine flexor carpi ulnaris (FCU) tendons in varying ionic environments were dynamically stretched to a set strain level and imaged through a plane polariscope with the polarizer and analyzer at a fixed angle. Analysis of the resulting images allowed for direct measurement of the crimp frequency and indirect measurement of the tendon thickness. Our findings demonstrate that collagen uncrimping and lateral contraction can occur independently and interstitial fluid impacts tendon mechanics directly. Furthermore, tensile stress, transverse contraction and degree of collagen uncrimping were all rate-dependent, suggesting that collagen uncrimping plays a role in tendon's dynamic mechanical response. This study is the first to characterize the time-dependence of collagen uncrimping in tendon, and establishes structure–function relationships for healthy tendons that can be used to better understand and assess changes in tendon mechanics after disease or injury.     

The effect of altered loading following rotator cuff tears in a rat model on the regional mechanical properties of the long head of the biceps tendon

Biceps tendon pathology is a common clinical problem often seen in conjunction with rotator cuff tears. A previous study found detrimental changes to biceps tendons in the presence of rotator cuff tears in a rat model. Therefore, the objective of this study was to utilize this model along with models of altered loading to investigate the effect of altered loading on the initiation of these detrimental changes. We created supraspinatus and infraspinatus rotator cuff tears in the rat and followed these tears with either increased or decreased loading. Mechanical properties were determined along the length of the biceps tendon 4 and 8 weeks following injury. At the insertion site, stiffness increased with decreased loading, while detrimental changes were seen with increased loading 4 weeks following detachments. Increased loading resulted in decreased mechanical properties along the entire tendon length at both time points. Decreased loading resulted in both increased and decreased tendon properties at different regions of the tendon at 4 weeks, but by 8 weeks, there were no differences between decreased loading and detachment alone. We could not conclude where changes begin in the tendon with altered loading, but did demonstrate that regional differences exist. These results support that there is an effect of altered loading, as decreased loading resulted in variable changes at 4 weeks that were no different from detachment alone by 8 weeks, and increased loading resulted in detrimental properties along the entire length at both 4 and 8 weeks.     

Transient decreases in forelimb gait and ground reaction forces following rotator cuff injury and repair in a rat model

Due to inadequate healing, surgical repairs of torn rotator cuff tendons often fail, limiting the recovery of upper extremity function. The rat is frequently used to study rotator cuff healing; however, there are few systems capable of quantifying forelimb function necessary to interpret the clinical significance of tissue level healing. We constructed a device to capture images, ground reaction forces and torques, as animals ambulated in a confined walkway, and used it to evaluate forelimb function in uninjured control and surgically injured/repaired animals. Ambulatory data were recorded before (D–1), and 3, 7, 14, 28 and 56 days after surgery. Speed as well as step width and length were determined by analyzing ventral images, and ground reaction forces were normalized to body weight. Speed averaged 22±6 cm/s and was not affected by repair or time. Step width and length of uninjured animals compared well to values measured with our previous system. Forelimbs were used primarily for braking (?1.6±1.5% vs +2.5±0.6%), bore less weight than hind limbs (49±5% vs 58±4%), and showed no differences between sides (49±5% vs 46±5%) for uninjured control animals. Step length and ground reaction forces of the repaired animals were significantly less than control initially (days 3, 7 and 14 post-surgery), but not by day 28. Our new device provided uninjured ambulatory data consistent with our previous system and available literature, and measured reductions in forelimb function consistent with the deficit expected by our surgical model.     

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.     

Decorin expression is important for age-related changes in tendon structure and mechanical properties

The aging population is at an increased risk of tendon injury and tendinopathy. Elucidating the molecular basis of tendon aging is crucial to understanding the age-related changes in structure and function in this vulnerable tissue. In this study, the structural and functional features of tendon aging are investigated. In addition, the roles of decorin and biglycan in the aging process were analyzed using transgenic mice at both mature and aged time points. Our hypothesis is that the increase in tendon injuries in the aging population is the result of altered structural properties that reduce the biomechanical function of the tendon and consequently increase susceptibility to injury. Decorin and biglycan are important regulators of tendon structure and therefore, we further hypothesized that decreased function in aged tendons is partly the result of altered decorin and biglycan expression. Biomechanical analyses of mature (day 150) and aged (day 570) patellar tendons revealed deteriorating viscoelastic properties with age. Histology and polarized light microscopy demonstrated decreased cellularity, alterations in tenocyte shape, and reduced collagen fiber alignment in the aged tendons. Ultrastructural analysis of fibril diameter distributions indicated an altered distribution in aged tendons with an increase of large diameter fibrils. Aged wild type tendons maintained expression of decorin which was associated with the structural and functional changes seen in aged tendons. Aged patellar tendons exhibited altered and generally inferior properties across multiple assays. However, decorin-null tendons exhibited significantly decreased effects of aging compared to the other genotypes. The amelioration of the functional deficits seen in the absence of decorin in aged tendons was associated with altered tendon fibril structure. Fibril diameter distributions in the decorin-null aged tendons were comparable to those observed in the mature wild type tendon with the absence of the subpopulation containing large diameter fibrils. Collectively, our findings provide evidence for age-dependent alterations in tendon architecture and functional activity, and further show that lack of stromal decorin attenuates these changes.     

