Brianne K. Connizzo

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

BACKGROUND Collagen 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 APPROACH A 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. RESULTS Decreases 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 tests 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. CONCLUSIONS Local 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.

Structure-function relationships of postnatal tendon development: a parallel to healing.

This review highlights recent research on structure-function relationships in tendon and comments on the parallels between development and healing. The processes of tendon development and collagen fibrillogenesis are reviewed, but due to the abundance of information in this field, this work focuses primarily on characterizing the mechanical behavior of mature and developing tendon, and how the latter parallels healing tendon. The role that extracellular matrix components, mainly collagen, proteoglycans, and collagen cross-links, play in determining the mechanical behavior of tendon will be examined in this review. Specifically, collagen fiber re-alignment and collagen fibril uncrimping relate mechanical behavior to structural alterations during development and during healing. Finally, attention is paid to a number of recent efforts to augment injured tendon and how future efforts could focus on recreating the important structure-function relationships reviewed here.

Determining the contribution of glycosaminoglycans to tendon mechanical properties with a modified shear-lag model.

Tendon has a complex hierarchical structure composed of both a collagenous and a non-collagenous matrix. Despite several studies that have aimed to elucidate the mechanism of load transfer between matrix components, the roles of glycosaminoglycans (GAGs) remain controversial. Thus, this study investigated the elastic properties of tendon using a modified shear-lag model that accounts for the structure and non-linear mechanical response of the GAGs. Unlike prior shear-lag models that are solved either in two dimensions or in axially symmetric geometries, we present a closed-form analytical model for three-dimensional periodic lattices of fibrils linked by GAGs. Using this approach, we show that the non-linear mechanical response of the GAGs leads to a distinct toe region in the stress-strain response of the tendon. The critical strain of the toe region is shown to decrease inversely with fibril length. Furthermore, we identify a characteristic length scale, related to microstructural parameters (e.g. GAG spacing, stiffness, and geometry) over which load is transferred from the GAGs to the fibrils. We show that when the fibril lengths are significantly larger than this length scale, the mechanical properties of the tendon are relatively insensitive to deletion of GAGs. Our results provide a physical explanation for the insensitivity for the mechanical response of tendon to the deletion of GAGs in mature tendons, underscore the importance of fibril length in determining the elastic properties of the tendon, and are in excellent agreement with computationally intensive simulations.

Effect of Age and Proteoglycan Deficiency on Collagen Fiber Re-Alignment and Mechanical Properties in Mouse Supraspinatus Tendon

Collagen fiber realignment is one mechanism by which tendon responds to load. Re-alignment is altered when the structure of tendon is altered, such as in the natural process of aging or with alterations of matrix proteins, such as proteoglycan expression. While changes in re-alignment and mechanical properties have been investigated recently during development, they have not been studied in (1) aged tendons, or (2) in the absence of key proteoglycans. Collagen fiber re-alignment and the corresponding mechanical properties are quantified throughout tensile mechanical testing in both the insertion site and the midsubstance of mouse supraspinatus tendons in wild type (WT), decorin-null (Dcn(-/-)), and biglycan-null (Bgn(-/-)) mice at three different ages (90 days, 300 days, and 570 days). Percent relaxation was significantly decreased with age in the WT and Dcn(-/-) tendons, but not in the Bgn(-/-) tendons. Changes with age were found in the linear modulus at the insertion site where the 300 day group was greater than the 90 day and 570 day group in the Bgn(-/-) tendons and the 90 day group was smaller than the 300 day and 570 day groups in the Dcn(-/-) tendons. However, no changes in modulus were found across age in WT tendons were found. The midsubstance fibers of the WT and Bgn(-/-) tendons were initially less aligned with increasing age. The re-alignment was significantly altered with age in the WT tendons, with older groups responding to load later in the mechanical test. This was also seen in the Dcn(-/-) midsubstance and the Bgn(-/-) insertion, but not in the other locations. Although some studies have found changes in the WT mechanical properties with age, this study did not support those findings. However, it did show fiber re-alignment changes at both locations with age, suggesting a breakdown of tendons ability to respond to load in later ages. In the proteoglycan-null tendons however, there were changes in the mechanical properties, accompanied only by location-dependent re-alignment changes, suggesting a site-specific role for these molecules in loading. Finally, changes in the mechanical properties did not occur in concert with changes in re-alignment, suggesting that typical mechanical property measurements alone are insufficient to describe how structural alterations affect tendons response to load.

