The Micromechanical Environment of the Impinged Achilles Tendon.

Although tendon predominantly experiences longitudinal tensile forces, transverse forces due to impingement from bone are implicated in both physiological and pathophysiological processes. However, prior studies have not characterized the micromechanical strain environment in the context of tendon impingement. To address this knowledge gap, mouse hindlimb explants are imaged on a multiphoton microscope, and image stacks of the same population of tendon cells are obtained in the Achilles tendon before and after dorsiflexion-induced impingement by the heel bone. Based on the acquired images, multiaxial strains are measured at the extracellular matrix (ECM), pericellular matrix (PCM), and cell scales. Impingement generated substantial transverse compression at the matrix-scale, which led to longitudinal stretching of cells, increased cell aspect ratio, and enormous volumetric compression of the PCM. These experimental results are corroborated by a finite element model, which further demonstrated that impingement produces high cell surface stresses and strains that greatly exceed those brought about by longitudinal tension. Moreover, in both experiments and simulations, impingement-generated microscale stresses and strains are highly dependent on initial cell-cell gap spacing. Identifying factors that influence the microscale strain environment generated by impingement could contribute to a more mechanistic understanding of impingement-induced tendinopathies.

Aged Tendons Exhibit Altered Mechanisms of Strain-Dependent Extracellular Matrix Remodeling.

Aging is a primary risk factor for degenerative tendon injuries, yet the etiology and progression of this degeneration are poorly understood. While aged tendons have innate cellular differences that support a reduced ability to maintain mechanical tissue homeostasis, the response of aged tendons to altered levels of mechanical loading has not yet been studied. To address this question, we subjected young and aged murine flexor tendon explants to various levels of in vitro tensile strain. We first compared the effect of static and cyclic strain on matrix remodeling in young tendons, finding that cyclic strain is optimal for studying remodeling in vitro. We then investigated the remodeling response of young and aged tendon explants after 7 days of varied mechanical stimulus (stress deprivation, 1%, 3%, 5%, or 7% cyclic strain) via assessment of tissue composition, biosynthetic capacity, and degradation profiles. We hypothesized that aged tendons would show muted adaptive responses to changes in tensile strain and exhibit a shifted mechanical setpoint, at which the remodeling balance is optimal. Interestingly, we found that 1% cyclic strain best maintains native physiology while promoting extracellular matrix (ECM) turnover for both age groups. However, aged tendons display fewer strain-dependent changes, suggesting a reduced ability to adapt to altered levels of mechanical loading. This work has a significant impact on understanding the regulation of tissue homeostasis in aged tendons, which can inform clinical rehabilitation strategies for treating elderly patients.

Aged Tendons Exhibit Altered Mechanisms of Strain-Dependent Extracellular Matrix Remodeling.

Aging is a primary risk factor for degenerative tendon injuries, yet the etiology and progression of this degeneration is poorly understood. While aged tendons have innate cellular differences that support a reduced ability to maintain mechanical tissue homeostasis, the response of aged tendons to altered levels of mechanical loading has not yet been studied. To address this question, we subjected young and aged murine flexor tendon explants to various levels of in vitro tensile strain. We first compared the effect of static and cyclic strain on matrix remodeling in young tendons, finding that cyclic strain is optimal for studying remodeling in vitro. We then investigated the remodeling response of young and aged tendon explants after 7 days of varied mechanical stimulus (stress-deprivation, 1%, 3%, 5%, or 7% cyclic strain) via assessment of tissue composition, biosynthetic capacity, and degradation profiles. We hypothesized that aged tendons would show muted adaptive responses to changes in tensile strain and exhibit a shifted mechanical setpoint, at which the remodeling balance is optimal. Interestingly, we found 1% cyclic strain best maintains native physiology while promoting ECM turnover for both age groups. However, aged tendons display fewer strain-dependent changes, suggesting a reduced ability to adapt to altered levels of mechanical loading. This work has significant impact in understanding the regulation of tissue homeostasis in aged tendons, which can inform clinical rehabilitation strategies for treating elderly patients.

Aged tendons lack adaptive response to acute compressive injury.

Rotator cuff tendinopathy has a multifactorial etiology, with both aging and external compression found to influence disease progression. However, tendon's response to these factors is still poorly understood and in vivo animal models make it difficult to decouple these effects. Therefore, we developed an explant culture model that allows us to directly apply compression to tendons and then observe their biological responses. Using this model, we applied a single acute compressive injury to C57BL/6J flexor digitorum longus tendon explants and observed changes in viability, metabolic activity, matrix composition, matrix biosynthesis, matrix structure, gene expression, and mechanical properties. We hypothesized that a single acute compressive load would result in an injury response in tendon and that this effect would be amplified in aged tendons. We found that young tendons had increased matrix turnover with a decrease in small leucine-rich proteoglycans, increase in compression-resistant proteoglycan aggrecan, increase in collagen synthesis, and an upregulation of collagen-degrading MMP-9. Aged tendons lacked any of these adaptive responses and instead had decreased metabolic activity and collagen synthesis. This implies that aged tendons lack the adaptation mechanisms required to return to homeostasis, and therefore are at greater risk for compression-induced injury. Overall, we present a novel compressive injury model that demonstrates lasting age-dependent changes and has the potential to examine the long-term response of tendon to a variety of compressive loading conditions.

Ultrasound strain mapping of the mouse Achilles tendon during passive dorsiflexion.

Immediately prior to inserting into bone, many healthy tendons experience impingement from nearby bony structures. However, super-physiological levels of impingement are implicated in insertional tendinopathies. Unfortunately, the mechanisms underlying the connection between impingement and tendon pathology remain poorly understood, in part due to the shortage of well-characterized animal models of impingement at clinically relevant sites. As a first step towards developing a model of excessive tendon impingement, the objective of this study was to characterize the mechanical strain environment in the mouse Achilles tendon insertion under passive dorsiflexion and confirm that - like humans - mice experience impingement of the tendon insertion from the calcaneus (heel bone) in dorsiflexed ankle positions. Based on previous work in humans, we hypothesized that during dorsiflexion, the mouse Achilles tendon insertion would experience high levels of transverse compressive strain due to calcaneal impingement. A custom-built loading platform was used to apply passive dorsiflexion, while an ultrasound transducer positioned over the Achilles tendon captured radiofrequency images. A non-rigid image registration algorithm was then used to map the transverse compressive strain based on the acquired ultrasound image sequences. Our results demonstrate that during passive dorsiflexion, transverse compressive strains were produced throughout the Achilles tendon, with significantly larger strain magnitudes at the tendon insertion than at the midsubstance. Furthermore, there was increasing transverse compressive strain observed within the Achilles tendon as a function of increasing dorsiflexion angle. This study enhances our understanding of the unique mechanical loading environment of the Achilles tendon under physiologically relevant conditions.