Rotator cuff tendon strain correlates with tear propagation

Rotator cuff tears are a common tendon injury often requiring surgical treatment. Understanding the relationships between tear size, tendon loading, and tendon strain adjacent to a rotator cuff tear can provide important insights into predicting the likelihood of propagation to larger tears which would influence clinical treatment. Previous studies assume that an increase in strain correlates with an increase in risk of tear propagation. However, these studies did not explicitly investigate these important relationships. Therefore, the objective of this study was to quantify two-dimensional strain fields adjacent to a rotator cuff tendon tear under loading to failure and to assess the relationship between tendon strain and tear size. Sheep infraspinatus tendons were used to evaluate the effect of tear size on principal strains in the region adjacent to the tear. The relationship between strain, tear propagation, and the direction of tear propagation was quantified. Results showed that principal strains linearly correlated with tear propagation and that tear propagation began at strains as low as 1.7%. In addition, tears propagated in the direction of highest maximum and lowest minimum principal strain. Finally, maximum and minimum principal strains were higher and lower, respectively, adjacent to larger tears compared to smaller tears. Findings from this study validate the use of local strain adjacent to a rotator cuff tear as an indicator of the risk and direction of tear propagation.     

Tendon healing in interleukin-4 and interleukin-6 knockout mice

Cytokines have been shown to play an important role in tendon and ligament healing by regulating cellular differentiation and activity. The majority of studies that have investigated the role of cytokines in tendon and ligament healing have added them to injured tissue and assessed their effect. Because the efficacy of exogenously applying cytokines is dependent upon many factors such as the correct dosage, timing, and frequency, conflicting results are often reported. To avoid these factors, this study used transgenic mice with knockouts of interleukin-4 (IL4 -/-) and interleukin-6 (IL6 -/-) to investigate their role in tendon healing. Because of the reported roles of both of these cytokines in inflammation and fibroplasia, it was hypothesized that the order of organizational, geometric, and mechanical properties would be (greatest to least) injured IL6 -/-, injured control, and injured IL4 -/- mice. In addition, it was hypothesized that specific cytokines would be upregulated in each knockout group, but not compensate for the lack of IL-4 or IL-6. Mechanical and organizational properties of injured tendons from IL6 -/- mice were inferior to that of control and IL4 -/- mice despite the upregulation of the pro-inflammatory cytokine TNF-alpha. Temporal levels of IL-10 and IL-13 in the IL4 -/- mice resulted in comparable and even superior properties when compared to CTL mice. This study shows that IL-6 could not be compensated for and plays an important role in tendon healing. This study also supports the use of this animal model to further investigate tendon healing.     

Biaxial tensile testing and constitutive modeling of human supraspinatus tendon

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon's two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R(2) ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.     

Characterizing local collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse supraspinatus tendon model

BackgroundCollagen fiber re-alignment and uncrimping are two postulated mechanisms of tendon structural response to load. Recent studies have examined structural changes in response to mechanical testing in a postnatal development mouse supraspinatus tendon model (SST), however, those changes in the mature mouse have not been characterized. The objective of this study was to characterize collagen fiber re-alignment and crimp behavior throughout mechanical testing in a mature mouse SST.Method of approachA tensile mechanical testing set-up integrated with a polarized light system was utilized for alignment and mechanical analysis. Local collagen fiber crimp frequency was quantified immediately following the designated loading protocol using a traditional tensile set up and a flash-freezing method. The effect of number of preconditioning cycles on collagen fiber re-alignment, crimp frequency and mechanical properties in midsubstance and insertion site locations were examined.ResultsDecreases in collagen fiber crimp frequency were identified at the toe-region of the mechanical test at both locations. The insertion site re-aligned throughout the entire test, while the midsubstance re-aligned during preconditioning and the test's linear-region. The insertion site demonstrated a more disorganized collagen fiber distribution, lower mechanical properties and a higher cross-sectional area compared to the midsubstance location.ConclusionsLocal collagen fiber re-alignment, crimp behavior and mechanical properties were characterized in a mature mouse SST model. The insertion site and midsubstance respond differently to mechanical load and have different mechanisms of structural response. Additionally, results support that collagen fiber crimp is a physiologic phenomenon that may explain the mechanical test toe-region.