Effect of Preconditioning and Stress Relaxation on Local Collagen Fiber Re-Alignment: Inhomogeneous Properties of Rat Supraspinatus Tendon

Repeatedly and consistently measuring the mechanical properties of tendon is important but presents a challenge. Preconditioning can provide tendons with a consistent loading history to make comparisons between groups from mechanical testing experiments. However, the specific mechanisms occurring during preconditioning are unknown. Previous studies have suggested that microstructural changes, such as collagen fiber re-alignment, may be a result of preconditioning. Local collagen fiber re-alignment is quantified throughout tensile mechanical testing using a testing system integrated with a polarized light setup, consisting of a backlight, 90 deg-offset rotating polarizer sheets on each side of the test sample, and a digital camera, in a rat supraspinatus tendon model, and corresponding mechanical properties are measured. Local circular variance values are compared throughout the mechanical test to determine if and where collagen fiber re-alignment occurred. The inhomogeneity of the tendon is examined by comparing local circular variance values, optical moduli and optical transition strain values. Although the largest amount of collagen fiber re-alignment was found during preconditioning, significant re-alignment was also demonstrated in the toe and linear regions of the mechanical test. No significant changes in re-alignment were seen during stress relaxation. The insertion site of the supraspinatus tendon demonstrated a lower linear modulus and a more disorganized collagen fiber distribution throughout all mechanical testing points compared to the tendon midsubstance. This study identified a correlation between collagen fiber re-alignment and preconditioning and suggests that collagen fiber re-alignment may be a potential mechanism of preconditioning and merits further investigation. In particular, the conditions necessary for collagen fibers to re-orient away from the direction of loading and the dependency of collagen reorganization on its initial distribution must be examined.

Diabetes alters mechanical properties and collagen fiber re-alignment in multiple mouse tendons.

Tendons function to transfer load from muscle to bone through their complex composition and hierarchical structure, consisting mainly of type I collagen. Recent evidence suggests that type II diabetes may cause alterations in collagen structure, such as irregular fibril morphology and density, which could play a role in the mechanical function of tendons. Using the db/db mouse model of type II diabetes, the diabetic skin was found to have impaired biomechanical properties when compared to the non-diabetic group. The purpose of this study was to assess the effect of diabetes on biomechanics, collagen fiber re-alignment, and biochemistry in three functionally different tendons (Achilles, supraspinatus, patellar) using the db/db mouse model. Results showed that cross-sectional area and stiffness, but not modulus, were significantly reduced in all three tendons. However, the tendon response to load (transition strain, collagen fiber re-alignment) occurred earlier in the mechanical test, contrary to expectations. In addition, the patellar tendon had an altered response to diabetes when compared to the other two tendons, with no changes in fiber re-alignment and decreased collagen content at the midsubstance of the tendon. Overall, type II diabetes alters tendon mechanical properties and the dynamic response to load.

The Detrimental Effects of Systemic Ibuprofen Delivery on Tendon Healing Are Time-Dependent

BackgroundCurrent clinical treatment after tendon repairs often includes prescribing NSAIDs to limit pain and inflammation. The negative influence of NSAIDs on bone repair is well documented, but their effects on tendon healing are less clear. While NSAIDs may be detrimental to early tendon healing, some evidence suggests that they may improve healing if administered later in the repair process.Questions/purposesWe asked whether the biomechanical and histologic effects of systemic ibuprofen administration on tendon healing are influenced by either immediate or delayed drug administration.MethodsAfter bilateral supraspinatus detachment and repair surgeries, rats were divided into groups and given ibuprofen orally for either Days 0 to 7 (early) or Days 8 to 14 (delayed) after surgery; a control group did not receive ibuprofen. Healing was evaluated at 1, 2, and 4 weeks postsurgery through biomechanical testing and histologic assessment.ResultsBiomechanical evaluation resulted in decreased stiffness and modulus at 4 weeks postsurgery for early ibuprofen delivery (mean ± SD [95% CI]: 10.8 ± 6.4 N/mm [6.7–14.8] and 8.9 ± 5.9 MPa [5.4–12.3]) when compared to control repair (20.4 ± 8.6 N/mm [16.3–24.5] and 15.7 ± 7.5 MPa [12.3–19.2]) (p = 0.003 and 0.013); however, there were no differences between the delayed ibuprofen group (18.1 ± 7.4 N/mm [14.2–22.1] and 11.5 ± 5.6 MPa [8.2–14.9]) and the control group. Histology confirmed mechanical results with reduced fiber reorganization over time in the early ibuprofen group.ConclusionsEarly administration of ibuprofen in the postoperative period was detrimental to tendon healing, while delayed administration did not affect tendon healing.Clinical RelevanceHistorically, clinicians have often prescribed ibuprofen after tendon repair, but this study suggests that the timing of ibuprofen administration is critical to adequate tendon healing. This research necessitates future clinical studies investigating the use of ibuprofen for pain control after rotator cuff repair and other tendon injuries.

Examining differences in local collagen fiber crimp frequency throughout mechanical testing in a developmental mouse supraspinatus tendon model.

Crimp morphology is believed to be related to tendon mechanical behavior. While crimp has been extensively studied at slack or nondescript load conditions in tendon, few studies have examined crimp at specific, quantifiable loading conditions. Additionally, the effect of the number of cycles of preconditioning on collagen fiber crimp behavior has not been examined. Further, the dependence of collagen fiber crimp behavior on location and developmental age has not been examined in the supraspinatus tendon. Local collagen fiber crimp frequency is quantified throughout tensile mechanical testing using a flash freezing method immediately following the designated loading protocol. Samples are analyzed quantitatively using custom software and semi-quantitatively using a previously established method to validate the quantitative software. Local collagen fiber crimp frequency values are compared throughout the mechanical test to determine where collagen fiber frequency changed. Additionally, the effect of the number of preconditioning cycles is examined compared to the preload and toe-region frequencies to determine if increasing the number of preconditioning cycles affects crimp behavior. Changes in crimp frequency with age and location are also examined. Decreases in collagen fiber crimp frequency were found at the toe-region at all ages. Significant differences in collagen fiber crimp frequency were found between the preload and after preconditioning points at 28 days. No changes in collagen fiber crimp frequency were found between locations or between 10 and 28 days old. Local collagen fiber crimp frequency throughout mechanical testing in a postnatal developmental mouse SST model was measured. Results confirmed that the uncrimping of collagen fibers occurs primarily in the toe-region and may contribute to the tendons nonlinear behavior. Additionally, results identified changes in collagen fiber crimp frequency with an increasing number of preconditioning cycles at 28 days, which may have implications on the measurement of mechanical properties and identifying a proper reference configuration.

Micromechanical poroelastic finite element and shear-lag models of tendon predict large strain dependent Poisson's ratios and fluid expulsion under tensile loading.

As tendons are loaded, they reduce in volume and exude fluid to the surrounding medium. Experimental studies have shown that tendon stretching results in a Poissons ratio greater than 0.5, with a maximum value at small strains followed by a nonlinear decay. Here we present a computational model that attributes this macroscopic observation to the microscopic mechanism of the load transfer between fibrils under stretch. We develop a finite element model based on the mechanical role of the interfibrillar-linking elements, such as thin fibrils that bridge the aligned fibrils or macromolecules such as glycosaminoglycans (GAGs) in the interfibrillar sliding and verify it with a theoretical shear-lag model. We showed the existence of a previously unappreciated structure-function mechanism whereby the Poissons ratio in tendon is affected by the strain applied and interfibrillar-linker properties, and together these features predict tendon volume shrinkage under tensile loading. During loading, the interfibrillar-linkers pulled fibrils toward each other and squeezed the matrix, leading to the Poissons ratio larger than 0.5 and fluid expulsion. In addition, the rotation of the interfibrillar-linkers with respect to the fibrils at large strains caused a reduction in the volume shrinkage and eventual nonlinear decay in Poissons ratio at large strains. Our model also predicts a fluid flow that has a radial pattern toward the surrounding medium, with the larger fluid velocities in proportion to the interfibrillar sliding.

Targeted Deletion of Collagen V in Tendons and Ligaments Results in a Classic Ehlers-Danlos Syndrome Joint Phenotype

Collagen V mutations underlie classic Ehlers-Danlos syndrome, and joint hypermobility is an important clinical manifestation. We define the function of collagen V in tendons and ligaments, as well as the role of alterations in collagen V expression in the pathobiology in classic Ehlers-Danlos syndrome. A conditional Col5a1(flox/flox) mouse model was bred with Scleraxis-Cre mice to create a targeted tendon and ligament Col5a1-null mouse model, Col5a1(Δten/Δten). Targeting was specific, resulting in collagen V-null tendons and ligaments. Col5a1(Δten/Δten) mice demonstrated decreased body size, grip weakness, abnormal gait, joint laxity, and early-onset osteoarthritis. These gross changes were associated with abnormal fiber organization, as well as altered collagen fibril structure with increased fibril diameters and decreased fibril number that was more severe in a major joint stabilizing ligament, the anterior cruciate ligament (ACL), than in the flexor digitorum longus tendon. The ACL also had a higher collagen V content than did the flexor digitorum longus tendon. The collagen V-null ACL and flexor digitorum longus tendon both had significant alterations in mechanical properties, with ACL exhibiting more severe changes. The data demonstrate critical differential regulatory roles for collagen V in tendon and ligament structure and function and suggest that collagen V regulatory dysfunction is associated with an abnormal joint phenotype, similar to the hypermobility phenotype in classic Ehlers-Danlos syndrome.